CN118715235A - Compositions and methods for preventing and/or treating Clostridium difficile infections caused by clade 2 strains - Google Patents

Abstract

The present application provides an isolated polypeptide that specifically binds to TcdB2 and/or TcdB4 (particularly TcdB4) from a branch 2 strain of Clostridium difficile, a pharmaceutical composition comprising the isolated polypeptide, and the use of the isolated polypeptide and the pharmaceutical composition in preventing and treating Clostridium difficile infections caused by branch 2 strains. The isolated polypeptide comprises the amino acid sequence of any one of SEQ ID NO:1‑5. The present application also provides a nucleic acid molecule encoding the isolated polypeptide, a vector or expression cassette and/or cell comprising the nucleic acid molecule, and a method for producing the isolated polypeptide. In addition, the present application provides a method for preventing and/or treating Clostridium difficile infections caused by branch 2 strains.

Description

Compositions and methods for preventing and/or treating clostridium difficile infection caused by a branch 2 strain

Technical Field

The present invention relates generally to isolated polypeptides that specifically bind to TcdB2 and/or TcdB4 (in particular TcdB 4) from clostridium difficile (Clostridioides difficile or c.diffiile, formerly Clostridium difficile) strain branch 2, pharmaceutical compositions comprising the isolated polypeptides and the use of the isolated polypeptides and pharmaceutical compositions in the prevention and/or treatment of Clostridium Difficile Infection (CDI) caused by branch 2 strain. The invention also relates to nucleic acid molecules encoding the isolated polypeptides, vectors or expression cassettes and/or cells comprising the nucleic acid molecules and methods of producing the isolated polypeptides. Furthermore, the present invention relates to a method for preventing and/or treating clostridium difficile infection caused by a strain of branch 2.

Background

Clostridium difficile (Clostridioides difficile or c.diffiile, formerly Clostridium Difficile) Infection (CDI) is a major cause of nosocomial and community-acquired diarrhea and gastroenteritis-related deaths in developed countries, with about 50 and 15,000 deaths per year in the united states (Guh et al 2020; lessa et al 2015). In recent years, the global burden of CDI has been exacerbated by the advent and widespread spread of virulent strains (Guh et al 2020; he et al 2013; hunt and Ballard, 2013). The emergence of clostridium difficile hyper-virulence branch 2 is associated with severe symptoms and accounts for over 20% of global infections.

Pathogenic clostridium difficile produces three known exotoxins: toxin a (TcdA), toxin B (TcdB) and Clostridium Difficile Transferase (CDT). Clostridium difficile uses these toxins to disrupt the intestinal epithelial barrier, cause tissue damage and gain nutrition, provoke or inhibit inflammation, and promote colonization (Aktories et al, 2017; coordin et al, 2016; fletcher et al, 2021; pruss and Sonnenburg,2021; vanlnsberghe et al, 2020; xu et al, 2014). Of the three toxins, tcdB plays a key role in causing gastrointestinal disease, as all pathogenic clostridium difficile contain a functional TcdB gene, and the lack of TcdB (rather than TcdA or CDT) can greatly attenuate virulence of clinical strains in animal models (Carter et al, 2015; kuehne et al, 2010; kuehne et al, 2014; lyras et al, 2009).

TcdB is a member of the Large Clostridial Toxin (LCT) family that enters target cells by receptor-mediated endocytosis and glycosylates small gtpase proteins, leading to cytoskeletal dysfunction and ultimately cell death (Aktories et al, 2017; chandrasekaran and Lacy,2017; voth and Ballard, 2005). Recent genomic and functional studies report that TcdB can be divided into at least eight natural variants/subtypes, of which types 1 to 4 are the major variants associated with human disease (Mansfield et al 2020; shen et al 2020). In the colon, prototypes (type 1, tcdB 1) and type 3 TcdB (TcdB 3) use Wnt receptor Frizzled (FZD) to disrupt the epithelial barrier and use chondroitin sulfate proteoglycan 4 (CSPG 4) to subsequently damage the subepithelial myofibroblast layer (Chen et al, 2018; chen et al, 2021; pan et al, 2021; tao et al, 2016; yuan et al, 2015). Clostridium difficile strains from multi-site sequence typing (MLST) branch 2, also known as the virulence branch (Stabler et al, 2006), express only two TcdB variants (TcdB 2 and TcdB 4) that do not recognize FZD (Chung et al, 2018; henkel et al, 2020; lopez-Urena et al, 2019; pan et al, 2021; simeon et al, 2019). How these TcdB variants target the intestinal epithelium to trigger lesions remains unknown.

Currently, the development of preventive and therapeutic methods for Clostridium Difficile Infection (CDI) is mainly focused on the following directions: antibiotic therapy, fecal microbiota transplantation, toxin-neutralizing antibodies/immunoglobulins, vaccine development, small molecule inhibitors, and the like. These methods are used to treat clostridium difficile infections in different situations, each of which has significant advantages and disadvantages in use. Antibiotic therapy is a traditional approach, and its effectiveness and recurrence of CDI are major problems associated with this therapy. Antibiotic therapy is not suitable for preventing CDI. Fecal microbiota transplantation is a novel method of treatment and intervention. At present, treatments related to fecal microbiota transplantation are in the phase III clinical trials in the united states, which have major problems of rejection, need for surgery, and public acceptance. Clostridium difficile expresses mainly two exotoxins TcdA and TcdB, with TcdB being the decisive causative agent. The use of toxin-neutralizing immune molecules is an effective method of prevention and treatment. The main purpose of toxin-neutralizing antibodies/immunoglobulins is to alleviate and prevent acute infectious diseases, and related technological routes are rapidly evolving. FDA approved drugs have been marketed in the united states for the prevention and treatment of clostridium difficile infection (ZINPLAVA ^TM developed by Merck corporation, a monoclonal antibody to the combination of aclostrobin Shu Shankang and belotoumab). Currently, there is no significant progress in the development of vaccines and small molecule inhibitors.

Although TcdB is the main virulence factor for clostridium difficile, the branch 2 strain expresses only two variants of TcdB (TcdB 2 and TcdB 4), which use unknown receptors other than classical TcdB. Thus, there is a need to identify receptors for TcdB variants from branch 2 strains and to develop new drugs and/or therapies for the prevention and treatment of CDI caused by branch 2 strains.

Disclosure of Invention

The object of the present invention is to identify receptors for the variants of TcdB expressed by clostridium difficile branch 2 strain (TcdB 2 and TcdB 4).

It is another object of the present invention to find inhibitors of TcdB variants expressed by clostridium difficile branch 2 strains (TcdB 2 and TcdB 4), and further to find effective therapies for preventing and/or treating CDI caused by branch 2 strains.

To achieve the above objective, the inventors performed CRISPR/Cas9 screening for TcdB4 and identified Tissue Factor Pathway Inhibitor (TFPI) as its receptor. Using cryo-electron microscopy, the inventors determined that full length TcdB4 and TFPIA common receptor binding region is defined for TcdB. Residue variation within this region divides the major TcdB variants into two classes: one class recognizes frizzled proteins (FZDs) and the other class recognizes TFPI. TFPI is highly expressed in the intestinal glands, whereas recombinant TFPI protects the colonic epithelium from TcdB 4/2. These findings establish TFPI as the colonic crypt receptor for TcdB from clostridium difficile branch 2 and reveal a new mechanism of CDI pathogenesis. Based on these findings, the inventors have completed the present invention.

In one aspect, the invention provides an isolated polypeptide that specifically binds to TcdB2 and/or TcdB4, wherein the isolated polypeptide comprises the amino acid sequence of SEQ ID No.1 or 2.

In some embodiments, the isolated polypeptide comprises any one of the amino acid sequences of SEQ ID NOs 3 to 5.

In some embodiments, the isolated polypeptide comprises R135, R140, I138, I133, M162, L159 (numbered according to SEQ ID NO:22 or 23, containing a signal peptide) of TFPI.

In some embodiments, the isolated polypeptide is soluble. In some embodiments, the isolated polypeptide is TFPI and derivatives thereof. In some embodiments, the TFPI is human TFPI or mouse Tfpi or a derivative thereof. Human TFPI includes two variants, TFPI alpha (SEQ ID NO: 3) and TFPI beta (SEQ ID NO: 4), without signal peptide, and mouse Tfpi is Tfpi beta (SEQ ID NO: 5).

In some embodiments, R135, R140, I138, I133, M162, L159 in TFPI are identified as critical residues that specifically bind to TcdB 4.

In some embodiments, the isolated polypeptide specifically binds to TcdB4, particularly the receptor binding domain (DRBD) TcdB ^4842-1834 fragment of TcdB4 (SEQ ID NO: 15) or the TcdB4 ^1285-1834 fragment (SEQ ID NO: 16). E1433、D1467、D1468、E1469、S1598、L1599、L1434、K1435、M1438、V1492、L1494、I1496、L1489、P1506、Y1510(SEQ ID NO:9) of TcdB4 is a key residue that recognizes the receptor. The receptor binding domain (DRBD) of TcdB4 is represented by SEQ ID NO. 14 (i.e., positions 841-1834 of SEQ ID NO. 9).

In some embodiments, the isolated polypeptide specifically binds to TcdB2, particularly the receptor binding domain (DRBD) of TcdB2, the TcdB2 ^841-1833 fragment (SEQ ID NO: 29) or the TcdB2 ^1284-1833 fragment (SEQ ID NO: 30). E1432, D1466, D1467, S1597, L1598, L1433, K1434, M1437, V1491, L1493 and L1488 of TcdB2 (SEQ ID NO: 7) are key residues that recognize receptors. The receptor binding domain (DRBD) of TcdB2 is represented by SEQ ID NO. 28 (i.e., positions 840-1833 of SEQ ID NO: 7).

In another aspect, the invention provides an isolated polypeptide comprising an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 99.5% identity to any one of SEQ ID NOs 1-5, wherein the isolated polypeptide retains specific binding to TcdB2 and/or TcdB4, similar to any one of SEQ ID NOs 1-5.

In another aspect, the invention provides an isolated polypeptide comprising an amino acid sequence having less than 10, less than 9, less than 8, less than 7, less than 6, less than 5, less than 4, less than 3, or less than 2 mutated amino acid residues in any of SEQ ID NOS 1-5, wherein the isolated polypeptide retains specific binding to TcdB2 and/or TcdB4, similar to any of SEQ ID NOS 1-5.

In some embodiments, the mutated amino acid residue results from a substitution, insertion or deletion of the amino acid residue of any one of SEQ ID NOs 1-5, preferably the substitution is a conservative substitution.

In some embodiments, the polypeptide is cross-linked, cyclized, conjugated, acylated, carboxylated, lipidated, acetylated, thioglycolated, alkylated, methylated, polyglycosylated, glycosylated, polysialized, phosphorylated, adenylated, pegylated, or any combination thereof. In some embodiments, the polypeptide comprises a modification at the C-terminus or the N-terminus.

In some embodiments, the polypeptide further comprises a fusion domain. In some embodiments, the fusion domain is selected from the group consisting of polyhistidine, glu-Glu, glutathione S Transferase (GST), thioredoxin, protein a, protein G, immunoglobulin heavy chain constant region (Fc), maltose Binding Protein (MBP), or human serum albumin. In some embodiments, the polypeptide further comprises an Fc portion of human IgG 1.

In a preferred embodiment, the isolated polypeptide consists of the amino acid sequence of any one of SEQ ID NOs 1 to 5.

In another aspect, the invention provides a fusion protein comprising:

(i) An isolated polypeptide comprising the amino acid sequence of any one of SEQ ID NOs 1-5 or an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 99.5% identity to any one of SEQ ID NOs 1-5; and

(Ii) Fusion domain.

In some embodiments, the fusion domain is selected from the group consisting of polyhistidine, glu-Glu, glutathione S Transferase (GST), thioredoxin, protein a, protein G, immunoglobulin heavy chain constant region (Fc), maltose Binding Protein (MBP), or human serum albumin. Preferably, the fusion domain is the Fc portion of an immunoglobulin, more preferably, the fusion domain is the Fc portion of human IgG 1. In some embodiments, the fusion protein comprises or consists of SEQ ID NO 26 or 27.

In another aspect, the invention provides conjugates comprising the isolated polypeptides disclosed herein.

In some embodiments, the isolated polypeptides disclosed herein are conjugated to a moiety having a desired activity. In some embodiments, the moiety extends the serum half-life of the isolated polypeptide. In some embodiments, the moiety extends the shelf life of the isolated polypeptide. In some embodiments, the moiety is a labeling molecule, imaging agent, toxin, radionuclide, or agent capable of preventing or treating CDI or other bacterial infection. In some embodiments, the moiety is an Fc fragment of human IgG, igA, igM, igD or IgE, preferably an Fc fragment of human IgG.

In some embodiments, an isolated polypeptide disclosed herein is covalently conjugated to any of the above moieties.

In another aspect, the invention provides a chimeric molecule comprising a first portion and a second portion, wherein the first portion comprises an isolated polypeptide disclosed herein, and wherein the second portion is a different molecule than the isolated polypeptide disclosed herein.

In some embodiments, the first moiety is a monomer or multimer of an isolated polypeptide disclosed herein. In some embodiments, the first moiety is an isolated polypeptide disclosed herein. In some embodiments, the first moiety is a dimer, trimer, tetramer, or pentamer of the isolated polypeptides disclosed herein.

In some embodiments, the second moiety may be a moiety capable of increasing the serum half-life of the isolated polypeptide. In some embodiments, the second portion may be a portion that increases the shelf life of the isolated polypeptide.

In some embodiments, the second moiety may be an antimicrobial agent. In some embodiments, the antimicrobial agent is an antibiotic, such as, but not limited to, vancomycin, metronidazole, and the like.

In some embodiments, the second moiety may be an anti-TcdB immune molecule, e.g., a binding agent or antibody that binds TcdB, or a nanobody against TcdB. In some embodiments, the second portion may be an ankyrin repeat.

In some embodiments, the second moiety is a CSPG4 polypeptide. In some embodiments, the second portion is a fragment of CSPG4, preferably CSPG4 ^410-550(CSPG4^R1, SEQ ID NO: 20).

In some embodiments, the first portion and the second portion are directly connected or indirectly connected through a linker.

In yet another aspect, the invention provides an isolated nucleic acid molecule comprising a polynucleotide encoding an isolated polypeptide disclosed herein.

In another aspect, the invention provides a vector or expression cassette comprising a polynucleotide encoding an isolated polypeptide disclosed herein.

In another aspect, the invention provides a recombinant host cell comprising a polynucleotide encoding an isolated polypeptide disclosed herein, or comprising a vector or expression cassette comprising a polynucleotide encoding an isolated polypeptide disclosed herein.

In some embodiments, the recombinant host cell is used to produce an isolated polypeptide disclosed herein. In some embodiments, the recombinant host cell is selected from, but is not limited to, a prokaryotic or eukaryotic cell, preferably a bacterial, yeast, insect or mammalian cell, such as an E.coli (E.coli) cell, 293F cell, or CHO cell, among others.

In another aspect, the invention provides a pharmaceutical composition comprising an isolated polypeptide, fusion protein, conjugate or chimeric molecule disclosed herein and a pharmaceutically acceptable carrier.

In some embodiments, the pharmaceutical composition further comprises an antibacterial agent (e.g., an antibiotic) or an anti-TcdB immune molecule, e.g., a monoclonal or nanobody directed against TcdB.

In some embodiments, the pharmaceutical composition is for use in the prevention and/or treatment of a disease caused by TcdB2 and/or TcdB4, for example clostridium difficile infection caused by clostridium difficile branch 2 strain.

In some embodiments, the pharmaceutical composition is provided as a pharmaceutical kit. The kit comprises a container containing a polypeptide or pharmaceutical composition disclosed herein.

In another aspect, the invention provides the use of an isolated polypeptide, fusion protein, conjugate or chimeric molecule disclosed herein in the preparation of a pharmaceutical composition for the prevention and/or treatment of a disease caused by TcdB2 and/or TcdB4, e.g. clostridium difficile infection caused by a strain of division 2, in a subject in need thereof.

In another aspect, the invention provides a method of preventing and/or treating a disease caused by TcdB2 and/or TcdB4, such as clostridium difficile infection caused by a branch 2 strain, in a subject in need thereof, wherein the method comprises administering to the subject a therapeutically effective amount of an isolated polypeptide, fusion protein, conjugate, chimeric molecule or pharmaceutical composition disclosed herein.

In some embodiments, the subject is a mammal, e.g., a human or a non-human primate.

In another aspect, the invention provides an immune molecule, e.g., an antibody to an isolated polypeptide disclosed herein. An immune molecule (e.g., an antibody) is capable of specifically binding to TFPI on the cell surface, thereby blocking binding between TFPI and TcdB2 or between TFPI and TcdB 4.

In some embodiments, the isolated polypeptides disclosed herein are used as targets for screening for immune molecules that compete with TcdB2 or TcdB4 for binding to TFPI on the cell surface, or block binding between TFPI and TcdB2 or between TFPI and TcdB 4. In some embodiments, the immune molecule is an anti-TFPI antibody.

In some embodiments, the isolated polypeptides disclosed herein are used as neutralizing proteins to prevent, block or inhibit binding between TcdB2 and/or TcdB4 and cell surface TFPI.

In some embodiments, the immune molecule is used to prevent and/or treat a disease caused by TcdB2 and/or TcdB4, such as clostridium difficile infection caused by a strain of branch 2, in a subject in need thereof.

In some particular embodiments, the isolated polypeptides disclosed herein are used as target antigens to screen antibodies that compete with TcdB2 or TcdB4 for binding to TFPI on the cell surface, or block binding between TFPI and TcdB2 or between TFPI and TcdB 4.

In some embodiments, the antibodies are used to prevent and/or treat a disease caused by TcdB2 and/or TcdB4, such as clostridium difficile infection caused by a strain of branch 2, in a subject in need thereof.

In another aspect, the invention provides a method of screening for a molecule that specifically binds TcdB4, wherein the method comprises: the candidate molecule is contacted with TcdB4, the binding affinity between the candidate molecule and TcdB4 is tested, and a molecule having the desired binding affinity is selected.

In some embodiments, the candidate molecule is contacted with a DRBD fragment of TcdB4 (SEQ ID NO: 14).

In some embodiments, the candidate molecule is contacted with a TcdB4 ^842-1834 fragment (SEQ ID NO: 15) or a TcdB4 ^{1285 -1834} fragment (SEQ ID NO: 16).

In another aspect, the invention provides a method of screening for a molecule that specifically binds TcdB2, wherein the method comprises: contacting the candidate molecule with TcdB2, testing the binding affinity between the candidate molecule and TcdB2, and selecting a molecule having the desired binding affinity.

In some embodiments, the candidate molecule is contacted with a DRBD fragment of TcdB2 (SEQ ID NO: 28).

In some embodiments, the candidate molecule is contacted with a TcdB2 ^841-1833 fragment (SEQ ID NO: 29) or a TcdB2 ^1284-1833 fragment (SEQ ID NO: 30).

In another aspect, the invention provides molecules that specifically bind to a DRBD fragment of TcdB 4.

In some embodiments, the molecule that specifically binds to a DRBD fragment of TcdB4 is a monoclonal antibody, a humanized antibody, or a chimeric antibody. In some embodiments, the molecule specifically binds to the TcdB4 ^842-1834 fragment (SEQ ID NO: 15) or the TcdB4 ^1285-1834 fragment (SEQ ID NO: 16).

In another aspect, the invention provides molecules that specifically bind to a DRBD fragment of TcdB 2.

In some embodiments, the molecule that specifically binds to a DRBD fragment of TcdB2 is a monoclonal antibody, a humanized antibody, or a chimeric antibody. In some embodiments, the molecule specifically binds to the TcdB2 ^841-1833 fragment (SEQ ID NO: 29) or the TcdB2 ^1284-1833 fragment (SEQ ID NO: 30).

In some embodiments, molecules that specifically bind TcdB4 or TcdB2 are used to prevent and treat clostridium difficile infection caused by clostridium difficile branch 2 strains.

In another aspect, the invention provides a neutralising molecule targeting one or more sites selected from E1433, D1467, D1468, E1469, S1598, L1599, L1434, K1435, M1438, V1492, L1494, I1496, L1489, P1506 or Y1510 of TcdB4 (SEQ ID NO: 9).

In some embodiments, the neutralizing molecule targets any combination of E1433, D1467, D1468, E1469, S1598, L1599, L1434, K1435, M1438, V1492, L1494, I1496, L1489, P1506, or Y1510 (SEQ ID NO: 9) of TcdB 4.

In another aspect, the invention provides a neutralising molecule targeting one or more sites selected from E1432, D1466, D1467, S1597, L1598, L1433, K1434, M1437, V1491, L1493 or L1488 of TcdB2 (SEQ ID NO: 7).

In some embodiments, the neutralizing molecule targets any combination of E1432, D1466, D1467, S1597, L1598, L1433, K1434, M1437, V1491, L1493, or L1488 of TcdB2 (SEQ ID NO: 7).

In some embodiments, the neutralizing molecule is an antibody, such as, but not limited to, a monoclonal antibody or a humanized antibody. In some embodiments, the neutralizing molecule is used to prevent and treat clostridium difficile infection caused by clostridium difficile branch 2 strain.

In some embodiments, the neutralizing molecule is formulated as a pharmaceutical composition. In some embodiments, the pharmaceutical composition comprises a therapeutically effective amount of a neutralizing molecule and a pharmaceutically acceptable carrier.

In some embodiments, the invention relates to a method of preventing and/or treating a disease caused by TcdB2 and/or TcdB4, in particular Clostridium Difficile Infection (CDI) caused by a strain of branch 2, in a subject in need thereof, wherein the method comprises administering to the subject a therapeutically effective amount of a neutralizing molecule.

In some embodiments, the invention relates to the use of a neutralizing molecule in the preparation of a pharmaceutical composition for the prevention and/or treatment of a disease caused by TcdB2 and/or TcdB4, in particular Clostridium Difficile Infection (CDI) caused by a strain of division 2, in a subject.

Drawings

The drawings are not intended to be drawn to scale. In the drawings, each identical or nearly identical component that is illustrated in various figures is represented by a like numeral. For purposes of clarity, not every component may be labeled in every drawing. In the drawings:

Figure 1.Crispr-Cas9 screen identifies host factors for TcdB 4.

(A) Sensitivity of several human cell lines (including HeLa, U2OS, A549, MCF-7, and HT-29) was measured using a cytopathic cell rounding assay. Data (n=6) are expressed as mean ± standard error (SD).

(B) Schematic of the procedure for screening HeLa cells transduced with GeCKO v2 lentiviral library using TcdB 4.

(C) The genes identified from the third round of screening (R3) were sequenced and mapped. The x-axis shows NGS reads for each gene. The y-axis represents the number of unique grnas per target gene.

(D) The identified genes were ranked and mapped based on fold enrichment of gRNA reads from start (R0) to three rounds of (R3) after toxin selection. The first 30 enriched candidate genes with at least three targeted grnas were labeled.

Fig. 2.Tfpi is the cellular receptor for TcdB 4.

(A) HeLa KO cells of the indicated genes were generated using CRISPR-Cas9 technology. For TFPI, PIGM, PIGX and PIGP KO cells, two individual clones were established. Sensitivity of these cells to TcdB4 was measured using cytopathic cell rounding experiments. Error bars (n=6) represent mean±sd.

(B) The sensitivity measured in (a) was quantified and expressed as fold-over resistance compared to HeLa WT cells. Error bars (n=6) represent mean ± SD, × P <0.001 vs WT (student t test).

(C) WT cells were pretreated with PI-PLC (i.e., phosphatidylinositol-specific phospholipase C) or not, and tested for sensitivity to TcdB 4. The percentage of cell rounding 2.5 hours after TcdB4 exposure is plotted as a bar graph. Data (n=6) are expressed as mean ± SD, × P <0.001 (student's t-test).

(D) The sensitivity of HeLa TFPI ^-/- cells to TcdB4 was compared to WT cells by analyzing the glycosylation level of RAC1 using immunoblotting assays.

(E) HeLa WT, CSPG4 ^-/-、FZD1/2/7^-/- or TFPI ^-/- cells were incubated with TcdB4 (10 nM) for 10 min on ice, washed with PBS, lysed, and subjected to immunoblot analysis. Cell surface bound TcdB4 was detected by anti-TcdB polyclonal antibody. Actin was the loading control.

(F) Schematic representation of TFPI alpha and TFPI beta (two major TFPI isoforms).

(G) Transient transfection of tfpia or tfpia restored TcdB4 entry into HeLa CSPG4 ^-/-/TFPI^-/- cells (0.14 nm TcdB4,3 hours). Scale bar, 50 μm.

(H) Quantifying cell rounding in (G). Error bars (n=6) represent mean±sd.

(I) HeLa WT or TFPI ^-/- cells transfected with TFPI alpha, TFPI beta or mock were incubated with TcdB4 (10 nM) for 10min on ice, washed with PBS, lysed, and analyzed by immunoblotting. Cell surface bound TcdB4 was detected by anti-TcdB polyclonal antibody. Actin was the loading control.

(J) Cytopathic effects of TcdB4 (0.14 nm,4 hours) induced CSPG4 ^-/- and CSPG4 ^-/-/TFPI^-/- cells in the presence or absence of TFPI alpha-GFP were quantified by cell rounding assays. Data (n=6) are expressed as mean ± SD.

FIG. 3. Interaction of TcdB4 with the Kunitz-2 domain of TFPI.

(A) Schematic representation of TFPI ^K1+K2-Fc、TFPI^K1 -Fc and TFPI ^K2 -Fc.

(B) TFPI ^K1+K2 protects HeLa CSPG4 ^-/- cells from TcdB4 but not TcdB1 as measured by cell rounding assays.

(C) Evolutionary relationship trees of closely related Kunitz domains from TFPI, TFPI2 and AMBP.

(D) Ectopic expression of GPI-anchored TFPI ^K2(TFPI^K2 -GPI) restored TcdB4 entry into CSPG4 ^-/-/TFPI^-/- cells.

(E) Binding of TcdB4 to Fc-labeled TFPI ^K1 or TFPI ^K2 was characterized using the BLI assay (see figure 11 for K _d analysis). Human IgG Fc fragments were used as negative controls.

FIG. 4. Frozen electron microscope structure of TcdB4-TFPI complex.

(A) Domain organization schematic for determining full length TcdB4 and TFPI ^K1+K2 for cryo-electron microscope structures.

(B) Frozen electron microscope structure of TcdB4-TFPI compound. The glucosyltransferase domain (GTD), cysteine Protease Domain (CPD), transmembrane Delivery and Receptor Binding Domain (DRBD) and the combined repeat oligopeptide (drop) domain of TcdB4 are shown in wheat, pink, light blue and green, respectively. TFPI binds at the periphery of TcdB4 through direct interaction between the Kunitz-2 domain (cyan) of TFPI and DRBD of TcdB 4.

(C) An enlarged view of the interaction between TcdB4 ^DRBD (light purple) and TFPI ^K2 (cyan). TFPI ^K2 is anchored to the hydrophobic surface of TcdB4 ^DRBD via two epitopes, loop 1 (residues 131-138) and loop 2 (residues 155-162).

(D) Close-up of the loop 1-TcdB4 (left panel) and loop 2-TcdB4 (right panel) combined interface.

(E) The mutation in TcdB4 ^1285-1834 was demonstrated by the pulldown experiments to disrupt its interaction with Fc-labeled TFPI ^K2.

(F) Preloading TcdB4 ^1285-1834 to TFPI ^K2 would prevent subsequent binding of factor Xa (FXa). The TFPI ^K2 -Fc loaded biosensor was first exposed to 300nM TcdB4 ^1285-1834 or control buffer, equilibrated, and then exposed to 100nM FXa.

Fig. 5.Tcdb contains two classes of RBI that identify FZD or TFPI.

(A) Schematic representation of RBI in TcdB (i.e. receptor binding interface) (upper panel) and phylogenetic split network covering RBI from known TcdB sequence (lower panel).

(B) 110 MLST-type maximum likelihood trees were created to isolate five major clostridium difficile branches (inner loop: branch 1 light blue, branch 2 purple, branch 3 yellow, branch 4 green, branch 5 orange, and gray other hidden branches). The different RBI categories in each MLST type are labeled outer-loop.

(C) Structural comparison between the TcdB1-FZD2 complex (PDB: 6C0B, left panel) and the TcdB4-TFPI complex (right panel). Only DRBD (residues 1285-1804) of TcdB1 (grey) and DRBD (residues 1285-1804) of TcdB4 (light purple) are shown.

(D) A close-up view of the TcdB1-FZD2 (left panel) and TcdB4-TFPI (right panel) interfaces. PAM molecules are inserted into the hydrophobic channel formed by residues of FZD2 and TcdB1 to stabilize the interaction of TcdB1-FZD 2. Key residues and PAM are shown as bar models.

(E) F1597 in TcdB1 stabilizes the middle part of PAM, while S1598 (corresponding residue) in TcdB4 forms a tight hydrogen bond with R140 from TFPI.

(F) Overlap of RBI from TcdB2 (green, PDB:6OQ 5) and from TcdB4-TFPI complex (light purple).

(G) Sensitivity of HeLa CSPG4 ^-/- and CSPG4 ^-/-/TFPI^-/- cells to TcdB2 was measured using a cell rounding assay and CR50 was plotted in a bar graph. P <0.001

(H) GPI-anchored Tfpi ^K2(Tfpi^K2 -GPI) ectopic expression restored TcdB2 entry into CSPG4 ^-/-/TFPI^-/- cells. The scale bar represents 50 μm.

(I) Quantitative use of mouse Tfpi ^K2 -Fc over time protected HeLa CSPG4 ^-/- cells from TcdB2 by cytopathic cell rounding assays. Data (n=6) are expressed as mean ± SD.

(J) Point mutations at TcdB4 ^1285-1834 were examined in pull-down experiments using TFPI ^K2 -Fc as a decoy. The bound TcdB4 ^1285-1834 mutant was co-precipitated with TFPI ^K2 -Fc using protein a resin and detected by immunoblot analysis.

Fig. 6.Tfpi is a physiologically relevant receptor for TcdB 4.

(A) IHC analysis was performed on sections of mouse ileum, jejunum, cecum and colonic paraffin for detection Tfpi. Blue staining indicates nuclei and brown staining indicates Tfpi. The scale bar represents 100 μm.

(B) TcdB4 (2 μg) of colon tissue of mice harvested after the colonic loop ligation assay was assessed by H & E staining for histopathology induced with or without TFPI ^K2 -Fc (50 μg). The eroded crypts are marked by hollow triangles. Representative images are displayed. The scale bar represents 100 μm.

(C and D) histopathological scores based on edema, inflammatory cell infiltration, epithelial destruction, and crypt damage assessment (B), respectively. The total scores are summarized in (C). n=6 mice, error bars represent mean ± Standard Error of Mean (SEM), single-factor analysis of variance for multiple comparisons using FISHER LSD test. P <0.0001, P <0.001, P <0.01, ns=no significance).

(E) Survival of WT, tfpi beta ^+/-、Tfpiβ^-/- mice after intraperitoneal injection of 1 μg/kg TcdB4 was shown by the Kaplan-Meier curve (7 days of monitoring).

(F) After intraperitoneal injection of 1. Mu.g/kg TcdB4, kidney tissue from WT and Tfpi β ^-/- mice was harvested and assessed by H & E staining. Normal (green arrow) and damaged (red arrow) glomeruli are shown. The scale bar represents 50. Mu.m.

Fig. 7. Simultaneous blocking of TFPI binding and CSPG4 binding provides optimal protection from clostridium difficile branch 2 TcdB.

(A) TcdB2 (2 μg) of colon tissue of mice harvested after the colonic loop ligation assay was assessed by H & E staining for induced histopathology with or without TFPIK-Fc (50 μg). Submucosal edema is highlighted by dashed lines. Representative images are displayed. The scale bar represents 100 μm.

(B and C) histopathological scores based on submucosal edema, mucosal edema, inflammatory cell infiltration, epithelial destruction, and crypt damage assessment (A), respectively. The total scores are summarized in (B). n=6 mice, error bars represent mean ± SEM, single-factor anova with multiple comparisons using FISHER LSD test. P <0.0001, P <0.001, P < 0.01).

(D) TcdB2 (2 μg) of colon tissue of mice harvested after the colonic loop ligation assay was assessed by H & E staining for histopathology induced with or without CSPG4 ^R1 (50 μg) or TFPI ^K2-CSPG4^R1 (50 μg). Submucosal edema is highlighted by dashed lines. Representative images are displayed. The scale bar represents 100 μm.

(E and F) histopathological scores based on submucosal edema, mucosal edema, inflammatory cell infiltration, epithelial destruction, and crypt damage assessment (D), respectively. The total scores are summarized in (F). n=3 mice, error bars represent mean ± SEM, single-factor anova with multiple comparisons using FISHER LSD test. P <0.0001, P <0.001, P <0.01, P <0.05, ns=insignificant).

FIG. 8 TcdB4 was effective against both HeLa WT and CSPG4 ^-/-/FZD1/2/7^-/- cells.

(A) Sensitivity of HeLa WT cells to eight known TcdB variants (including TcdB1, tcdB2, tcdB3, tcdB4, tcdB5, tcdB6, tcdB7, tcdB 8) was measured using cytopathic cell rounding experiments (n=6, error bars represent mean ± SD).

(B) Sensitivity of HeLa CSPG4 ^-/-/FZD1/2/7^-/- cells to eight known TcdB variants (including TcdB1, tcdB2, tcdB3, tcdB4, tcdB5, tcdB6, tcdB7, tcdB 8) was measured using cytopathic cell rounding experiments (n=6, error bars represent mean ± SD).

FIG. 9 PI-PLC treatment protects cells from TcdB 4.

HeLa CSPG4 ^-/- cells were pretreated for 30min with or without PI-PLC. The cells were then exposed to 0.28nM TcdB4 and incubated at 37 ℃. Representative images of cell rounding 2.5 hours and 3.5 hours after exposure to TcdB4 are shown. The scale bar represents 50 μm.

Fig. 10. Exogenous TFPI alpha-sensitized TFPI KO cells, but protected TFPI positive cells from TcdB 4.

(A) Representative fluorescent images showed that supplemental TFPI alpha-GFP, but not TFPI ^K1+K2 -GFP, bound to the surface of CSPG4 ^-/-/TFPI^-/- cells.

(B) HeLa CSPG4 ^-/- and CSPG4 ^-/-/FZD1/2/7^-/- cells were exposed to 0.14nM TcdB4 and gradient concentrations of TFPI alpha-GFP. Cells were then incubated at 37 ℃ and examined for percent cell rounding. Representative bright field images of cell rounding 4 hours after toxin exposure are shown. The scale bar is 50 μm.

Figure 11 tcdb4 specifically binds TFPI.

(A) TFPI ^K1+K2 -Fc protects HeLa CSPG 4-/-cells from TcdB4 but not TcdB1 as measured by cell rounding assays.

(B) Pull-down experiments showed that TcdB4, but not TcdB1, binds to Fc-labeled TFPI ^K1+K2.

(C) Purified TFPI ^K2 -Fc, but not TFPI ^K1 -Fc, protected CSPG4 ^-/- cells from TcdB4 as measured by cell rounding assays. The scale bar represents 50 μm.

FIG. 12 kinetic analysis of TcdB4-TFPI interactions.

(A) A representative binding curve of TcdB4 to Fc-labeled human TFPI ^K1+K2 was examined by BLI analysis.

(B) A representative binding curve of TcdB4 to Fc-labeled human TFPI ^K2.

(C) Representative binding curve of TcdB4 to Fc-tagged mice Tfpi ^K2.

Purification and functional characterization of tcdb4 and TFPI.

(A) SDS-PAGE of the size exclusion chromatogram and peak fractions of TFPI are shown in the inset.

(B) SDS-PAGE of the size exclusion chromatogram and peak fractions of TcdB4 are shown in the inset.

(C) SDS-PAGE of the size exclusion chromatogram and peak fractions of the TcdB4-TFPI complex are shown in the inset.

Fig. 14 image processing of single particle cryoelectron microscopy data.

(A) A flow chart of the data processing of the frozen electron microscope. For details, please refer to "cryo-electron-microscope data processing" in the section "method details".

(B) Representative cryoelectron microscopy images. Some individual particles of TcdB4-TFPI are highlighted by a cyan circle.

(C) 2D classification average of frozen electron microscopy particle images of TcdB 4.

Figure 15 single particle cryoelectron microscopy analysis of tcdb 4.

(A) The angular distribution of the particle image contained in the final 3D reconstruction.

(B) TcdB4 local resolution of the final cryo-electron micrograph.

(C) The Fourier Shell Correlation (FSC) curve of TcdB4 has the indicated resolution at fsc=0.5.

(D) The FSC curve of TcdB4 between the atomic model and the final plot, with indicated resolution at fsc=0.5 (black); FSC curves between half fig. 1 (red) or half fig. 2 (green) and the atomic model refined for half fig. 1.

Figure 16 tcdb4 and FXa competitively bind TFPI.

(A) Fc-labeled TFPI ^K2 binds to TcdB4 ^842-1834, but not TcdB4 ^1-841 and TcdB4 ^1801-2367, as shown by immunoblot analysis.

(B) Comparison between the structures of TFPI-FXa/trypsin (PDB: 1TFX and 1FAX, left panel) and the TFPI-TcdB4 complex (right panel). Their interfaces are marked with red circles.

(C) Preloading FXa to TFPI ^K2 prevents subsequent binding of TcdB4 ^1285-1834. The TFPI ^K2 -Fc loaded biosensor was first exposed to 100nM FXa or control buffer for equilibration and then 300nM TcdB4 ^{1285 -1834}.

Fig. 17 tcdb2 binds TFPI ^K2. Fig. 17 is related to fig. 5.

(A) HeLa CSPG4 ^-/- cells were exposed to 100pm TcdB2 with or without Fc labeled mice Tfpi ^K2. Cells were then incubated at 37 ℃ and examined for percent cell rounding. Representative bright field images of cell rounding 4 hours after toxin exposure are shown. The scale bar represents 100 μm.

(B) Binding of TcdB2 to Fc-labeled TFPI ^K1 or TFPI ^K2 was characterized using the BLI assay.

(C) Representative binding curves for TcdB2 ^1285-1834 and Fc-labeled human TFPI ^K2.

(D) Representative binding curves for TcdB2 ^1285-1834 and Fc-labeled mice Tfpi ^K2.

FIG. 18S 1496 is an important residue affecting TcdB2-TFPI interactions.

(A) Sequence alignment between RBI of TcdB2 and TcdB 4. Non-conserved residues are labeled in red and similar residues are labeled in blue. Residues contributing to TFPI binding are indicated by green triangles.

(B) Representative binding curves of TcdB2 ^1285-1834 S1496I mutant to Fc-tagged mice Tfpi.

(C) Representative binding curves of TcdB2 ^1285-1834 S1496I mutant to Fc-labeled human TFPI.

Fig. 19.Tfpi is the colonic crypt receptor for TcdB 4.

(A) The low magnification image of immunochemical staining shows an overview of mouse Tfpi distribution in the ileum, jejunum, cecum and colon. The enlarged area shown in fig. 19A is framed. The scale bar represents 100 μm.

(B) In the colon tissue of mice harvested after the colonic loop ligation assay, epithelial cells were closely adhered with immunofluorescent staining of the binding marker ZO-1 (red). Nuclei were stained with Hoechst (blue). The scale bar represents 100 μm.

Figure 20.Tfpi is the physiologically relevant receptor for TcdB4 in systemic infection.

(A) Schematic of experimental design for knock-out Tfpi beta isoforms from mouse genome using CRISPR-Cas9 method.

(B) After intraperitoneal injection of 1. Mu.g/kg TcdB4 (7 days of monitoring), kaplan-Meier survival curves of WT, tfpi beta ^+/-、Tfpiβ^-/- mice were separated according to sex.

Fig. 21 kidneys are susceptible to systemic TcdB4 exposure.

(A) Immunoblot analysis of Tfpi expression in various organs from WT and Tfpi β ^-/- mice.

(B) Histological analysis showed that intraperitoneal injection of 1 μg/kg TcdB4 resulted in acute kidney injury in WT mice, but not Tfpi β ^-/- mice. All mice appeared normal in liver, lung, spleen and heart. The scale bar represents 100 μm.

Detailed Description

While this invention may be embodied in many different forms, there are disclosed herein specific illustrative embodiments thereof which illustrate the principles of the invention. It should be emphasized that the invention is not limited to the specific embodiments shown. Furthermore, any section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

Unless defined otherwise herein, scientific and technical terms related to the present application shall have the meanings commonly understood by one of ordinary skill in the art. Furthermore, unless the context requires otherwise, singular terms shall include the plural and plural terms shall include the singular. More specifically, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a protein" includes a plurality of proteins; reference to "a cell" includes mixtures of cells and the like. In the present application, the use of "or" means "and/or" unless stated otherwise. Furthermore, the use of the term "include" and other forms (e.g., "contain" and "include") is not limited. Furthermore, the scope provided in the specification and the appended claims includes endpoints and all points between endpoints.

Generally, the terms and techniques described herein in connection with cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization are well known and commonly used in the art. Unless otherwise indicated, the methods and techniques of the present invention are generally performed according to conventional methods well known in the art and as described in the various general and more specific references cited and discussed in this specification. The terms and laboratory procedures and techniques described herein in connection with analytical chemistry, synthetic organic chemistry, and pharmaceutical and medicinal chemistry are well known and commonly used in the art. Moreover, any section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

The emergence of the virulence branch 2 of clostridium difficile is associated with severe symptoms, accounting for over 20% of global infections. TcdB is the main virulence factor for clostridium difficile, whereas the branch 2 strain expresses only two TcdB variants (TcdB 2 and TcdB 4) using unknown receptors other than classical TcdB. Here we performed CRISPR/Cas9 screening for TcdB4 and identified Tissue Factor Pathway Inhibitor (TFPI) as its receptor. Using cryo-electron microscopy, we determined full length TcdB4 and TFPIA common receptor binding region is defined for TcdB. Residue variation within this region divides the major TcdB variants into two classes: one class recognizes frizzled proteins (FZDs) and the other class recognizes TFPI. TFPI is highly expressed in the intestinal glands, whereas recombinant TFPI protects the colonic epithelium from TcdB 4/2. These findings establish TFPI as the colonic crypt receptor for TcdB from clostridium difficile branch 2 and reveal a new mechanism of CDI pathogenesis.

Without wishing to be bound by any particular mechanism or theory, it is believed that certain aspects of the invention rely at least in part on the novel mechanism of clostridium difficile infection. This mechanism involves identifying TFPI as the colonic crypt receptor for TcdB from clostridium difficile branch 2. TcdB alone can cause various diseases. However, how TcdB targets the colonic epithelium remains largely uncertain due to the lack of established receptors. Chondroitin sulfate proteoglycan 4 (CSPG 4, also known as glial antigen 2 (NG 2) in rodents) has been identified as a functional receptor for TcdB in HeLa cells and colorectal cell line HT-29. However, CSPG4 is not expressed in colonic epithelial cells. Poliovirus receptor like 3 (PVRL 3) has recently been considered as a cytokine leading to necrotic cell death processes (cytotoxicity) following exposure to high concentrations of TcdB in HeLa cells and colorectal cell line Caco-2, but whether PVRL3 is the relevant TcdB receptor in the colonic epithelium remains unknown and its role in directly mediating TcdB entry into cells has not been established.

The identification and validation of TcdB receptors in colonic epithelial cells using a CRISPR/Cas9 mediated knockout screening system is described in the examples and figures of the present invention. CRISPR/Cas9 systems and their use are known in the art, see for example US20140357530. In the present invention TFPI and its K2 domain are identified and validated as novel and pathologically related TcdB receptors, particularly colonic crypt receptors for TcdB2 and/or TcdB4 (particularly TcdB 4) from clostridium difficile branch 2 strain. Binding of TcdB to cell surface TFPI or the K2 domain mediates entry of TcdB toxin into cells. TcdB catalyzes the glycosylation of small gtpases within epithelial cells, leading to cell rounding and death. Thus, shown herein is a new mechanism independent of the intracellular mechanism of TcdB pathogenesis, involving inhibition of the binding between TFPI or its K2 domain and TcdB (e.g. TcdB2 and/or TcdB4, in particular TcdB 4) from clostridium difficile branch 2 strain.

The region of TcdB4 that interacts with TFPI or its K2 domain is identified as being the amino acid sequence at positions 842 to 1834 (SEQ ID NO: 15), particularly at positions 1285 to 1834 (SEQ ID NO: 16) of the TcdB4 protein (SEQ ID NO:9, full length TcdB4 protein). E1433, D1467, D1468, E1469, S1598, L1599, L1434, K1435, M1438, V1492, L1494, I1496, L1489, P1506, Y1510 of TcdB4 are key residues for receptor recognition.

The region of TcdB2 interacting with TFPI or its K2 domain was identified as the amino acid sequence at positions 841 to 1833 (SEQ ID NO: 29), particularly at positions 1284 to 1833 (SEQ ID NO: 30) of the TcdB2 protein (SEQ ID NO:7, full length TcdB2 protein). E1432, D1466, D1467, S1597, L1598, L1433, K1434, M1437, V1491, L1493 or L1488 of TcdB2 are key residues for receptor recognition.

R135, R140, I138, I133, M162, L159 in TFPI are the key residues (SEQ ID NO:22 or 23, containing signal peptides) that bind specifically to TcdB4. There are two variants of human TFPI, TFPI alpha (SEQ ID NO: 3) and TFPI beta (SEQ ID NO: 4), with NO signal peptide, and mouse TFPI beta represented by SEQ ID NO: 5. The K2 domain of human TFPI is represented by SEQ ID NO. 1 and the K2 domain of mouse Tfpi is represented by SEQ ID NO. 2. The K2 domain is a functional fragment of TFPI for specifically binding TcdB2 and/or TcdB4.

Isolated polypeptides that specifically bind TcdB2 and/or TcdB4

In some embodiments, the invention provides isolated polypeptides that specifically bind TcdB2 and/or TcdB 4. As used herein, an "isolated polypeptide" refers to a polypeptide that is isolated from or substantially free (e.g., at least 80%, 90%, 95%, 97%, 99% or 99.5% free) of other proteins and/or other polypeptides. In some embodiments, the isolated polypeptide is 100% free of other proteins and/or other polypeptides.

The term "specific binding" or "specifically binding" as used herein refers to a non-random binding reaction between two molecules, such as a binding reaction between TcdB4 and TFPI.

The isolated polypeptides of the invention prevent, block or inhibit binding of TcdB2 and/or TcdB4 to TFPI or a K2 domain thereof on the cell surface. The terms "prevent", "block" and "inhibit" are used interchangeably herein. The preventing, blocking and inhibiting effects of the isolated polypeptides of the invention were measured compared to the binding of TcdB2 and/or TcdB4 to TFPI or its K2 domain on the cell surface in the absence of the isolated polypeptides. Thus, the isolated polypeptides of the invention may themselves be used as efficient TcdB (e.g. TcdB2 and/or TcdB 4) neutralizing proteins, thereby preventing or treating diseases caused by TcdB (e.g. TcdB2 and/or TcdB 4), in particular clostridium difficile infection caused by the strain branch 2. Thus, the present invention also contemplates methods of preventing or treating such diseases.

In some embodiments, the isolated polypeptide comprises the amino acid sequence of SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4 or SEQ ID NO. 5. In some embodiments, the isolated polypeptide comprises R135, R140, I138, I133, M162, L159 of TFPI, numbering according to SEQ ID NO. 22 or 23.

In some embodiments, the isolated polypeptide comprises an amino acid sequence having at least 85% identity to any one of SEQ ID NOs 1-5. For example, an isolated polypeptide comprises an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 99.5% identity to any one of SEQ ID NOs 1-5.

The "percent identity" of two amino acid sequences was determined using the algorithm of Karlin and Altschul, proc.Natl. Acad.Sci.USA 87:2264-68,1990 (modified in Karlin and Altschul, proc.Natl. Acad.Sci.USA 90:5873-77,1993). Such algorithms are incorporated into the NBLAST and XBLAST programs of Altschul, et al J.mol.biol.215:403-10,1990 (version 2.0). BLAST protein searches can be performed using the XBLAST program with a score=50 and a word length=3 to obtain amino acid sequences homologous to the protein molecule of interest. If a gap exists between the two sequences, gap BLAST can be used, as described in Altschul et al, nucleic Acids Res.25 (17): 3389-3402, 1997. When using BLAST and gapped BLAST programs, default parameters for the respective programs (e.g., XBLAST and NBLAST) can be used.

Conjugate or fusion protein

The polypeptides described herein may be conjugated or otherwise covalently linked to other molecules (e.g., using chemical linkers). One such form of attachment is through a non-amide linkage (e.g., disulfide linkage). In some embodiments, the polypeptide is covalently linked (e.g., by a linker molecule) to an antibody or domain thereof suitable for enhancing the half-life of the molecule (e.g., one or more constant domains in the Fc domain). In some embodiments, the polypeptide is linked to an Fc domain disclosed herein (e.g., igG, igA, igM, igD or IgE).

In some embodiments, the isolated polypeptides of the invention further comprise a fusion domain. Thus, also provided herein are functional variants or modified forms of polypeptide fragments having one or more fusion domains. Well-known examples of such fusion domains include, but are not limited to, polyhistidine, glu-Glu, glutathione S Transferase (GST), thioredoxin, protein A, protein G, immunoglobulin heavy chain constant region (Fc), maltose Binding Protein (MBP), or human serum albumin. The fusion domain may be selected to confer a desired property. For example, some fusion domains are particularly useful for isolating fusion proteins by affinity chromatography. For affinity purification purposes, the relevant matrices for affinity chromatography, such as glutathione, amylase and nickel or cobalt conjugated resins, are used. Many such matrices are provided in "kit" form, such as PHARMACIA GST purification systems and the useful QIAexpress ^TM system (Qiagen) with a (HIS 6) fusion partner. In some embodiments, the isolated polypeptide fragment is fused to a domain that stabilizes the isolated polypeptide fragment in vivo ("stabilizer" domain). As used herein, "stabilization" refers to an increase in the in vivo half-life of a polypeptide, whether this is caused by reduced disruption, reduced renal clearance, or other pharmacokinetic effects. Fusion to the Fc portion of an immunoglobulin is known to confer desirable pharmacokinetic properties to a variety of proteins. Likewise, fusion with human serum albumin can confer desirable properties. Other types of fusion domains that may be selected include poly (e.g., dimeric, tetrameric) domains and functional domains.

In some embodiments, the isolated polypeptides of the invention further comprise an Fc portion of human IgG 1. Thus, fusion proteins comprising an immunoglobulin Fc portion are also contemplated herein. In some embodiments, the fusion protein comprises or consists of SEQ ID NO. 26 or SEQ ID NO. 27.

Optionally, the Fc domain may have one or more mutations at residues, such as Asp-265, lysine 322 and Asn-434. In some cases, a mutant Fc domain having one or more of these mutations (e.g., an Asp-265 mutation) has reduced binding capacity to an Fc receptor relative to the wild-type Fc domain. In other cases, a mutant Fc domain having one or more of these mutations (e.g., asn-434 mutation) has increased ability to bind to MHC class I-related Fc receptors (FcRN) relative to the wild-type Fc domain.

It is understood that the different elements of the fusion protein may be arranged in any manner consistent with the desired function.

As used herein, the term "immunoglobulin Fc region" or simply "Fc" is understood to refer to the carboxy-terminal portion of an immunoglobulin chain constant region, preferably the carboxy-terminal portion of an immunoglobulin heavy chain constant region or a portion thereof. For example, the immunoglobulin Fc region may comprise: 1) a CH1 domain, a CH2 domain, and a CH3 domain, 2) a CH1 domain and a CH2 domain, 3) a CH1 domain and a CH3 domain, 4) a CH2 domain and a CH3 domain, or 5) a combination of two or more domains and an immunoglobulin hinge region. In a preferred embodiment, the immunoglobulin Fc region comprises at least an immunoglobulin hinge region, a CH2 domain and a CH3 domain, and preferably lacks a CH1 domain.

In some embodiments, the immunoglobulin class from which the heavy chain constant region is derived is IgG (igγ) (subclass 1,2, 3, or 4). Other classes of immunoglobulins may be used: igA (Igalpha), igD (Igdelta), igE (Igepsilon) and IgM (Igmu). The selection of appropriate immunoglobulin heavy chain constant regions is discussed in detail in U.S. Pat. nos. 5,541,087 and 5,726,044. It is considered within the skill of the art to select specific immunoglobulin heavy chain constant region sequences from certain classes and subclasses of immunoglobulins to achieve specific results. The portion of the DNA construct encoding the Fc region of an immunoglobulin preferably comprises at least a portion of a hinge domain, and preferably at least a portion of the CH3 domain of fcγ or IgA, igD, igE or any homologous domain in IgM.

Furthermore, substitution or deletion of amino acids within the immunoglobulin heavy chain constant region is contemplated for use in the practice of the methods and compositions disclosed herein. One example is the introduction of amino acid substitutions in the upper CH2 region to produce Fc variants with reduced affinity for Fc receptors (Cole et al, (1997) J.Immunol.159:3613). Optionally, an isolated polypeptide of the invention may comprise a modification. The polypeptides comprising the modifications have other characteristics not possessed by the reference polypeptide. As used herein, a "modification" or "derivatization" of a peptide results in a modified or derivatized polypeptide that is in the form of a given peptide that is chemically modified relative to a reference peptide, including, but not limited to, oligomerization or polymerization, modification of amino acid residues or peptide backbones, crosslinking, cyclization, conjugation, glycosylation, acetylation, phosphorylation, acylation, carboxylation, lipidation, thioglycolation amidation, alkylation, methylation, glycation, glycosylation, polysialization, adenylation, pegylation, fusion with other heterologous amino acid sequences, or other modifications that substantially alter stability, solubility, or other properties of the peptide while substantially preserving the activity of the polypeptides herein. It will be appreciated that the isolated polypeptides comprising these modifications are crosslinked, cyclized, conjugated, acylated, carboxylated, lipidated, acetylated, thioglycolated, alkylated, methylated, polyglylated, glycosylated, polysialised, phosphorylated, adenylated, pegylated or any combination thereof. As a result, modified polypeptide fragments of the invention may contain non-amino acid elements such as polyethylene glycol, lipids, polysaccharides or monosaccharides, as well as phosphates. An isolated polypeptide of the invention can comprise modifications disclosed herein at the C-terminus (e.g., C-terminal amidation) or the N-terminus (e.g., N-terminal acetylation).

Terminal modifications are useful and well known to reduce susceptibility to protease digestion and thus help to extend the half-life of the polypeptide in solution, particularly in biological fluids where proteases may be present. In some embodiments, the polypeptides or fusion proteins described herein are further modified within the sequence, for example by terminal NH2 acylation (e.g., acetylation) or thioglycolaldehyde amidation, by terminal carboxyamidation (e.g., terminal modification with ammonia, methylamine, etc.).

Amino-terminal modifications include methylation (e.g., -NHCH3 or-N (CH 3) 2), acetylation (e.g., with acetic acid or halogenated derivatives thereof such as α -chloroacetic acid, α -bromoacetic acid, or α -iodoacetic acid), addition of benzyloxycarbonyl (Cbz) groups, or blocking of the amino-terminal with any blocking group containing carboxylate functionality defined by RCOO-or sulfonyl functionality defined by R-SO2-, where R is selected from the group consisting of alkyl, aryl, heteroaryl, alkylaryl, and the like. One can also incorporate a deaminated acid at the N-terminus (and thus no N-terminal amino group present) to reduce susceptibility to proteases or limit the conformation of the polypeptide. In certain embodiments, the N-terminus is acetylated with acetic acid or acetic anhydride.

Carboxyl-terminal modifications include substitution of the free acid with a carboxamide group or formation of a cyclic lactam at the carboxyl terminus to introduce structural constraints. The peptides described herein can also be cyclized, or deaminated or decarboxylated residues incorporated at the ends of the peptide, such that no terminal amino or carboxyl groups are present, to reduce susceptibility to proteases or limit the conformation of the peptide. Methods of cyclic peptide synthesis are known in the art, for example, in U.S. patent application number 20090035814; muralidharan and Muir,2006,Nat Methods,3:429-38; and Lockless and Muir,2009,Proc Natl Acad Sci USA.6 months 18 days, epub. The C-terminal functional groups of the peptides described herein include amides, amide lower alkyl groups, amide di (lower alkyl groups), lower alkoxy groups, hydroxy and carboxy groups, and lower ester derivatives thereof, and pharmaceutically acceptable salts thereof.

In some embodiments, the polypeptides or fusion proteins described herein are phosphorylated. Peptides can also be readily modified by phosphorylation and other methods (e.g., as described in Hruby, et al, (1990) Biochem J.268:249-262).

In another embodiment, the linker used in the fusion protein, conjugate or chimeric molecule of the invention is a chemical linker, e.g. linked by disulfide bonds between cysteine amino acid residues or chemical bonds formed by amine cross-linkers, e.g. glutaraldehyde, bis (imidoester), bis (succinimidoester), diisocyanate and diacyl chloride. A large amount of data on chemical crosslinkers can be found in section 5.2 of Molecular probes of INVITROGEN.

In certain embodiments, an isolated polypeptide described herein (as a monomer) dimerizes or multimerizes by covalent attachment to at least one linker moiety. Methods for preparing dimers or multimers from polypeptides are well known in the art.

The polypeptides, fusion proteins, and polypeptide multimers described herein can be linked to one or more polymeric moieties. Preferably, these polymers are covalently linked to the polypeptides of the invention. Preferably, the polymer is pharmaceutically acceptable for therapeutic use in the final product formulation. Those skilled in the art will be able to select a desired polymer based on factors such as whether the polymer-peptide conjugate will be used therapeutically and, if so, the desired dosage, circulation time, resistance to proteolysis and other considerations.

Suitable polymers include, for example, polyethylene glycol (PEG), polyvinylpyrrolidone, polyvinyl alcohol, polyamino acids, divinyl ether maleic anhydride, N- (2-hydroxypropyl) -methacrylamide, dextran derivatives (including dextran sulfate), polypropylene glycol, polyoxyethylated polyols, heparin fragments, polysaccharides, cellulose and cellulose derivatives (including methylcellulose and carboxymethylcellulose), starch and starch derivatives, polyalkyl glycols and derivatives thereof, copolymers of polyalkyl glycols and derivatives thereof, polyvinyl ethyl ether and α, β -poly [ (2-hydroxyethyl) -DL-asparagine, and the like or mixtures thereof. Such polymers may or may not have their own biological activity. The polymer may be conjugated covalently or non-covalently to the polypeptide. Conjugation methods for increasing serum half-life are known in the art, see for example U.S. patent nos. 5,180,816, 6,423,685, 6,884,780 and 7,022,673.

In some embodiments, the polymer, when attached to an isolated polypeptide, extends the serum half-life of the isolated polypeptide. In some embodiments, the polymer, when attached to an isolated polypeptide, extends the shelf life of the isolated polypeptide. As used herein, the term "serum half-life" of an isolated polypeptide refers to the period of time required for the concentration or amount of the polypeptide to be reduced by half in vivo. The serum half-life of a polypeptide depends on the rate at which it is eliminated from the serum. The longer the serum half-life, the more stable the polypeptide in vivo. The term "shelf life" refers to the period of time from the date of manufacture that the product is expected to remain within its approved product specifications when stored under specified conditions. There is a need for therapeutic agents (e.g., isolated polypeptides of the invention) that have a longer shelf life.

An isolated polypeptide of the invention may comprise conservative amino acid substitutions. The term "conservative amino acid substitution" refers to an amino acid substitution that changes an amino acid to a different amino acid that has similar biochemical properties (e.g., charge, hydrophobicity, and size). Conservative substitutions of amino acids include, for example, substitutions made between amino acids in the following groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D. Conservative amino acid substitutions do not alter the relative charge or size characteristics of the protein in which the amino acid substitution is performed. Conservative amino acid substitutions typically do not alter the overall structure of the peptide and/or the type of amino acid side chains that are available to form van der Waals bonds with the binding partner.

Amino acid substitutions can be achieved during chemical synthesis of the peptide by adding the desired substitution amino acids in the appropriate order during synthesis. Alternatively, molecular biological methods may be used. Non-conservative substitutions are also included to the extent that they substantially retain the activity of those polypeptides described herein. The amino acid substituted polypeptide will substantially retain the activity of the unsubstituted polypeptide. By "substantially retained" is meant that the variant has at least 50% of one or more activities as compared to the activity of the original polypeptide in a similar assay under similar conditions; preferably the activity is at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, at least 100%, at least 2-fold, at least 5-fold, at least 10-fold, at least 100-fold or more compared to the original polypeptide.

Chimeric molecules

Other aspects of the invention provide chimeric molecules comprising a first portion and a second portion, wherein the first portion comprises an isolated polypeptide disclosed herein, and wherein the second portion is a different molecule than the isolated polypeptide disclosed herein.

In some embodiments, the second portion of the chimeric molecule comprises a therapeutic agent. In some embodiments, the therapeutic agent may be an antibacterial agent. In some embodiments, the therapeutic agent may be an antibiotic, such as, but not limited to, vancomycin, metronidazole, and the like. Classes of antimicrobial agents that may be used according to the present invention include, but are not limited to, aminoglycoside antibiotics, ansamycins, carbacephem, carbapenems, cephalosporins, glycopeptides, lincomamides, lipopeptides, macrolides, monocyclic beta-lactams, nitrofurans, oxazolidinones, penicillins, quinolones, sulfonamides, and tetracyclines. It should be understood that any known antimicrobial agent that can be linked to polypeptides in the art can be used herein.

In some embodiments, the second portion of the chimeric molecule may be an anti-TcdB immune molecule, e.g., a binding agent or antibody that binds TcdB, or a nanobody against TcdB. In some embodiments, the second portion may be an ankyrin repeat.

In some embodiments, the second moiety is a CSPG4 polypeptide. In some embodiments, the second portion is a fragment of CSPG4, preferably CSPG4 ^410-550(CSPG4^R1, SEQ ID NO: 20).

Nucleic acid molecules, vectors or expression cassettes or recombinant host cells

Other aspects of the invention provide an isolated nucleic acid molecule comprising a polynucleotide encoding an isolated polypeptide described herein, a vector or expression cassette comprising a polynucleotide encoding an isolated polypeptide described herein, or a recombinant host cell comprising a polynucleotide encoding an isolated polypeptide described herein or a vector or expression cassette comprising a polynucleotide encoding an isolated polypeptide described herein. In a polynucleotide, vector or expression cassette, some regulatory elements may include, for example, promoters, enhancers, selectable markers, or purification tags, and the like.

Method for producing isolated polypeptides

In another aspect, the invention provides a method of producing an isolated polypeptide, wherein the method comprises obtaining a cell as described herein and expressing a nucleic acid as described herein in the cell. In some embodiments, the method further comprises isolating and purifying the polypeptides described herein.

Neutralizing molecules

Another aspect of the invention provides neutralizing molecules targeting one or more sites selected from E1433, D1467, D1468, E1469, S1598, L1599, L1434, K1435, M1438, V1492, L1494, I1496, L1489, P1506 or Y1510 of TcdB4 (SEQ ID NO: 9).

In some embodiments, the neutralizing molecule targets any combination of E1433, D1467, D1468, E1469, S1598, L1599, L1434, K1435, M1438, V1492, L1494, I1496, L1489, P1506, or Y1510 of TcdB4 (SEQ ID NO: 9).

Another aspect of the invention provides neutralizing molecules targeting one or more sites selected from E1432, D1466, D1467, S1597, L1598, L1433, K1434, M1437, V1491, L1493 or L1488 of TcdB2 (SEQ ID NO: 7).

In some embodiments, the neutralizing molecule targets any combination of E1432, D1466, D1467, S1597, L1598, L1433, K1434, M1437, V1491, L1493, or L1488 of TcdB2 (SEQ ID NO: 7).

In some embodiments, the neutralizing molecule is an antibody, such as, but not limited to, a monoclonal antibody or a humanized antibody. In some embodiments, the neutralizing molecule is used to prevent and treat clostridium difficile infection caused by clostridium difficile strain branch 2 strain.

In some embodiments, the neutralizing molecule is formulated as a pharmaceutical composition. In some embodiments, the pharmaceutical composition comprises a therapeutically effective amount of a neutralizing molecule and a pharmaceutically acceptable carrier.

In some embodiments, the invention relates to a method of preventing and/or treating a disease caused by TcdB2 and/or TcdB4, in particular Clostridium Difficile Infection (CDI) caused by a strain of branch 2, in a subject in need thereof, wherein the method comprises administering to the subject a therapeutically effective amount of a neutralizing molecule.

In some embodiments, the invention relates to the use of a neutralizing molecule in the preparation of a pharmaceutical composition for the prevention and/or treatment of a disease caused by TcdB2 and/or TcdB4 in a subject, in particular Clostridium Difficile Infection (CDI) caused by strain 2.

Pharmaceutical compositions or kits

Other aspects of the invention provide a pharmaceutical composition comprising an isolated polypeptide, fusion protein, conjugate, chimeric molecule or neutralizing molecule targeting a specific site of TcdB2 and/or TcdB4 as disclosed herein, and a pharmaceutically acceptable carrier. The pharmaceutical composition may further comprise additional agents (e.g., for specific delivery, to increase half-life, or other therapeutic agents). For example, the pharmaceutical compositions of the present invention may further comprise other therapeutic agents suitable for the particular disease for which such compositions are designed.

As used herein, the term "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, talc magnesium, calcium stearate or zinc stearate or stearic acid), or solvent encapsulating material, which involves carrying or transporting the polypeptide from one site (e.g., delivery site) to another site (e.g., organ, tissue or part of the body). Pharmaceutically acceptable carriers are "acceptable" in the sense of being compatible with the other ingredients of the formulation and not damaging the subject's tissues (e.g., physiological compatibility, sterility, physiological pH, etc.). Some examples of materials that can be used as pharmaceutically acceptable carriers are well known in the art and can be suitably selected by those skilled in the art as desired. Wetting agents, colorants, release agents, coating agents, sweeteners, flavoring agents, flavorants, preservatives, and antioxidants may also be present in the formulation.

In some embodiments, the isolated polypeptide of the invention in the composition is administered by injection, by catheter, by suppository, or by implant that is a porous, non-porous, or gel-like material, including membranes or fibers. In general, materials that are not absorbed by the polypeptides of the invention are used when the composition is administered.

In other embodiments, the isolated polypeptides of the invention are delivered by a controlled release system.

In typical embodiments, the pharmaceutical composition is formulated according to conventional procedures into a pharmaceutical composition suitable for intravenous or subcutaneous administration to a subject (e.g., a human). Typically, the composition for injection administration is a solution in a sterile isotonic aqueous buffer. If desired, the drug may also include solubilizing agents and local anesthetics, such as lidocaine, to relieve pain at the injection site. Typically, the ingredients are provided separately or mixed together in unit dosage form, e.g., as a dry lyophilized powder or anhydrous concentrate, in a sealed container (e.g., ampoule or sachet) to indicate the amount of active agent. If the drug is administered by infusion, the dosage may be made using an infusion bottle containing sterile pharmaceutical grade water or saline. If the drug is to be administered by injection, an ampoule of sterile water for injection or saline may be provided to mix the ingredients prior to administration.

The pharmaceutical composition for systemic administration may be a liquid, such as sterile saline, ringer's lactate solution or Hank's solution. In addition, the pharmaceutical composition may be in solid form and redissolved or suspended immediately prior to use. Lyophilized forms are also contemplated.

The pharmaceutical composition may be contained within a lipid particle or vesicle, such as a liposome or microcrystal, which is also suitable for parenteral administration.

For example, the pharmaceutical compositions of the present invention may be administered or packaged as unit doses. When the term "unit dose" is used in reference to the pharmaceutical compositions of the present invention, it refers to physically discrete units suitable as unitary dosages for subjects, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the desired diluent (e.g., carrier or vehicle).

In some embodiments, the isolated polypeptides described herein can be conjugated to a therapeutic moiety (e.g., an antibiotic). Techniques for conjugating such therapeutic moieties to polypeptides (including, for example, fc domains) are well known; see, for example, amon et al ,"Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy",in Monoclonal Antibodies And Cancer Therapy,Reisfeld (eds.), 1985, pp.243-56,Alan R.Liss,Inc); hellstrom et al, "Antibodies For Drug Delivery", in Controlled Drug Delivery (2 nd edition), robinson et al ,(eds.),1987,pp.623-53,Marcel Dekker,Inc.);Thorpe,"Antibody Carriers Of Cytotoxic Agents In Cancer Therapy:A Review",in Monoclonal Antibodies'84:Biological And Clinical Applications,Pinchera et al ,(eds.),1985,pp.475-506);"Analysis,Results,And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy",in Monoclonal Antibodies For Cancer Detection And Therapy,Baldwin, (eds.), 1985, pp.303-16,Academic Press; and Thorpe et al ,(1982)"The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates,"Immunol.Rev.,62:119-158.

Furthermore, the pharmaceutical composition may be provided as a pharmaceutical kit. The kit comprises a container containing a polypeptide or pharmaceutical composition disclosed herein.

Method for preventing and/or treating CDI

The isolated polypeptides, chimeric molecules and pharmaceutical compositions comprising the isolated polypeptides of the invention are useful for the prevention and/or treatment of various diseases caused by TcdB2 and/or TcdB 4. In some embodiments, the disease is Clostridium Difficile Infection (CDI), e.g., CDI caused by clostridium difficile branch 2 strain. Accordingly, further provided herein is a method of preventing and/or treating clostridium difficile infection comprising administering to a subject in need thereof a therapeutically effective amount of an isolated polypeptide or a pharmaceutical composition comprising such an isolated polypeptide disclosed herein, or a neutralizing molecule targeting a specific site of TcdB2 and/or TcdB4 disclosed herein. An isolated polypeptide or pharmaceutical composition comprising such an isolated polypeptide may be effective to block the binding of TcdB2 and/or TcdB4 to cell surface TFPI.

As used herein, the term "clostridium difficile branch 2 strain" refers to a class of clostridium difficile strains classified as branch 2 according to multi-site sequencing typing (MLST) (Griffiths, d. Et al, multilocus sequence typing of Clostridium difficilie j. Clin. Microbiol.48,770-778 (2010)).

Clostridium difficile MLST classification can be found in an internet-accessed database (http:// pubmlst. Org).

Clostridium difficile branch 2 expresses only two TcdB variants (TcdB 2 and TcdB 4). TcdB2 and TcdB4 do not recognize Wnt receptor Frizzled (FZD) (Chung et al, 2018; henkel et al, 2020; lopez-Urena et al, 2019; pan et al, 2021; simeon et al, 2019), but recognize TFPI as demonstrated by the present invention.

The "Clostridium difficile branch 2 strain" contains several epidemic and clinically important strains, belonging to the group consisting of ST01 (RT 027), ST67, ST41, ST48, etc., (Knight,D.R.,Imwattana,K.,Kullin,B.,Guerrero-Araya,E.,Paredes-Sabja,D.,Didelot,X.,Dingle,K.E.,Eyre,D.W.,Rodriguez,C., and Riley,T.V.(2021).Major genetic discontinuity and novel toxigenic species in Clostridioides difficile taxonomy.Elife 10).

In some embodiments, the pharmaceutical compositions of the invention for preventing and/or treating CDI further comprise additional therapeutic agents or polypeptides, such as CSPG4 polypeptides or fragments thereof, preferably CSPG4 ^410-550(CSPG4^R1, SEQ ID NO: 20.

However, in other embodiments, the pharmaceutical compositions of the invention for preventing and/or treating CDI further comprise an agent that facilitates blocking binding of TcdB2 and/or TcdB4 to cell surface TFPI. For example, these agents may be antibodies to TcdB2 and/or TcdB 4.

As used herein, a "therapeutically effective amount" refers to the amount of each therapeutic agent of the invention (e.g., an isolated polypeptide fragment, an additional isolated polypeptide fragment, and an agent that blocks binding of TcdB2 and/or TcdB4 to cell surface TFPI) required to produce a therapeutic effect on a subject when used alone or in combination with one or more other therapeutic agents. As will be appreciated by those of skill in the art, the effective amount will vary depending upon the particular condition being treated, the severity of the condition, the individual subject parameters (including age, physical condition, body shape, sex, and weight), the duration of the treatment, the nature of concurrent therapy (if any), the particular route of administration, and like factors within the knowledge and expertise of a health practitioner. These factors are well known to those of ordinary skill in the art and can be addressed by no more than routine experimentation. It is generally preferred to use the maximum dose of the individual components or combinations thereof, i.e. the highest safe dose according to sound medical judgment. However, one of ordinary skill in the art will appreciate that the subject may adhere to lower or tolerable doses for medical reasons, psychological reasons, or almost any other reason.

Empirical considerations, such as half-life, often aid in determining the dosage. For example, therapeutic agents compatible with the human immune system, such as polypeptides comprising regions from humanized antibodies or fully humanized antibodies, can be used to extend the half-life of the polypeptide and prevent the polypeptide from being attacked by the host immune system. The frequency of administration may be determined and adjusted during the course of treatment, typically but not necessarily based on treatment and/or inhibition and/or amelioration and/or delay of disease. Or a sustained continuous release formulation of the polypeptide may be suitable. Various formulations and devices for achieving sustained release (sustained release) are known in the art.

In some embodiments, the dose is daily, every other day, every third day, every fourth day, every fifth day, or every sixth day. In some embodiments, dosing frequency is weekly, every 2 weeks, every 4 weeks, every 5 weeks, every 6 weeks, every 7 weeks, every 8 weeks, every 9 weeks, or every 10 weeks; or once a month, once every 2 months, or once every 3 months, or longer. The progress of this therapy is readily monitored by conventional techniques and assays. The dosing regimen (including the polypeptide used) may vary over time.

For the purposes of the present invention, the appropriate dosage of the therapeutic agents described herein will depend on the particular agent (or composition thereof) used, the formulation and route of administration, the type and severity of the disease, whether the polypeptide is used for prophylactic or therapeutic purposes, previous therapies, the subject's clinical history and response to the antagonist, and the discretion of the attendant physician. Typically, the clinician will administer the polypeptide until a dose of the desired result is reached. The administration of one or more polypeptides may be continuous or intermittent, depending on, for example, the physiological condition of the recipient, whether the purpose of the administration is therapeutic or prophylactic, and other factors known to the skilled practitioner. The administration of the polypeptide may be substantially continuous over a preselected period of time, or may be a series of spaced doses, for example, before, during or after the occurrence of the disease.

As used herein, the term "prophylactic" or "preventing" in connection with a disease refers to preventing the appearance or onset of the disease, or preventing the appearance or onset of one or more symptoms of the disease. As used herein, the term "treating" or "treatment" refers to the application or administration of a polypeptide or a composition comprising the polypeptide to a subject in need thereof. By "subject in need thereof" is meant an individual suffering from a disease, a symptom of a disease, or having a susceptibility to a disease, with the aim of treating, curing, alleviating, altering, remediating, ameliorating, improving, or affecting a disease, a symptom of a disease, or susceptibility to a disease. In some embodiments, the subject has CDI, e.g., CDI caused by a strain of branch 2. In some embodiments, the subject is a mammal. In some embodiments, the subject is a non-human primate. In some embodiments, the subject is a human.

Alleviation of a disease includes delaying the progression or progression of a disease, or reducing the severity of a disease. Alleviating the disease does not necessarily require a cure. As used herein, "delaying" the progression of a disease means to delay, hinder, slow, delay, stabilize and/or delay the progression of the disease. This delay may vary in length of time depending on the disease history and/or the individual being treated. A method of "delaying" or alleviating the progression of a disease or delaying the onset of a disease is a method of reducing the likelihood of developing one or more symptoms of a disease over a given time frame and/or reducing the extent of symptoms over a given time frame, as compared to a control not using the method. Such comparisons are typically based on clinical studies, using a large number of subjects sufficient to produce statistically significant results.

"Progression" or "progression" of a disease means the initial manifestation and/or subsequent progression of the disease. The progression of the disease can be detected and assessed using standard clinical techniques well known in the art. However, development also refers to progress that may not be detectable. For the purposes of the present invention, development or progression refers to the biological process of symptoms. "progression" includes occurrence, recurrence and onset. As used herein, a "episode" or "appearance" of a disease includes an initial episode and/or recurrence.

Conventional methods known to those of ordinary skill in the medical arts may be used to administer an isolated polypeptide or pharmaceutical composition to a subject, depending on the type of disease or the site of the disease to be treated. The composition may also be administered by other conventional routes, such as orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally, or by an implanted reservoir. The term "parenteral" as used herein includes subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intralesional and intracranial injection or infusion techniques. In addition, it may be administered by an injectable depot route of administration, such as using 1 month, 3 months or 6 months of depot injectable or biodegradable materials and methods.

Sequence summary

The main sequences mentioned in this disclosure are summarized below:

the K2 domain of human TFPI (SEQ ID NO:1, positions 125-175 of human TFPI)

CFLEEDPGICRGYITRYFYNNQTKQCERFKYGGCLGNMNNFETLEECKNIC

The K2 domain of mouse Tfpi (SEQ ID NO:2, positions 121-171 of mouse Tfpi)

CFLEEDPGLCRGYMKRYLYNNQTKQCERFVYGGCLGNRNNFETLDECKKIC

Human TFPI alpha (SEQ ID NO: 3)

DSEEDEEHTIITDTELPPLKLMHSFCAFKADDGPCKAIMKRFFFNIFTRQCEEFIYGGCEGNQNRFESLEECKKMCTRDNANRIIKTTLQQEKPDFCFLEEDPGICRGYITRYFYNNQTKQCERFKYGGCLGNMNNFETLEECKNICEDGPNGFQVDNYGTQLNAVNNSLTPQSTKVPSLFEFHGPSWCLTPADRGLCRANENRFYYNSVIGKCRPFKYSGCGGNENNFTSKQECLRACKKGFIQRISKGGLIKTKRKRKKQRVKIAYEEIFVKNM

Human TFPI beta (SEQ ID NO: 4)

DSEEDEEHTIITDTELPPLKLMHSFCAFKADDGPCKAIMKRFFFNIFTRQCEEFIYGGCEGNQNRFESLEECKKMCTRDNANRIIKTTLQQEKPDFCFLEEDPGICRGYITRYFYNNQTKQCERFKYGGCLGNMNNFETLEECKNICEDGPNGFQVDNYGTQLNAVNNSLTPQSTKVPSLFVTKEGTNDGWKNAAHIYQVFLNAFCIHASMFFLGLDSISCLC

Mouse Tfpi β (SEQ ID NO: 5)

MTYKMKKEYAFWATVCLLLSLVPEFLNALSEEADDTDSELGSMKPLHTFCAMKADDGPCKAMIRSYFFNMYTHQCEEFIYGGCEGNENRFDTLEECKKTCIPGYEKTAVKAASGAERPDFCFLEEDPGLCRGYMKRYLYNNQTKQCERFVYGGCLGNRNNFETLDECKKICENPVHSPSPVNEVQMSDYVTDGNTVTDRSTVNNIVVPQSPKVPRRRVTKEETNGGWKNADYTYQGFLSSVYIHVLYFVFRIG

TcdB2 (full length sequence of Clostridium difficile strain R20291, SEQ ID NO:7; underlined is SEQ ID NO:29, positions 841-1833 corresponding to SEQ ID NO:7; bold is SEQ ID NO:30, positions 1284-1833 corresponding to SEQ ID NO:7; receptor binding domain (DRBD) of TcdB2 corresponds to positions 840-1833 of SEQ ID NO: 7)

MSLVNRKQLEKMANVRFRVQEDEYVAILDALEEYHNMSENTVVEKYLKLKDINSLTDIYIDTYKKSGRNKALKKFKEYLVTEVLELKNNNLTPVEKNLHFVWIGGQINDTAINYINQWKDVNSDYNVNVFYDSNAFLINTLKKTIVESATNDTLESFRENLNDPRFDYNKFYRKRMEIIYDKQKNFINYYKTQREENPDLIIDDIVKIYLSNEYSKDIDELNSYIEESLNKVTENSGNDVRNFEEFKGGESFKLYEQELVERWNLAAASDILRISALKEVGGVYLDVDMLPGIQPDLFESIEKPSSVTVDFWEMVKLEAIMKYKEYIPGYTSEHFDMLDEEVQSSFESV

TcdB4 (full length sequence of Clostridium difficile strain 8864, SEQ ID NO:9; underlined: SEQ ID NO:15, positions 842-1834 of SEQ ID NO:9; bold: SEQ ID NO:16, positions 1285-1834 of SEQ ID NO:9; receptor binding domain (DRBD) of TcdB4, positions 841-1834 of SEQ ID NO: 9)

CSPG4^R1(SEQ ID NO:20)

ELPEPCVPEPGLPPVFANFTQLLTISPLVVAEGGTAWLEWRHVQPTLDLMEAELRKSQVLFSVTRGARHGELELDIPGAQARKMFTLLDVVNRKARFIHDGSEDTSDQLVLEVSVTARVPMPSCLRRGQTYLLPIQVNPVND

TFPI ^K2-CSPG4^R1 (SEQ ID NO:21, capitalized is linker)

cfleedpgicrgyitryfynnqtkqcerfkyggclgnmnnfetleecknicGGGGSGGGGSGGGGSelpepcvpepglppvfanftqlltisplvvaeggtawlewrhvqptldlmeaelrksqvlfsvtrgarhgeleldipgaqarkmftlldvvnrkarfihdgsedtsdqlvlevsvtarvpmpsclrrgqtyllpiqvnpvnd

TFPI ^K2 -Fc (SEQ ID NO:26, fc portion shown in lower case, TFPI ^K2 underlined) )MGILPSPGMPALLSLVSLLSVLLMGCVAETGEKPDFCFLEEDPGICRGYITRYFYNNQTKQCERFKYGGCLGNMNNFETLEECKNICEDGPNGFQVDNYGTQLNAVNNSLTPQSTKVPSLFGTLEVLFQgpkscdkthtcppcpapellggpsvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhqdwlngkeykckvsnkalpapiektiskakgqprepqvytlppsrdeltknqvsltclvkgfypsdiavewesngqpennykatppvldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspgkhhhhhh

Tfpi ^K2 -Fc (SEQ ID NO:27, fc portion shown in lowercase, tfpi ^K2 underlined) )MGILPSPGMPALLSLVSLLSVLLMGCVAETGYEKTAVKAASGAERPDFCFLEEDPGLCRGYMKRYLYNNQTKQCERFVYGGCLGNRNNFETLDECKKICENPVHSGTLEVLFQgpkscdkthtcppcpapellggpsvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhqdwlngkeykckvsnkalpapiektiskakgqprepqvytlppsrdeltknqvsltclvkgfypsdiavewesngqpennykatppvldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspgkhhhhhh

Examples

Accordingly, the invention as generally described will be more readily understood by reference to the following examples, which are provided for illustrative purposes only and are not intended to limit the invention. These examples are not meant to be all or the only experiments performed.

All cell lines, plasmids, reagents used in the examples below are commercially available unless otherwise indicated.

Experimental model and topic details

Cell lines

All cell lines are commercially available. All cell lines were cultured in DMEM medium with 10% Fetal Bovine Serum (FBS) and 1% penicillin-streptomycin in a humid environment of 95% air and 5% co2 at 37 ℃. Mycoplasma contamination of HeLa and 293T cells was detected as negative and identified by STR analysis (Shanghai wing and applied Biotechnology Co., ltd. (Shanghai Biowing Biotechnology Co., LTD), shanghai, china).

A mouse

All animal procedures reported herein were approved by the Institutional animal care and Use Committee (Institutional ANIMAL CARE AND Use Committee) (IACUC protocol # 19-010-TL) of the West lake university study. C57BL/6J mice (male and female, 6-8 weeks) were purchased from Shanghai Ji Hui laboratory animal feeding limited (Shanghai Jihui Laboratory ANIMAL CARE co., ltd.) (Shanghai, china). Tfpi. Beta. KO mice were generated at the laboratory animal resource center (Laboratory Animal Resources Center) at the university of West lake. Mice were kept in cages, food and water were unrestricted, and monitored under the care of full-time staff.

TABLE 1 Main resources

Method details

CDNA constructs

The genes encoding TcdB5 (reference sequence: tcdB _ES130, SEQ ID NO: 10) and TcdB6 (reference sequence: tcdB _CD160, SEQ ID NO: 11) were codon optimized, synthesized by Genscript (Nanjing, china), and cloned into the pHT01 vector. The DNA fragments encoding TcdB4 ^1-841、TcdB4^842-1834、TcdB4^1801-2367、TcdB4^1285-1834 and TcdB2 ^1285-1834 were PCR amplified and cloned into pET28a vector and HA-His tag was introduced at its C-terminus. The genes encoding human TFPIα(SEQ ID NO:3)、TFPIβ(SEQ ID NO:4)、TFPI^K1-GPI、TFPI^K2-GPI、TFPI2^K1-GPI、TFPI2^K2-GPI、AMBP^K3-GPI and mouse Tfpi beta (SEQ ID NO: 5) were codon optimized, synthesized by Genscript (Nanjing, china), and cloned into the PLVX-IRES-Cherry vector. The DNA fragments encoding TFPI ^K1+K2、TFPI^K1、TFPI^K2、Tfpi^K1、Tfpi^K2、CSPG4^R1 and TFPI ^K2-CSPG4^R1 were PCR amplified and cloned into pCAG or PHLsec vectors with FLAG, fc-His or GFP-His tags fused to their C-termini.

Recombinant proteins

Recombinant full length TcdB protein was expressed in Bacillus subtilis SL401 as described previously (Shen et al 2020). TcdB4 ^1-841、TcdB4^842-1834、TcdB4^1801-2367、TcdB4^1285-1834 and TcdB2 ^1285-1834 were expressed in e.coli BL21 (DE 3) and purified as His-tagged proteins. Recombinant TFPIα-GFP、TFPI^K1+K2-GFP、TFPI^K1+K2、TFPI^K1+K2-Fc、TFPI^K1-Fc、TFPI^K2-Fc、Tfpi^K1-Fc、Tfpi^K2-Fc、CSPG4^R1 and TFPI ^K2-CSPG4^R1 were expressed in 293F cells and purified as His or FLAG tagged proteins.

Cytopathic cell rounding assay

Cytopathic effects of TcdB (cell rounding) were analyzed using a gold standard cell rounding assay. Briefly, cells are exposed to the toxin for a specified period of time (or 12 hours if not noted). The phase contrast image was captured by an Olympus IX73 microscope system (×10-20 objective). The number of round and normal shape cells was counted manually. The percentage of round cells was analyzed using GRAPHPAD PRISM and Origin software.

Whole genome CRISPR screening

A HELA CRISPR/Cas9 whole genome KO library was generated as described previously (Tao et al, 2019; tao et al, 2016). The GeCKO v2 library consists of two sub-libraries (a and B) and contains six grnas targeting each gene. 293T cells are used to package lentiviruses. The supernatant of 293T cultures was collected 48 hours after transfection. HeLa-Cas9 cells were then transduced with a lentiviral library at a multiplicity of infection (MOI) of 0.3 and selected with 2.5ug/mL puromycin for 4 days. For each CRISPR sub-library, at least 6.7x10 ⁷ cells were seeded onto 15 cm cell culture dishes to ensure adequate gRNA coverage. TcdB4 was then added to the cell library at the indicated concentration and incubated for 18 hours. The plates were then washed with Phosphate Buffered Saline (PBS) to remove loosely attached cells. The remaining cells were cultured with non-toxic medium, allowed to grow to about 70% confluence, and subjected to the next round of screening. Three rounds of screening were performed with increasing concentrations of TcdB4 (0.045 pM, 0.15pM and 0.45pM, respectively). Cells from the last round of screening were collected and their genomic DNA was extracted using blood and cell culture DNA mini kit (Qiagen). The DNA fragment containing the gRNA sequence was amplified by PCR using primers lentiGP1_ F (AATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCG) and lentiGP-3_R (ATGAATACTGCCATTTGTCTCAAGATCTAGTTACGC). Next Generation Sequencing (NGS) is performed by commercial suppliers (Novogene). CRISPR screening data is not shown.

Production of KO cell lines

To generate HeLa TFP ^I-/-、PIGM^-/-、PIGP^-/- and PIGX ^-/- cells, the following gRNA sequences were cloned into LentiGuide-Puro vectors to target the indicated genes: 5'-CCTGACTCCGCAATCAACCA-3' (TFPI), 5'-GTATACGGACATCGACTACC-3' (PIGM), 5'-CTACAGTACTTTACCTCGTG-3' (PIGP) and 5'-CGATGATAGCGGCAGTGCAC-3' (PIGX). HeLa CSPG4 ^-/-/TFPI^-/- cells were generated from HeLa CSPG4 ^-/- cells by CRISPR/Cas9 mediated KO using the TFPI-targeted gRNAs described above. Lentiviruses were generated by co-transfecting LentiGuide-puro plasmids containing each of gRNA, pSPAX2 and pmd2.G into 293T cells. HeLa-Cas9 cells were transduced with lentiviruses expressing target gRNAs. A mixed population of infected cells was selected with puromycin (2.5. Mu.g/ml). For all KO cells, monoclonal cells were then isolated and verified by sequencing.

Cell surface binding of TcdB4

Cells were pre-washed with PBS and incubated with 50nm TcdB4 for 15 min on ice. Cells were then washed three times with ice-cold PBS, scraped from the plate, lysed with RIPA, and subjected to immunoblot analysis.

Immunoblot analysis

Protein content in cell lysates was determined by BCA assay (Beyotime, P0012S). SDS sample buffer was added to the cell lysate, heated for 10min, separated on SDS-PAGE gels, and transferred to nitrocellulose membranes (GE HEALTHCARE, 10600002). Membranes were blocked with tris buffered saline containing 0.5% Bovine Serum Albumin (BSA) and 0.1% Tween-20 for 30 minutes at Room Temperature (RT). The membrane was then incubated with primary antibody overnight (4 ℃), washed, and incubated with secondary antibody for 30 minutes. The signals were detected using an enhanced chemiluminescent substrate (Thermo FISHER SCIENTIFIC, 1863096) and a Fuji LAS3000 imaging system.

Cell-based toxin competition assay

The toxins were pre-incubated with TFPI ^K1+K2-Fc、TFPI^K1 -Fc or TFPI ^K2 -Fc at room temperature for 10 minutes at the indicated toxin/protein ratio. The mixture was added to the medium. Cells were then incubated at 37 ℃ and examined for percent cell rounding.

Pull-down measurement

Pull-down assays were performed using protein a sepharose beads (Thermo FISHER SCIENTIFIC). Briefly, fc-labeled TFPI domains were mixed with full length TcdB1/TcdB4 or TcdB4 fragments at the indicated concentrations in 1ml PBS. The mixture was incubated at 4℃for 30 min and co-precipitated by protein A agarose gel beads. The beads were washed, pelleted, boiled in SDS sample buffer, and subjected to SDS-PAGE or immunoblot analysis.

Biofilm interferometry

Binding affinity between recombinant TcdB4 and human/mouse TFPI domains was measured by BLI assay using the Octet RED96 system (ForteBio). All proteins were diluted in PBS. Briefly, fc-labeled human/mouse TFPI domains (10. Mu.g/mL) were immobilized on Dip and Read anti-human IgG-Fc biosensors (ForteBio) and equilibrated with PBS. The biosensor was then exposed to full length TcdB4, tcdB4 ^1285-1834, or TcdB2 ^1285-1834, and then washed (dissociated) with PBS. Binding affinity (K _d) was calculated using data analysis software (ForteBio).

To analyze the sequential binding of factors Xa (FXa) and TcdB4 to TFPI, TFPI ^K2 -Fc (10 μg/mL) was immobilized on a Dip and Read anti-human IgG-Fc biosensor and equilibrated with HNBSACa buffer (50 mM HEPES, 100mM NaCl, 5mM CaCl2, 0.1% BSA, pH 7.3). The loaded biosensor was first exposed to 100nM FXa, equilibrated again with HNBSACa buffer, and then exposed to 300nM TcdB4 ^1285-1834. Or first exposing the loaded biosensor to 300nM TcdB4 ^1285-1834, equilibrated with HNBSACa buffer, and then to 100nM FXa. All biosensors were then washed with HNBSACa buffer.

Preparation and data acquisition of frozen electron microscope samples

To prepare a cryo-electron microscope sample, 4. Mu.L of purified TcdB4-TFPI ^K1+K2 complex at a concentration of 9.2mg/mL was placed on a glow discharge porous carbon film (Quantifoil Au R2.1/3.1,300 mesh). The carbon film was blotted with filter paper in a chamber set at 8 ℃ at 100% humidity for 4 seconds to remove excess sample, and then flash frozen in liquid nitrogen cooled ethane using a vitro Mark IV system (Thermo FISHER SCIENTIFIC).

The cryo-electron microscope specimen was imaged on a 300kV Titan Krios electron microscope (Thermo FISHER SCIENTIFIC) at a normal magnification of 81,000 x. Film was recorded in counter mode using a Gatan K3 detector (Thermo FISHER SCIENTIFIC) equipped with a GIF quantum energy filter (slit width 20 eV), pixel sizeEach stack of 32 frames is exposed for 2.56 seconds, the dose rate per frame is 23 counts/second/physical pixelAutoEMation II (Lei and Frank, 2005) are used for efficient (-3000 stack/24 hours) fully automated data collection. All 32 frames in each stack are aligned and summed using the full image motion correction program MotionCor (Zheng et al, 2017) and combined into oneIs larger than the pixel size of the pixel. The defocus value of each image varies from 1.5 μm to 2.0 μm, as determined by Gctf (Zhang, 2016).

Data processing of frozen electron microscope

A total of 4,816 micrographs were collected, of which 4,251 were manually selected for further processing. A total of 2,317,113 particles were automatically selected and two-dimensional (2D) classified using Gautomatch (developed by Kai Zhang, https:// www.mrc-lmb.cam.ac. uk/kzhang/Gautomatch /), yielding 1,244,165 good particles. Then, the inventors used 4x binning particles (pixel size: ) (round 1) single reference 3D classification was performed for two parallel runs. After round 1, good particles were selected and combined and duplicate particles were removed. The remaining 953,332 particles were applied to the other two parallel running single reference 3D classifications (round 2), but with 2x bin particles (pixel size: ). After local classification, the remaining 536,277 particles were re-centered and re-extracted (pixel size: ) To automatically refine, thereby The TcdB4-TFPI complex is reconstructed from the average resolution of (a). An additional round (round 3) of 3D classification is then performed. The remaining particles are classified with a soft mask of the composite core region. Good class containing 227825 particles yields an average resolution ofIs performed in the reconstruction of (a). Further local classification and refinement of these particles using a soft mask at the interface between TFPI and TcdB4, thereby providing a new and improved resolutionAdditional reconstructions of the interface are generated.

The angular distribution of the particles used for the final reconstruction is reasonable and the refinement of the atomic coordinates is not affected by a severe overfitting. The resulting electron microscopy density map shows the clear characteristics of the amino acid side chains in the core region and TcdB4-TFPI interface. The reported resolution limit is calculated according to the FSC 0.143 standard and the high resolution noise substitution method (Chen et al, 2013). Prior to visualization, all electron micrographs were corrected for the Modulation Transfer Function (MTF) of the detector and then sharpened by applying a negative B factor estimated using an automated procedure (Rosenthal and Henderson, 2003). Local resolution changes were estimated using ResMap (Swint-Kruse and Brown, 2005).

Model construction and optimization

Using the crystal structure of clostridium difficile toxin B (PDB code: 6OQ 5) as a template, a homology model of TcdB4 was generated using CHAINSAW (Stein, 2008). Homology models were fit to the cryo-electron microscopy images of TcdB4-TFPI complexes using UCSF chimeras (PETTERSEN et al, 2004). The model is manually adjusted in COOT (Emsley and Cowtan, 2004), then the real space optimization is performed for several iterations in PHENIX, and the manual adjustment is performed in COOT. Similarly, the crystal structure of the K2 domain of TFPI was fitted into the cryo-electron micrograph and adjusted manually in COOT. For statistical information on 3D reconstruction and model optimization, see table 2.

Table 2.3 statistical data for D reconstruction and model optimization

Site-directed mutagenesis

Mutations in TcdB or TcdB fragments were generated using oligonucleotides containing the mutation of interest using QuickChange II site-directed mutagenesis kit (Agilent Technologies, 200523) or Q5 site-directed mutagenesis kit (NEW ENGLAND Biolabs, E0554S).

Evolutionary analysis

A first order sequence of 2,691 TcdB was obtained directly from previous studies (Shen et al 2020). The evolutionary relationship tree was generated using SPLITSTREE v.4.17.0 software (Huson and Bryant, 2006).

Tfpi. Beta. KO mice

SpCas9 protein and sgRNA were microinjected into fertilized eggs of C57BL/6 mice, and the sequence of the sgRNA is shown in Table 3. Fertilized eggs were transplanted to obtain positive F0 mice, which were confirmed by PCR and sequencing. Stable F1 mice models were obtained by mating positive F0 mice with C57BL/6 mice. Mice were genotyped using a mouse tissue direct PCR kit (10185es50, yeasen). The PCR products were analyzed by agarose gel electrophoresis. Table 3 summarizes the primers used for genotyping.

TABLE 3 oligonucleotides used in the study

Colonic loop ligation assay

All procedures were performed according to guidelines approved by the institutional animal care and use committee of western lake university (IACUC Protocol # 19-010-TL). Female mice of 6-8 weeks old were anesthetized by intraperitoneal injection of 1% sodium pentobarbital. Midline laparotomy was performed to locate the ascending colon and ligate the seal with silk threadIs a ring of (a). Mu.g of TCDB or TcdB2 with or without 100. Mu.g of TFPI ^K2 -Fc, 50. Mu.g of CSPG4 ^R1 or 100. Mu.g of TFPI ^K2-CSPG4^R1 were injected into 100. Mu.l of 0.9% saline or 100. Mu.l of 0.9% saline into the sealed colon segment using an insulin syringe and then the skin incision was sutured. Mice were allowed to recover in a 37 ℃ thermostated plate. After 6 hours, the mice were euthanized and the ligated colon segments were excised. The colon segments were fixed, paraffin embedded, sectioned, and then subjected to histopathological analysis.

H & E staining, immunohistochemistry and histopathological analysis

Colon specimens were fixed in formalin for 12 hours, then dehydrated using an alcohol gradient, permeabilized with xylene, and then embedded in paraffin. Paraffin blocks were cut into 5 μm thick sections. Sections were stained with hematoxylin and eosin or detected by immunohistochemical analysis with anti-TFPI antibodies (ab 180619, abcam). H & E stained sections were scored by two pathologists for edema, inflammatory cell infiltration, epithelial destruction, and crypt damage, ranging from 0 to 3 (mild to severe). The average score is plotted on a graph.

Quantification and statistical analysis

Quantitative TcdB-induced cell rounding

Phase contrast images of cells were taken (Olympus IX73, 10-20x objective). Randomly selecting a300×300 μm region comprisingIndividual cells. Round and normal shaped cells were counted manually and the percentage of round cells was calculated. Data are expressed as mean ± SD from six independent replicates and analyzed using student t test in the OriginPro or GRAPHPAD PRISM software. ns, not significant; * P <0.05; * P <0.01; * P < 0.001.

Statistical analysis of histopathological analysis

H & E stained sections were scored blindly and plotted on a chart. Data are expressed as mean ± SEM and analyzed in the OriginPro or GRAPHPAD PRISM software using FISHER LSD test for multiple comparisons of one-way analysis of variance. ns, not significant; * P <0.05; * P <0.01; * P <0.001; * P < 0.0001.

Results

Whole genome CRISPR-Cas9 screening for identifying host factors of TcdB4

The inventors began a gold standard cytopathic cell rounding assay on HeLa-Cas9 cells (parental HeLa cell line, hereafter wild-type) and CSPG4 ^-/-FZD1/2/7^-/- cells to monitor the activity of reference TcdB from each of the eight known subtypes. The inventors observed that TcdB4 (reference sequence from strain 8864) is a very potent variant of toxin for both HeLa WT and CSPG4 ^-/-/FZD1/2/7^-/- cells (fig. 8). The inventors then tested several human cell lines from different tissue sources for sensitivity to TcdB 4. Some cell lines, including U2OS and MCF-7, were much less sensitive to TcdB4 than others, such as HeLa, A549 and HT-29 (FIG. 1A). These findings indicate that uncharacterized cell type specific receptors can efficiently mediate cell entry of TcdB 4.

To identify the receptor for TcdB4, the inventors performed a whole genome CRISPR-Cas9 screen in HeLa cells. Briefly, cells were transduced with a whole genome gRNA library (GeCKO v 2) targeting 19,052 human genes (Sanjana et al, 2014) with lentiviruses and three rounds of selection with increasing concentrations of TcdB4 (fig. 1B). The gRNA sequences from the surviving cell population were decoded by Next Generation Sequencing (NGS). The inventors assessed the identified genes based on fold enrichment of the gRNA reads, NGS reads per gene, and the number of unique grnas (fig. 1C and 1D).

The inventors focused on top-grade enrichment genes with multiple unique gRNA targets. UGP2 and CSPG4 are two contemplated hot spots. UGP2 is a UDP-glucose producing cytosolic enzyme (Flores-Diaz et al, 1997), a cofactor required for the glycosylation of small GTPases by most LCTs (Chaves-Olarte et al, 1996; jank and Aktories, 2008). This gene often appears as a hotspot in previous whole genome CRISPR-Cas9 screens LCT (including TcdA, tcdB and TcsL) (Lee et al, 2020; tao et al, 2019; tao et al, 2016), but not tcnα (Zhou et al, 2021), which uses UDP-N-acetylglucosamine as a co-substrate for glycosylation (Selzer et al, 1996). CSPG4 is a characterized receptor for TcdB1 and TcdB2 (Chen et al, 2021; tao et al, 2016; yuan et al, 2015). Previous studies have also shown that CSPG4 KO cells are mildly resistant to TcdB4 (Pan et al, 2021).

TFPI was targeted by five different grnas and emerged from the screen. TFPI modulates the tissue factor-dependent pathway of coagulation, which exists primarily on the cell membrane and in the extracellular space (Broze and Girard,2012; wood et al, 2014). Five genes, including PIGX, PIGM, PIGP, PIGB and PIGV, encode key proteins in the Glycosyl Phosphatidylinositol (GPI) anchored biosynthetic pathway. GPI anchors are posttranslational modifications that allow the modified protein to attach to the cell membrane via a glycolipid structure. In addition, many other GPI-anchored biosynthetic genes, such as PIGA, DPM1, PIGW, PIGQ, PIGS, PIGY, PIGC, and PIGF, have been enriched but do not meet the stringent criteria described above. RNF41 is a cytoplasmic E3 ubiquitin protein ligase (Qiu and Goldberg, 2002), and ZNF619 is a predicted nucleoprotein with nucleic acid binding properties.

Validation of receptor candidates

To verify the receptor candidates, the inventors generated TFPI ^-/-、PIGX^-/-、PIGM^-/- and PIGP ^-/- HeLa cell lines using the CRISPR-Cas9 method. For each candidate gene, two single KO clones were established and targeted disruption of the open reading frame was confirmed by DNA sequencing. These cell lines, as well as FZD1/2/7 ^-/- and CSPG4 ^-/- cells, were tested by cytopathic cell rounding experiments (toxin concentration inducing 50% cell rounding defined as CR ₅₀) using TcdB4 (fig. 2A).

HeLa FZD1/2/7 ^-/- cells were as sensitive as WT cells to TcdB4, while CSPG4 ^-/- cells exhibited slightly increased resistance (based on CR ₅₀ hereafter). HeLa PIGX ^-/-、PIGM^-/- and PIGP ^-/- cells had moderately reduced sensitivity (-7 to 20-fold) to TcdB4 compared to WT cells (fig. 2B). Furthermore, pretreatment of phosphatidylinositol-specific phospholipase C (PI-PLC), an enzyme that cleaves phosphatidylinositol and releases GPI-anchored proteins from the cell membrane, reduced cytopathic effects of TcdB4 induced in HeLa cells (fig. 2C and 9). These results indicate that the GPI anchored proteins are involved in TcdB 4-induced toxicity to host cells.

Notably, heLa TFPI ^-/- cells were highly resistant to TcdB4, with a-260 fold reduced sensitivity compared to WT cells (fig. 2B). Increased resistance in TFPI ^-/- cells was further confirmed by immunoblotting of RAC1 glycosylation (fig. 2D). In addition, the inventors monitored surface binding of TcdB4 in HeLa WT, FZD1/2/7 ^-/-、CSPG4^-/-, and TFPI ^-/- cells using immunoblotting assays. TcdB4 binds equally to the cell surface of WT and FZD1/2/7 ^-/- cells. The binding of TcdB4 was slightly reduced in CSPG4 ^-/- cells, and drastically reduced in TFPI ^-/- cells (fig. 2E). These results indicate that TFPI promotes the toxic effects of TcdB4 by mediating cell surface binding of the toxin.

The membrane-attached TFPI isoform acts as a receptor for TcdB4

TFPI has multiple splice isoforms because its mRNA precursor undergoes alternate splicing events (Broze and Girard,2012; maroney et al, 2010). In humans, TFPI has two major isoforms, TFPI alpha and TFPI beta, which are also produced by all mammals (Wood et al, 2014). TFPI alpha contains three tandem Kunitz-type protease inhibition (Kunitz-1, kunitz-2 and Kunitz-3, or K1, K2 and K3) domains followed by an alkaline carboxy-terminal (C-terminal) region. Tfpiβ lacks the K3 and basic C-terminal domains of tfpiα. In contrast, it contains a C-terminal signal peptide that directs cleavage and ligation of GPI anchors (Broze and Girard,2012; girard et al 2012; maroney and Mast,2015; zhang et al 2003) (FIG. 2F).

Tfpiβ is a GPI-anchored protein, consistent with screening results targeting multiple GPI-anchored biosynthetic genes. Because TcdB4 also binds CSPG4 (but is inefficient) into cells and CSPG4 is highly expressed in HeLa cells (Gupta et al, 2017; tao et al, 2016), heLa CSPG4 ^-/- cells were used to minimize the effect of CSPG4 when studying the role of TFPI in HeLa cells. The inventors found that HeLa CSPG4 ^-/-/TFPI^-/- cells were more resistant to TcdB4 than CSPG4 ^-/- cells, whereas the susceptibility of CSPG4 ^-/-/TFPI^-/- cells could be restored by transient transfection of TFPI beta (fig. 2G and 2H).

TFPI alpha is a secreted isoform located on the cell surface or in plasma (Novotny et al, 1989; piro and Broze,2005; wood et al, 2014). Cell membrane localization of tfpia is achieved primarily by binding of its K3 domain to protein S and the basic C-terminal region to cell surface glycosaminoglycans (Donahue et al, 2006; ndonwi et al, 2012; ndonwi et al, 2010; piro and Broze,2004; sandset et al, 1988). When supplemented into the medium, GFP-fused TFPI alpha, but not TFPI ^K1+K2, was firmly bound to the surface of CSPG4 ^-/-/TFPI^-/- cells (fig. 10A). The inventors showed that transient transfection of TFPI alpha restored the susceptibility of CSPG4 ^-/-/TFPI^-/- cells to TcdB4 (fig. 2G and 2H), indicating that TFPI alpha may also mediate the entry of TcdB 4. Consistently, immunoblot analysis showed that overexpression of TFPI alpha or TFPI beta mediated firm binding of TcdB4 on the cell surface (fig. 2I). Taken together, these data indicate that membrane-linked TFPI can act as a cellular receptor for TcdB4 in both the α and β isoforms.

Interestingly, the inventors found that "free" TFPI alpha had a binary effect on mediating TcdB4 entry into cells, depending on the presence of cell surface TFPI. Cultured CSPG4 ^-/-/TFPI^-/- cells did not express TFPI alpha but allowed exogenous TFPI alpha binding. Thus, the inventors observed that supplemental TFPI α -GFP binds to the surface of CSPG4 ^-/-/TFPI^-/- cells and mediates entry of TcdB4 (fig. 2J, fig. 10A and 10B). In contrast, the cell surface of CSPG4 ^-/- cells would be expected to be saturated with endogenously expressed TFPI alpha. Indeed, the inventors showed that added TFPI alpha-GFP acts as a neutralizing agent and protects CSPG4 ^-/- cells from TcdB4 in a dose-dependent manner (fig. 2J and 10B).

Kunitz-2 domain of TFPI interactions with TcdB4

Because both TFPI alpha and TFPI beta can mediate the binding/entry of TcdB4, the inventors speculate that TcdB4 interacts with Kunitz-1 and Kunitz-2 domains of TFPI (TFPI ^K1+K2). Thus, the inventors expressed and purified recombinant TFPI ^K1+K2 fused to a human Fc fragment (fig. 3A) and performed competition assays on HeLa CSPG4 ^-/- cells. TFPI ^K1+K2 was effective in protecting cells from TcdB4, but failed to mitigate the poisoning of TcdB1 (fig. 3B and 11A), supporting the notion that TFPI ^K1+K2 specifically binds TcdB 4. In addition, tcdB4, but not TcdB1, binds to recombinant TFPI ^K1+K2 -Fc and is co-precipitated by protein a beads (fig. 11B).

The Kunitz domain is a small, disulfide-rich, and alpha/beta sheet domain that is useful as a protease inhibitor (Ascenzi et al, 2003). Kunitz domains can be found in a number of proteins, including TFPI, TFPI2, alpha-1-microglobulin/Bikunin precursor (AMBP) and Amyloid Precursor Protein (APP). Evolutionary analysis showed that the primary sequence of TFPI ^K2 was most closely related to TFPI ^K1, with sequence similarity of 67.9%, followed by TFPI2 ^K1 (64.1% similarity), while TFPI ^K3 was less similar to TFPI ^K1 (56.6% similarity) and TFPI ^K2 (58.5% similarity) (fig. 3C).

To determine whether TcdB4 binds to TFPI ^K1 or to TFPI ^K2 and the binding selectivity, the inventors expressed GPI-anchored TFPI ^K1、TFPI^K2、TFPI2^K1 or AMBP ^K3 in HeLa CSPG ^4-/-/TFP^I-/- cells and then tested for their sensitivity to TcdB 4. The inventors showed that transient transfection of GPI-anchored TFPI ^K2 alone could restore the susceptibility of CSPG4 ^-/-/TFPI^-/- cells (fig. 3D), indicating that TcdB4 specifically recognizes the Kunitz-2 domain of TFPI. Consistently, TFPI ^K2 -Fc, but not TFPI ^K1 -Fc, protected HeLa CSPG4 ^-/- cells in competition experiments (fig. 11C).

The inventors further quantified the binding kinetics between TFPI ^K1+K2 -Fc and TcdB4 using a biological membrane interference (BLI) assay that showed high affinity with a dissociation constant (K _d) of-13 nM (fig. 12A). BLI assays also show TcdB4 toIs bound to TFPI ^K2 -Fc but not to TFPI ^K1 -Fc or the human Fc fragment (fig. 3E and 12B). Similarly, tcdB4 binds to Fc-labeled mice Tfpi ^K2 with K _d of-4 nM (fig. 12C).

Freezing electron microscope structure of TcdB4-TFPI compound

TcdBIs one of the largest bacterial toxins consisting of four functional domains, including the glucosyltransferase domain (GTD), the Cysteine Protease Domain (CPD), the transmembrane Delivery and Receptor Binding Domain (DRBD), and the C-terminal combined repeat oligopeptide (drop) domain (fig. 4A). To further characterize the detailed interaction between TcdB4 and TFPI, full length TcdB4 was mixed with TFPI ^K1+K2 and the complex was then isolated by size-exclusion FPLC. Fractions of the complexes were confirmed by SDS-PAGE and concentrated for preparing frozen electron microscopy samples (fig. 13). The two-dimensional classification average of the TcdB4-TFPI complex showed significant structural features (fig. 14). At neutral pH, the resolution of the full-length TcdB4 core region isAnd the resolution of the TcdB4-TFPI complex isThe final pattern of (c) reveals most of the side chain density and secondary structural elements (fig. 4B and 15).

The overall architecture of TcdB4 is similar to the previously determined TcdB structure (Chen et al, 2019; simeon et al, 2019). CPD and GTD form an integrated region, while the CROP domain module bends around the core region by forming a hook (FIG. 4B). The Kunitz-2 domain of TFPI binds to the convex edge of DRBD primarily through two flexible loops in TFPI: loop-1 (residues 131-138) and loop-2 (residues 155-162) (FIGS. 4B and 4C). Each ring binds TcdB4 through a broad network consisting of hydrogen bonds and hydrophobic interactions (fig. 4D). The interaction between TcdB4 ^DRBD and TFPI ^K2 was further verified by pull-down assay (fig. 16A). The point mutations at L1599, K1435, L1434, V1492, L1494, M1438 and D1468 in TcdB4 abrogate TFPI binding capacity, indicating that these positions are important for receptor recognition (fig. 4E).

Furthermore, previous studies have shown that the K2 domain of TFPI binds to trypsin-like substrates such as factor Xa (FXa) (Brandstetter et al, 1996; burgering et al, 1997). Structurally, the K2 domain of TFPI interacts with FXa and TcdB4 through the same loop (fig. 16B). Using the BLI assay, the inventors further demonstrated that pre-binding of TcdB4 ^1285-1834 to TFPI blocked FXa binding and vice versa (fig. 4F and 16C).

Determination of receptor binding specificity of TcdB variants

The analytical structure of TcdB4 TFPI and the previously reported structure of TcdB1-FZD2 (Chen et al, 2018) reveal evolutionary functional regions in TcdB for receptor recognition. To predict receptor specificity for different TcdB sequences, the inventors performed an evolutionary analysis of the Receptor Binding Interface (RBI) of TcdB (residues 1431-1606, fig. 5A). The evolutionary relationship tree is aggregated into two main branches (denoted as class I and class II). Class I RBI is derived from TcdB1, tcdB3, and TcdB5, which prefer to bind FZD. Class II RBI consists of sequences from TcdB2, tcdB4, tcdB6 and TcdB7 (fig. 5A), mainly present in clostridium difficile branch 2 (fig. 5B). A small number of RBI from TcdB2 and TcdB4 are found in class I or as outliers, possibly due to historically frequent recombination events between TcdB variants (Knight et al 2021; mansfield et al 2020; shen et al 2020).

Although FZD2 and TFPI are very different in structure, they all interface with the same hydrophobic interface located at the TcdB ^RBI convex side (fig. 5C). Class I and class II RBIs share several residues, including but not limited to L1434, M1438, L1494, and Y1510 (positions in TcdB 4; residues in TcdB1 move one position to the left in the alignment) and facilitate FZD and TFPI interactions. In the TcdB1-FZD2 complex, the interaction is bridged by Palmitoleic Acid (PAM), with its tail protruding into the hydrophobic pocket of TcdB 1. In the TcdB4-TFPI complex, the side chain of TFPI R135 is deeply embedded in the same pocket, but forms multiple hydrogen bonds with adjacent residues of TcdB4 (including E1433, D1467, and E1469) (fig. 5D). F1597 in TcdB1 is a key residue in the middle part of stable PAM and interacts with nearby F130 in FZD2 (Chen et al 2018; peng et al 2019), while Phe-to-Ser substitutions mimicking TcdB2 and TcdB4 eliminate FZD binding (Henkel et al 2020). Notably, S1598 in TcdB4 forms tight hydrogen bonds with nearby R140 in TFPI (fig. 5E). Thus, tcdB with F1598S substitution may impair FZD binding, but may promote TFPI binding. Accordingly, phe is conserved in all class I RBIs, while Ser is conserved in all class II RBIs at this position.

The potential TFPI binding interface in TcdB2 is similar to TcdB4 (fig. 5F), which provides a structural clue that TcdB2 may also recognize TFPI. As expected, heLa CSPG4 ^-/-/TFPI^-/- cells were more resistant to TcdB2 than their parent CSPG4 ^-/- cells (fig. 5G), whereas their susceptibility to TcdB2 could be restored by ectopic expression of GPI-anchored Tfpi ^K2 (fig. 5H). Furthermore, tfpi ^K2 -Fc effectively protected HeLa CSPG4 ^-/- cells from TcdB2 poisoning (fig. 5I and 17A). The inventors also performed BLI analysis to detect TcdB2-TFPI interactions (reference sequence from R20291, a ST01/RT027 strain) and observed that TcdB2 bound to Fc-tagged mice Tfpi ^K2 and human Tfpi ^K2, K _d was respectivelyAnd 0.4. Mu.M (FIGS. 17B-17D).

There are some differences between TcdB2 and TcdB4 in RBI (fig. 18A). To find out the residues in TcdB2 that lead to reduced TFPI binding, the inventors generated a series of TcdB4 ^1285-1834 mutants by replacing the residues with the corresponding residues in TcdB 2. Pull down experiments showed that I1496S is a key mutation that attenuated TFPI binding, whereas substitutions P1506L, Y1510C and K1597N/K1600Q slightly reduced interactions (fig. 5J). On the other hand, the S1496I substitution in TcdB2 ^1285-1834 enhanced its binding affinity to human and mouse TFPI (fig. 18B and 18C).

TFPI is a physiologically relevant receptor for TcdB4

TFPI is produced primarily by endothelial cells, megakaryocytes, monocytes and smooth muscle cells (Maroney and post, 2015; wood et al, 2014). TFPI is highly expressed in endothelial cells and glandular cells in the human gut (Uhlen et al, 2015). The inventors also confirmed by Immunohistochemical (IHC) analysis that Tfpi was highly expressed in the basal gland of the mouse intestinal tract (including ileum, jejunum, cecum and colon) (fig. 6A and 19A).

To investigate the role of TFPI in TcdB 4-induced colon lesions, the inventors performed a mouse colon loop ligation experiment, which was often used to examine clostridium difficile toxin-induced tissue lesions (Sun et al, 2010). Saline, tcdB4 (2 μg), or TcdB4 (2 μg) premixed with TFPI ^K2 -Fc (100 μg) was injected into the lumen of the ligated colon segment. After 6 hours, mice injected with TcdB4 alone exhibited significant toxic symptoms including reduced mobility, humpback posture and squinting, while mice injected with saline or TcdB4 premixed with TFPI ^K2 -Fc exhibited normal activity. By using hematoxylin and eosin staining and immunofluorescent labeling of ZO-1, the inventors found that the most collapsed structure of the colonic crypt and partially destroyed crypt epithelium in the TcdB4 group, indicating that the colonic crypt is a fragile target of TcdB. As expected, co-injection of TFPI ^K2 -Fc effectively prevented TcdB 4-induced colonic crypt erosion (fig. 6B-6D and fig. 19B).

To further investigate the role of TFPI in vivo, the inventors turned to Tfpi knockout mouse model. Although complete disruption of Tfpi in mice was intrauterine lethal (Huang et al, 1997), mice retaining part Tfpi appeared to have no obvious developmental defect (Girard et al, 2018). Thus, the inventors generated Tfpi β isoform KO mice (more precisely, β+γ isoform KO) using the CRISPR-Cas9 method (fig. 20A). When TcdB4 (1 μg/kg) was injected intraperitoneally (i.p.), all wild-type (Tfpi β ^+/+) and heterozygous (Tfpi β ^+/-) mice tested died rapidly within 12 hours. Surprisingly, 63.6% (7 out of 11) homozygous KO (Tfpi β ^-/-) mice survived (fig. 6E), with no significant lethal sex differences observed (fig. 20B). These results indicate Tfpi is the physiological receptor for TcdB4 during systemic toxin exposure. Immunoblot analysis showed that Tfpi β ^-/- mice had reduced Tfpi levels compared to WT mice, especially in the kidneys, presumably the kidneys expressed predominantly Tfpi β isoform (fig. 21A). According to histopathological analysis, intraperitoneal injection of TcdB4 in WT mice resulted in acute kidney injury, including glomerular and retrobulbar telangiectasia, erythrocyte structural injury, hemoglobin deposition in blood vessels, and renal interstitial congestion and bleeding. All injected mice appeared to be normal in liver, lung, spleen and heart. In contrast, tfpi β ^-/- mice injected with TcdB4 showed normal kidneys (fig. 6F and 21B). These data indicate that TcdB4 enters the circulatory system, which is common in severe infections, and can severely damage the kidneys and cause death.

Blocking both TFPI binding and CSPG4 binding provides optimal protection from clostridium difficile branch 2TcdB

TcdB2 is another major subtype of clostridium difficile branch 2 expression, in addition to TcdB 4. To examine whether blocking TFPI binding also protects colon epithelium from TcdB2, the inventors further performed a colon loop ligation assay using TcdB2 and TFPI ^K2 -Fc. TcdB2 causes severe intestinal damage, including oedema, epithelial destruction and inflammatory cell infiltration, consistent with previous studies (Chen et al, 2021; pan et al, 2021). All of the above pathological features, particularly submucosal edema, were alleviated in mice co-injected with TcdB2 and TFPI ^K2 -Fc (fig. 7A-7C). However, this protection still seems to be suboptimal for TcdB 2. Since TcdB2 also utilizes CSPG4 as its high affinity receptor, residual tissue damage may be due to CSPG 4-mediated toxin entry.

On the other hand, blocking CSPG4 binding also partially protects intestinal epithelium from TcdB2, previous studies used CSPG4 ^410-550(CSPG4^R1) as an inhibitor to reduce TcdB lesions in a mouse model (Chen et al, 2021; peng et al, 2019). Based on the structural features of toxin receptor binding, it is possible to block both TFPI and CSPG4 binding sites on TcdB 2. Thus, the inventors generated TFPI ^K2-CSPG4^R1 fusion proteins and evaluated their ability to inhibit TcdB 2-induced toxicity in vivo using a mouse colon loop model. Briefly, tcdB2 (2 μg), tcdB2 premixed with CSPG4 ^R1 (50 μg), or TcdB2 premixed with TFPI ^K2-CSPG4^R1 (50 μg) was injected into the lumen of the ligated mouse colon segment, 6 hours after injection, and then colon tissue was dissected for histopathological analysis. CSPG4 ^R1 partially reduced TcdB 2-induced colon lesions, including inflammatory cell infiltration and submucosal edema, consistent with previous reports. TFPI ^K2-CSPG4^R1 showed further enhanced protection of colon tissue compared to CSPG4 ^R1, although the molar ratio of toxin inhibitor employed was indeed lower, particularly in terms of reduction of mucosal oedema, epithelial destruction and crypt damage (fig. 7D-7F). Taken together, these results suggest a possible therapeutic approach to simultaneously inhibit TFPI and CSPG4 binding capacity, which provides optimal protection against clostridium difficile branch 2 TcdB.

Discussion of the invention

Clinical strains belonging to Clostridium difficile branch 2 are often isolated in North America, europe and Australia, accounting for over 20% of global CDI (Badilla-Lobo and Rodriguez,2021; he et al, 2013; knight et al, 2021). The branch 2 lineages are of general interest not only because they are associated with severe pandemic but also because they exclusively produce variant forms of TcdB that exhibit a variety of biological activities including receptor recognition (Lanis et al, 2010; pan et al, 2021; quesada-Gomez et al, 2016; stabler et al, 2008). These TcdB variants do not bind FZD but retain a different CSPG4 recognition capacity (Pan et al 2021). However, as demonstrated by several studies, CSPG4 is absent from the intestinal epithelium (Mileto et al 2020; tao et al 2016; terda et al 2006). Here, the inventors show that TFPI is a functional receptor for TcdB from clostridium difficile branch 2. More importantly, TFPI is highly expressed in human and mouse intestinal glandular epithelium (Uhlen et al, 2015). We found that TFPI is a physiologically relevant receptor for TcdB4 and TcdB2, filling the gap in how TcdB of clostridium difficile branch 2 targets the colonic epithelium to induce tissue damage.

The cryo-electron microscope structure of TcdB4-TFPI reveals a similar interaction pattern as TcdB1-FZD2 and TcsL-SEMA6A (Chen et al, 2018; lee et al, 2020). This finding consolidated the notion that LCTs have evolved to bind different receptors through similar interaction regions. Notably, this is the first co-structure of the full length LCT in complex with its receptor. The structure of the toxin moiety is similar to the full length TcdB structure previously reported (Chen et al, 2019; simeon et al, 2019), indicating that TFPI binding does not require or trigger conformational changes of the toxin. Based on co-structure, the inventors also concluded that TcdB2 expressed by several notoriously difficult clostridium genotypes (including ST01/RT 027) can also recognize TFPI. The inventors have shown that the reference sequence for TcdB2 combines mouse Tfpi with human TFPI, K _d is AndAttenuation of affinity (TcdB 4-TFPI K _d is) Mainly due to residue changes of I1496S in TcdB2, whereas substitutions at other positions may also involve attenuation of affinity. Notably, I1496 is found in certain TcdB2 sequences, which TcdB2 may exhibit strong binding to TFPI.

The discovery that TcdB variants can utilize both types of cell receptors TFPI and FZD to target the intestinal barrier of the host greatly expands our understanding of CDI pathogenesis. TcdB variants differ in sequence; the primary sequence identity between TcdB1 and TcdB4 is only 85.6%. We found that TcdB1 and TcdB4 employ different classes of proteins as intestinal epithelial receptors, and that these variants showed significant enzymatic activity (Genth et al, 2014; quesada-Gomez et al, 2016; von Eichel-Streiber et al, 1995) the fact that functionally supporting the TcdB subfamily might be a view of the toxin cluster from multiple ancestral lineages (Knight et al, 2021; mansfield et al, 2020; screen et al, 2020).

The presence of TFPI in various cell types of the gut including glandular cells, endothelial cells, intestinal cells, megakaryocytes and monocytes is consistent with pathological observations of CDI. Colonic glands, also known as colonic crypts, are critical for self-renewal of intestinal epithelium, production of mucus and secretion of antimicrobial molecules (Clevers, 2013). Severe intestinal damage caused by clostridium difficile often results in crypt erosion and loss of glandular cells (Carter et al 2015; smits et al 2016). A recent study showed that TcdB2 destroyed the crypt base of the mouse colon in a FZD-independent manner (Mileto et al, 2020), which could be elucidated by our findings that the colon crypt expressed high levels of TFPI and was therefore a vulnerable target for TcdB2 and TcdB 4. Remarkably, over 60% Tfpi β ^-/- mice survived the TcdB4 challenge assay, while all WT mice died rapidly within 12 hours. Tfpi beta ^-/- mice are only partially Tfpi depleted, but have shown high resistance to TcdB4, strongly supporting Tfpi the notion that the system is exposed to the physiologically relevant receptor of clostridium difficile branch 2TcdB of toxins, which is common in severe infections. Previous toxin challenge assays showed that TcdB1 was the same mortality for WT and CSPG4 KO mice (Yuan et al, 2015), meaning CSPG4 may not be critical for system exposure to TcdB. The inventors also determined that kidney was a fragile target in this case, as acute kidney injury was observed in WT mice, as well as rapid death, but not in Tfpi β ^-/- mice. Consistent with our results, clostridium difficile infection is reported to be clinically accompanied by acute kidney injury (Charilaou et al, 2018).

TFPI inhibits the onset of coagulation by inhibiting the TF factor VIIa (TF-FVIIa) complex, FXa and prothrombinase (Maroney and post, 2015; post, 2016; wood et al, 2013). Under structural view, the K2 domain of TFPI binds to TcdB and its trypsin-like substrate via the same loop (Brandstetter et al, 1996; burgering et al, 1997) (i.e., FXa). anti-TFPI therapy has recently been proposed as a new strategy for treating coagulation disorders such as hemophilia (Chowdary, 2020; sidonio and Zimowski, 2019). TcdB4 ^RBI, an effective TFPI binding agent, may be a potential candidate for the development of a therapeutic agent for hematological disorders. Furthermore, the inventors hypothesize that affected coagulation may bring additional advantages to clostridium difficile during infection, while the exact relevance and contribution to disease progression remains to be studied.

Soluble TFPI and its derivatives are potential drugs that neutralize toxins and alleviate poisoning caused by clostridium difficile branch 2 TcdB. Indeed, the inventors utilized TFPI ^K2 as an effective decoy to effectively reduce TcdB4 and TcdB2 induced tissue damage in vivo. Furthermore, the inventors demonstrate that simultaneous inhibition of TFPI and CSPG4 binding provides optimal protection from TcdB, while protection by inhibition of TFPI or CSPG4 binding alone is not optimal. Interestingly, a similar strategy to simultaneously eliminate FZD and CSPG4 binding provides ideal protection for TcdB1 (Simeon et al, 2019). The inventors believe that blocking both receptor binding sites simultaneously will be the best strategy to neutralize TcdB, thereby developing next generation therapies for the prevention and treatment of CDI.

Those skilled in the art will further appreciate that the invention may be embodied in other specific forms without departing from its spirit or central attributes. Since the foregoing description of the invention discloses only exemplary embodiments thereof, it should be understood that other variations are considered to be within the scope of the invention. Therefore, the present invention is not limited to the specific embodiments described in detail herein. Rather, reference should be made to the appended claims for indicating the scope and content of the invention.

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Sequence listing

<110> University of West lake (WESTLAKE UNIVERSITY)

<120> Compositions and methods for preventing and/or treating clostridium difficile infection caused by a branch 2 strain

<130> IEC216065PCT

<160> 30

<170> PatentIn version 3.5

<210> 1

<211> 51

<212> PRT

<213> Artificial sequence

<220>

<223> K2 Domain of human TFPI

<400> 1

Cys Phe Leu Glu Glu Asp Pro Gly Ile Cys Arg Gly Tyr Ile Thr Arg

1 5 10 15

Tyr Phe Tyr Asn Asn Gln Thr Lys Gln Cys Glu Arg Phe Lys Tyr Gly

20 25 30

Gly Cys Leu Gly Asn Met Asn Asn Phe Glu Thr Leu Glu Glu Cys Lys

35 40 45

Asn Ile Cys

50

<210> 2

<211> 51

<212> PRT

<213> Artificial sequence

<220>

<223> K2 Domain of mouse Tfpi

<400> 2

Cys Phe Leu Glu Glu Asp Pro Gly Leu Cys Arg Gly Tyr Met Lys Arg

1 5 10 15

Tyr Leu Tyr Asn Asn Gln Thr Lys Gln Cys Glu Arg Phe Val Tyr Gly

20 25 30

Gly Cys Leu Gly Asn Arg Asn Asn Phe Glu Thr Leu Asp Glu Cys Lys

35 40 45

Lys Ile Cys

50

<210> 3

<211> 276

<212> PRT

<213> Artificial sequence

<220>

<223> TFPIα

<400> 3

Asp Ser Glu Glu Asp Glu Glu His Thr Ile Ile Thr Asp Thr Glu Leu

1 5 10 15

Pro Pro Leu Lys Leu Met His Ser Phe Cys Ala Phe Lys Ala Asp Asp

20 25 30

Gly Pro Cys Lys Ala Ile Met Lys Arg Phe Phe Phe Asn Ile Phe Thr

35 40 45

Arg Gln Cys Glu Glu Phe Ile Tyr Gly Gly Cys Glu Gly Asn Gln Asn

50 55 60

Arg Phe Glu Ser Leu Glu Glu Cys Lys Lys Met Cys Thr Arg Asp Asn

65 70 75 80

Ala Asn Arg Ile Ile Lys Thr Thr Leu Gln Gln Glu Lys Pro Asp Phe

85 90 95

Cys Phe Leu Glu Glu Asp Pro Gly Ile Cys Arg Gly Tyr Ile Thr Arg

100 105 110

Tyr Phe Tyr Asn Asn Gln Thr Lys Gln Cys Glu Arg Phe Lys Tyr Gly

115 120 125

Gly Cys Leu Gly Asn Met Asn Asn Phe Glu Thr Leu Glu Glu Cys Lys

130 135 140

Asn Ile Cys Glu Asp Gly Pro Asn Gly Phe Gln Val Asp Asn Tyr Gly

145 150 155 160

Thr Gln Leu Asn Ala Val Asn Asn Ser Leu Thr Pro Gln Ser Thr Lys

165 170 175

Val Pro Ser Leu Phe Glu Phe His Gly Pro Ser Trp Cys Leu Thr Pro

180 185 190

Ala Asp Arg Gly Leu Cys Arg Ala Asn Glu Asn Arg Phe Tyr Tyr Asn

195 200 205

Ser Val Ile Gly Lys Cys Arg Pro Phe Lys Tyr Ser Gly Cys Gly Gly

210 215 220

Asn Glu Asn Asn Phe Thr Ser Lys Gln Glu Cys Leu Arg Ala Cys Lys

225 230 235 240

Lys Gly Phe Ile Gln Arg Ile Ser Lys Gly Gly Leu Ile Lys Thr Lys

245 250 255

Arg Lys Arg Lys Lys Gln Arg Val Lys Ile Ala Tyr Glu Glu Ile Phe

260 265 270

Val Lys Asn Met

275

<210> 4

<211> 223

<212> PRT

<213> Artificial sequence

<220>

<223> TFPIβ

<400> 4

Asp Ser Glu Glu Asp Glu Glu His Thr Ile Ile Thr Asp Thr Glu Leu

1 5 10 15

Pro Pro Leu Lys Leu Met His Ser Phe Cys Ala Phe Lys Ala Asp Asp

20 25 30

Gly Pro Cys Lys Ala Ile Met Lys Arg Phe Phe Phe Asn Ile Phe Thr

35 40 45

Arg Gln Cys Glu Glu Phe Ile Tyr Gly Gly Cys Glu Gly Asn Gln Asn

50 55 60

Arg Phe Glu Ser Leu Glu Glu Cys Lys Lys Met Cys Thr Arg Asp Asn

65 70 75 80

Ala Asn Arg Ile Ile Lys Thr Thr Leu Gln Gln Glu Lys Pro Asp Phe

85 90 95

Cys Phe Leu Glu Glu Asp Pro Gly Ile Cys Arg Gly Tyr Ile Thr Arg

100 105 110

Tyr Phe Tyr Asn Asn Gln Thr Lys Gln Cys Glu Arg Phe Lys Tyr Gly

115 120 125

Gly Cys Leu Gly Asn Met Asn Asn Phe Glu Thr Leu Glu Glu Cys Lys

130 135 140

Asn Ile Cys Glu Asp Gly Pro Asn Gly Phe Gln Val Asp Asn Tyr Gly

145 150 155 160

Thr Gln Leu Asn Ala Val Asn Asn Ser Leu Thr Pro Gln Ser Thr Lys

165 170 175

Val Pro Ser Leu Phe Val Thr Lys Glu Gly Thr Asn Asp Gly Trp Lys

180 185 190

Asn Ala Ala His Ile Tyr Gln Val Phe Leu Asn Ala Phe Cys Ile His

195 200 205

Ala Ser Met Phe Phe Leu Gly Leu Asp Ser Ile Ser Cys Leu Cys

210 215 220

<210> 5

<211> 253

<212> PRT

<213> Artificial sequence

<220>

<223> Mouse Tfpi beta

<400> 5

Met Thr Tyr Lys Met Lys Lys Glu Tyr Ala Phe Trp Ala Thr Val Cys

1 5 10 15

Leu Leu Leu Ser Leu Val Pro Glu Phe Leu Asn Ala Leu Ser Glu Glu

20 25 30

Ala Asp Asp Thr Asp Ser Glu Leu Gly Ser Met Lys Pro Leu His Thr

35 40 45

Phe Cys Ala Met Lys Ala Asp Asp Gly Pro Cys Lys Ala Met Ile Arg

50 55 60

Ser Tyr Phe Phe Asn Met Tyr Thr His Gln Cys Glu Glu Phe Ile Tyr

65 70 75 80

Gly Gly Cys Glu Gly Asn Glu Asn Arg Phe Asp Thr Leu Glu Glu Cys

85 90 95

Lys Lys Thr Cys Ile Pro Gly Tyr Glu Lys Thr Ala Val Lys Ala Ala

100 105 110

Ser Gly Ala Glu Arg Pro Asp Phe Cys Phe Leu Glu Glu Asp Pro Gly

115 120 125

Leu Cys Arg Gly Tyr Met Lys Arg Tyr Leu Tyr Asn Asn Gln Thr Lys

130 135 140

Gln Cys Glu Arg Phe Val Tyr Gly Gly Cys Leu Gly Asn Arg Asn Asn

145 150 155 160

Phe Glu Thr Leu Asp Glu Cys Lys Lys Ile Cys Glu Asn Pro Val His

165 170 175

Ser Pro Ser Pro Val Asn Glu Val Gln Met Ser Asp Tyr Val Thr Asp

180 185 190

Gly Asn Thr Val Thr Asp Arg Ser Thr Val Asn Asn Ile Val Val Pro

195 200 205

Gln Ser Pro Lys Val Pro Arg Arg Arg Val Thr Lys Glu Glu Thr Asn

210 215 220

Gly Gly Trp Lys Asn Ala Asp Tyr Thr Tyr Gln Gly Phe Leu Ser Ser

225 230 235 240

Val Tyr Ile His Val Leu Tyr Phe Val Phe Arg Ile Gly

245 250

<210> 6

<211> 2366

<212> PRT

<213> Artificial sequence

<220>

<223> TcdB1

<400> 6

Met Ser Leu Val Asn Arg Lys Gln Leu Glu Lys Met Ala Asn Val Arg

1 5 10 15

Phe Arg Thr Gln Glu Asp Glu Tyr Val Ala Ile Leu Asp Ala Leu Glu

20 25 30

Glu Tyr His Asn Met Ser Glu Asn Thr Val Val Glu Lys Tyr Leu Lys

35 40 45

Leu Lys Asp Ile Asn Ser Leu Thr Asp Ile Tyr Ile Asp Thr Tyr Lys

50 55 60

Lys Ser Gly Arg Asn Lys Ala Leu Lys Lys Phe Lys Glu Tyr Leu Val

65 70 75 80

Thr Glu Val Leu Glu Leu Lys Asn Asn Asn Leu Thr Pro Val Glu Lys

85 90 95

Asn Leu His Phe Val Trp Ile Gly Gly Gln Ile Asn Asp Thr Ala Ile

100 105 110

Asn Tyr Ile Asn Gln Trp Lys Asp Val Asn Ser Asp Tyr Asn Val Asn

115 120 125

Val Phe Tyr Asp Ser Asn Ala Phe Leu Ile Asn Thr Leu Lys Lys Thr

130 135 140

Val Val Glu Ser Ala Ile Asn Asp Thr Leu Glu Ser Phe Arg Glu Asn

145 150 155 160

Leu Asn Asp Pro Arg Phe Asp Tyr Asn Lys Phe Phe Arg Lys Arg Met

165 170 175

Glu Ile Ile Tyr Asp Lys Gln Lys Asn Phe Ile Asn Tyr Tyr Lys Ala

180 185 190

Gln Arg Glu Glu Asn Pro Glu Leu Ile Ile Asp Asp Ile Val Lys Thr

195 200 205

Tyr Leu Ser Asn Glu Tyr Ser Lys Glu Ile Asp Glu Leu Asn Thr Tyr

210 215 220

Ile Glu Glu Ser Leu Asn Lys Ile Thr Gln Asn Ser Gly Asn Asp Val

225 230 235 240

Arg Asn Phe Glu Glu Phe Lys Asn Gly Glu Ser Phe Asn Leu Tyr Glu

245 250 255

Gln Glu Leu Val Glu Arg Trp Asn Leu Ala Ala Ala Ser Asp Ile Leu

260 265 270

Arg Ile Ser Ala Leu Lys Glu Ile Gly Gly Met Tyr Leu Asp Val Asp

275 280 285

Met Leu Pro Gly Ile Gln Pro Asp Leu Phe Glu Ser Ile Glu Lys Pro

290 295 300

Ser Ser Val Thr Val Asp Phe Trp Glu Met Thr Lys Leu Glu Ala Ile

305 310 315 320

Met Lys Tyr Lys Glu Tyr Ile Pro Glu Tyr Thr Ser Glu His Phe Asp

325 330 335

Met Leu Asp Glu Glu Val Gln Ser Ser Phe Glu Ser Val Leu Ala Ser

340 345 350

Lys Ser Asp Lys Ser Glu Ile Phe Ser Ser Leu Gly Asp Met Glu Ala

355 360 365

Ser Pro Leu Glu Val Lys Ile Ala Phe Asn Ser Lys Gly Ile Ile Asn

370 375 380

Gln Gly Leu Ile Ser Val Lys Asp Ser Tyr Cys Ser Asn Leu Ile Val

385 390 395 400

Lys Gln Ile Glu Asn Arg Tyr Lys Ile Leu Asn Asn Ser Leu Asn Pro

405 410 415

Ala Ile Ser Glu Asp Asn Asp Phe Asn Thr Thr Thr Asn Thr Phe Ile

420 425 430

Asp Ser Ile Met Ala Glu Ala Asn Ala Asp Asn Gly Arg Phe Met Met

435 440 445

Glu Leu Gly Lys Tyr Leu Arg Val Gly Phe Phe Pro Asp Val Lys Thr

450 455 460

Thr Ile Asn Leu Ser Gly Pro Glu Ala Tyr Ala Ala Ala Tyr Gln Asp

465 470 475 480

Leu Leu Met Phe Lys Glu Gly Ser Met Asn Ile His Leu Ile Glu Ala

485 490 495

Asp Leu Arg Asn Phe Glu Ile Ser Lys Thr Asn Ile Ser Gln Ser Thr

500 505 510

Glu Gln Glu Met Ala Ser Leu Trp Ser Phe Asp Asp Ala Arg Ala Lys

515 520 525

Ala Gln Phe Glu Glu Tyr Lys Arg Asn Tyr Phe Glu Gly Ser Leu Gly

530 535 540

Glu Asp Asp Asn Leu Asp Phe Ser Gln Asn Ile Val Val Asp Lys Glu

545 550 555 560

Tyr Leu Leu Glu Lys Ile Ser Ser Leu Ala Arg Ser Ser Glu Arg Gly

565 570 575

Tyr Ile His Tyr Ile Val Gln Leu Gln Gly Asp Lys Ile Ser Tyr Glu

580 585 590

Ala Ala Cys Asn Leu Phe Ala Lys Thr Pro Tyr Asp Ser Val Leu Phe

595 600 605

Gln Lys Asn Ile Glu Asp Ser Glu Ile Ala Tyr Tyr Tyr Asn Pro Gly

610 615 620

Asp Gly Glu Ile Gln Glu Ile Asp Lys Tyr Lys Ile Pro Ser Ile Ile

625 630 635 640

Ser Asp Arg Pro Lys Ile Lys Leu Thr Phe Ile Gly His Gly Lys Asp

645 650 655

Glu Phe Asn Thr Asp Ile Phe Ala Gly Phe Asp Val Asp Ser Leu Ser

660 665 670

Thr Glu Ile Glu Ala Ala Ile Asp Leu Ala Lys Glu Asp Ile Ser Pro

675 680 685

Lys Ser Ile Glu Ile Asn Leu Leu Gly Cys Asn Met Phe Ser Tyr Ser

690 695 700

Ile Asn Val Glu Glu Thr Tyr Pro Gly Lys Leu Leu Leu Lys Val Lys

705 710 715 720

Asp Lys Ile Ser Glu Leu Met Pro Ser Ile Ser Gln Asp Ser Ile Ile

725 730 735

Val Ser Ala Asn Gln Tyr Glu Val Arg Ile Asn Ser Glu Gly Arg Arg

740 745 750

Glu Leu Leu Asp His Ser Gly Glu Trp Ile Asn Lys Glu Glu Ser Ile

755 760 765

Ile Lys Asp Ile Ser Ser Lys Glu Tyr Ile Ser Phe Asn Pro Lys Glu

770 775 780

Asn Lys Ile Thr Val Lys Ser Lys Asn Leu Pro Glu Leu Ser Thr Leu

785 790 795 800

Leu Gln Glu Ile Arg Asn Asn Ser Asn Ser Ser Asp Ile Glu Leu Glu

805 810 815

Glu Lys Val Met Leu Thr Glu Cys Glu Ile Asn Val Ile Ser Asn Ile

820 825 830

Asp Thr Gln Ile Val Glu Glu Arg Ile Glu Glu Ala Lys Asn Leu Thr

835 840 845

Ser Asp Ser Ile Asn Tyr Ile Lys Asp Glu Phe Lys Leu Ile Glu Ser

850 855 860

Ile Ser Asp Ala Leu Cys Asp Leu Lys Gln Gln Asn Glu Leu Glu Asp

865 870 875 880

Ser His Phe Ile Ser Phe Glu Asp Ile Ser Glu Thr Asp Glu Gly Phe

885 890 895

Ser Ile Arg Phe Ile Asn Lys Glu Thr Gly Glu Ser Ile Phe Val Glu

900 905 910

Thr Glu Lys Thr Ile Phe Ser Glu Tyr Ala Asn His Ile Thr Glu Glu

915 920 925

Ile Ser Lys Ile Lys Gly Thr Ile Phe Asp Thr Val Asn Gly Lys Leu

930 935 940

Val Lys Lys Val Asn Leu Asp Thr Thr His Glu Val Asn Thr Leu Asn

945 950 955 960

Ala Ala Phe Phe Ile Gln Ser Leu Ile Glu Tyr Asn Ser Ser Lys Glu

965 970 975

Ser Leu Ser Asn Leu Ser Val Ala Met Lys Val Gln Val Tyr Ala Gln

980 985 990

Leu Phe Ser Thr Gly Leu Asn Thr Ile Thr Asp Ala Ala Lys Val Val

995 1000 1005

Glu Leu Val Ser Thr Ala Leu Asp Glu Thr Ile Asp Leu Leu Pro

1010 1015 1020

Thr Leu Ser Glu Gly Leu Pro Ile Ile Ala Thr Ile Ile Asp Gly

1025 1030 1035

Val Ser Leu Gly Ala Ala Ile Lys Glu Leu Ser Glu Thr Ser Asp

1040 1045 1050

Pro Leu Leu Arg Gln Glu Ile Glu Ala Lys Ile Gly Ile Met Ala

1055 1060 1065

Val Asn Leu Thr Thr Ala Thr Thr Ala Ile Ile Thr Ser Ser Leu

1070 1075 1080

Gly Ile Ala Ser Gly Phe Ser Ile Leu Leu Val Pro Leu Ala Gly

1085 1090 1095

Ile Ser Ala Gly Ile Pro Ser Leu Val Asn Asn Glu Leu Val Leu

1100 1105 1110

Arg Asp Lys Ala Thr Lys Val Val Asp Tyr Phe Lys His Val Ser

1115 1120 1125

Leu Val Glu Thr Glu Gly Val Phe Thr Leu Leu Asp Asp Lys Ile

1130 1135 1140

Met Met Pro Gln Asp Asp Leu Val Ile Ser Glu Ile Asp Phe Asn

1145 1150 1155

Asn Asn Ser Ile Val Leu Gly Lys Cys Glu Ile Trp Arg Met Glu

1160 1165 1170

Gly Gly Ser Gly His Thr Val Thr Asp Asp Ile Asp His Phe Phe

1175 1180 1185

Ser Ala Pro Ser Ile Thr Tyr Arg Glu Pro His Leu Ser Ile Tyr

1190 1195 1200

Asp Val Leu Glu Val Gln Lys Glu Glu Leu Asp Leu Ser Lys Asp

1205 1210 1215

Leu Met Val Leu Pro Asn Ala Pro Asn Arg Val Phe Ala Trp Glu

1220 1225 1230

Thr Gly Trp Thr Pro Gly Leu Arg Ser Leu Glu Asn Asp Gly Thr

1235 1240 1245

Lys Leu Leu Asp Arg Ile Arg Asp Asn Tyr Glu Gly Glu Phe Tyr

1250 1255 1260

Trp Arg Tyr Phe Ala Phe Ile Ala Asp Ala Leu Ile Thr Thr Leu

1265 1270 1275

Lys Pro Arg Tyr Glu Asp Thr Asn Ile Arg Ile Asn Leu Asp Ser

1280 1285 1290

Asn Thr Arg Ser Phe Ile Val Pro Ile Ile Thr Thr Glu Tyr Ile

1295 1300 1305

Arg Glu Lys Leu Ser Tyr Ser Phe Tyr Gly Ser Gly Gly Thr Tyr

1310 1315 1320

Ala Leu Ser Leu Ser Gln Tyr Asn Met Gly Ile Asn Ile Glu Leu

1325 1330 1335

Ser Glu Ser Asp Val Trp Ile Ile Asp Val Asp Asn Val Val Arg

1340 1345 1350

Asp Val Thr Ile Glu Ser Asp Lys Ile Lys Lys Gly Asp Leu Ile

1355 1360 1365

Glu Gly Ile Leu Ser Thr Leu Ser Ile Glu Glu Asn Lys Ile Ile

1370 1375 1380

Leu Asn Ser His Glu Ile Asn Phe Ser Gly Glu Val Asn Gly Ser

1385 1390 1395

Asn Gly Phe Val Ser Leu Thr Phe Ser Ile Leu Glu Gly Ile Asn

1400 1405 1410

Ala Ile Ile Glu Val Asp Leu Leu Ser Lys Ser Tyr Lys Leu Leu

1415 1420 1425

Ile Ser Gly Glu Leu Lys Ile Leu Met Leu Asn Ser Asn His Ile

1430 1435 1440

Gln Gln Lys Ile Asp Tyr Ile Gly Phe Asn Ser Glu Leu Gln Lys

1445 1450 1455

Asn Ile Pro Tyr Ser Phe Val Asp Ser Glu Gly Lys Glu Asn Gly

1460 1465 1470

Phe Ile Asn Gly Ser Thr Lys Glu Gly Leu Phe Val Ser Glu Leu

1475 1480 1485

Pro Asp Val Val Leu Ile Ser Lys Val Tyr Met Asp Asp Ser Lys

1490 1495 1500

Pro Ser Phe Gly Tyr Tyr Ser Asn Asn Leu Lys Asp Val Lys Val

1505 1510 1515

Ile Thr Lys Asp Asn Val Asn Ile Leu Thr Gly Tyr Tyr Leu Lys

1520 1525 1530

Asp Asp Ile Lys Ile Ser Leu Ser Leu Thr Leu Gln Asp Glu Lys

1535 1540 1545

Thr Ile Lys Leu Asn Ser Val His Leu Asp Glu Ser Gly Val Ala

1550 1555 1560

Glu Ile Leu Lys Phe Met Asn Arg Lys Gly Asn Thr Asn Thr Ser

1565 1570 1575

Asp Ser Leu Met Ser Phe Leu Glu Ser Met Asn Ile Lys Ser Ile

1580 1585 1590

Phe Val Asn Phe Leu Gln Ser Asn Ile Lys Phe Ile Leu Asp Ala

1595 1600 1605

Asn Phe Ile Ile Ser Gly Thr Thr Ser Ile Gly Gln Phe Glu Phe

1610 1615 1620

Ile Cys Asp Glu Asn Asp Asn Ile Gln Pro Tyr Phe Ile Lys Phe

1625 1630 1635

Asn Thr Leu Glu Thr Asn Tyr Thr Leu Tyr Val Gly Asn Arg Gln

1640 1645 1650

Asn Met Ile Val Glu Pro Asn Tyr Asp Leu Asp Asp Ser Gly Asp

1655 1660 1665

Ile Ser Ser Thr Val Ile Asn Phe Ser Gln Lys Tyr Leu Tyr Gly

1670 1675 1680

Ile Asp Ser Cys Val Asn Lys Val Val Ile Ser Pro Asn Ile Tyr

1685 1690 1695

Thr Asp Glu Ile Asn Ile Thr Pro Val Tyr Glu Thr Asn Asn Thr

1700 1705 1710

Tyr Pro Glu Val Ile Val Leu Asp Ala Asn Tyr Ile Asn Glu Lys

1715 1720 1725

Ile Asn Val Asn Ile Asn Asp Leu Ser Ile Arg Tyr Val Trp Ser

1730 1735 1740

Asn Asp Gly Asn Asp Phe Ile Leu Met Ser Thr Ser Glu Glu Asn

1745 1750 1755

Lys Val Ser Gln Val Lys Ile Arg Phe Val Asn Val Phe Lys Asp

1760 1765 1770

Lys Thr Leu Ala Asn Lys Leu Ser Phe Asn Phe Ser Asp Lys Gln

1775 1780 1785

Asp Val Pro Val Ser Glu Ile Ile Leu Ser Phe Thr Pro Ser Tyr

1790 1795 1800

Tyr Glu Asp Gly Leu Ile Gly Tyr Asp Leu Gly Leu Val Ser Leu

1805 1810 1815

Tyr Asn Glu Lys Phe Tyr Ile Asn Asn Phe Gly Met Met Val Ser

1820 1825 1830

Gly Leu Ile Tyr Ile Asn Asp Ser Leu Tyr Tyr Phe Lys Pro Pro

1835 1840 1845

Val Asn Asn Leu Ile Thr Gly Phe Val Thr Val Gly Asp Asp Lys

1850 1855 1860

Tyr Tyr Phe Asn Pro Ile Asn Gly Gly Ala Ala Ser Ile Gly Glu

1865 1870 1875

Thr Ile Ile Asp Asp Lys Asn Tyr Tyr Phe Asn Gln Ser Gly Val

1880 1885 1890

Leu Gln Thr Gly Val Phe Ser Thr Glu Asp Gly Phe Lys Tyr Phe

1895 1900 1905

Ala Pro Ala Asn Thr Leu Asp Glu Asn Leu Glu Gly Glu Ala Ile

1910 1915 1920

Asp Phe Thr Gly Lys Leu Ile Ile Asp Glu Asn Ile Tyr Tyr Phe

1925 1930 1935

Asp Asp Asn Tyr Arg Gly Ala Val Glu Trp Lys Glu Leu Asp Gly

1940 1945 1950

Glu Met His Tyr Phe Ser Pro Glu Thr Gly Lys Ala Phe Lys Gly

1955 1960 1965

Leu Asn Gln Ile Gly Asp Tyr Lys Tyr Tyr Phe Asn Ser Asp Gly

1970 1975 1980

Val Met Gln Lys Gly Phe Val Ser Ile Asn Asp Asn Lys His Tyr

1985 1990 1995

Phe Asp Asp Ser Gly Val Met Lys Val Gly Tyr Thr Glu Ile Asp

2000 2005 2010

Gly Lys His Phe Tyr Phe Ala Glu Asn Gly Glu Met Gln Ile Gly

2015 2020 2025

Val Phe Asn Thr Glu Asp Gly Phe Lys Tyr Phe Ala His His Asn

2030 2035 2040

Glu Asp Leu Gly Asn Glu Glu Gly Glu Glu Ile Ser Tyr Ser Gly

2045 2050 2055

Ile Leu Asn Phe Asn Asn Lys Ile Tyr Tyr Phe Asp Asp Ser Phe

2060 2065 2070

Thr Ala Val Val Gly Trp Lys Asp Leu Glu Asp Gly Ser Lys Tyr

2075 2080 2085

Tyr Phe Asp Glu Asp Thr Ala Glu Ala Tyr Ile Gly Leu Ser Leu

2090 2095 2100

Ile Asn Asp Gly Gln Tyr Tyr Phe Asn Asp Asp Gly Ile Met Gln

2105 2110 2115

Val Gly Phe Val Thr Ile Asn Asp Lys Val Phe Tyr Phe Ser Asp

2120 2125 2130

Ser Gly Ile Ile Glu Ser Gly Val Gln Asn Ile Asp Asp Asn Tyr

2135 2140 2145

Phe Tyr Ile Asp Asp Asn Gly Ile Val Gln Ile Gly Val Phe Asp

2150 2155 2160

Thr Ser Asp Gly Tyr Lys Tyr Phe Ala Pro Ala Asn Thr Val Asn

2165 2170 2175

Asp Asn Ile Tyr Gly Gln Ala Val Glu Tyr Ser Gly Leu Val Arg

2180 2185 2190

Val Gly Glu Asp Val Tyr Tyr Phe Gly Glu Thr Tyr Thr Ile Glu

2195 2200 2205

Thr Gly Trp Ile Tyr Asp Met Glu Asn Glu Ser Asp Lys Tyr Tyr

2210 2215 2220

Phe Asn Pro Glu Thr Lys Lys Ala Cys Lys Gly Ile Asn Leu Ile

2225 2230 2235

Asp Asp Ile Lys Tyr Tyr Phe Asp Glu Lys Gly Ile Met Arg Thr

2240 2245 2250

Gly Leu Ile Ser Phe Glu Asn Asn Asn Tyr Tyr Phe Asn Glu Asn

2255 2260 2265

Gly Glu Met Gln Phe Gly Tyr Ile Asn Ile Glu Asp Lys Met Phe

2270 2275 2280

Tyr Phe Gly Glu Asp Gly Val Met Gln Ile Gly Val Phe Asn Thr

2285 2290 2295

Pro Asp Gly Phe Lys Tyr Phe Ala His Gln Asn Thr Leu Asp Glu

2300 2305 2310

Asn Phe Glu Gly Glu Ser Ile Asn Tyr Thr Gly Trp Leu Asp Leu

2315 2320 2325

Asp Glu Lys Arg Tyr Tyr Phe Thr Asp Glu Tyr Ile Ala Ala Thr

2330 2335 2340

Gly Ser Val Ile Ile Asp Gly Glu Glu Tyr Tyr Phe Asp Pro Asp

2345 2350 2355

Thr Ala Gln Leu Val Ile Ser Glu

2360 2365

<210> 7

<211> 2366

<212> PRT

<213> Artificial sequence

<220>

<223> Full-length sequence of TcdB2 from Clostridium difficile (Clostridioides difficile) strain R20291

<400> 7

Met Ser Leu Val Asn Arg Lys Gln Leu Glu Lys Met Ala Asn Val Arg

1 5 10 15

Phe Arg Val Gln Glu Asp Glu Tyr Val Ala Ile Leu Asp Ala Leu Glu

20 25 30

Glu Tyr His Asn Met Ser Glu Asn Thr Val Val Glu Lys Tyr Leu Lys

35 40 45

Leu Lys Asp Ile Asn Ser Leu Thr Asp Ile Tyr Ile Asp Thr Tyr Lys

50 55 60

Lys Ser Gly Arg Asn Lys Ala Leu Lys Lys Phe Lys Glu Tyr Leu Val

65 70 75 80

Thr Glu Val Leu Glu Leu Lys Asn Asn Asn Leu Thr Pro Val Glu Lys

85 90 95

Asn Leu His Phe Val Trp Ile Gly Gly Gln Ile Asn Asp Thr Ala Ile

100 105 110

Asn Tyr Ile Asn Gln Trp Lys Asp Val Asn Ser Asp Tyr Asn Val Asn

115 120 125

Val Phe Tyr Asp Ser Asn Ala Phe Leu Ile Asn Thr Leu Lys Lys Thr

130 135 140

Ile Val Glu Ser Ala Thr Asn Asp Thr Leu Glu Ser Phe Arg Glu Asn

145 150 155 160

Leu Asn Asp Pro Arg Phe Asp Tyr Asn Lys Phe Tyr Arg Lys Arg Met

165 170 175

Glu Ile Ile Tyr Asp Lys Gln Lys Asn Phe Ile Asn Tyr Tyr Lys Thr

180 185 190

Gln Arg Glu Glu Asn Pro Asp Leu Ile Ile Asp Asp Ile Val Lys Ile

195 200 205

Tyr Leu Ser Asn Glu Tyr Ser Lys Asp Ile Asp Glu Leu Asn Ser Tyr

210 215 220

Ile Glu Glu Ser Leu Asn Lys Val Thr Glu Asn Ser Gly Asn Asp Val

225 230 235 240

Arg Asn Phe Glu Glu Phe Lys Gly Gly Glu Ser Phe Lys Leu Tyr Glu

245 250 255

Gln Glu Leu Val Glu Arg Trp Asn Leu Ala Ala Ala Ser Asp Ile Leu

260 265 270

Arg Ile Ser Ala Leu Lys Glu Val Gly Gly Val Tyr Leu Asp Val Asp

275 280 285

Met Leu Pro Gly Ile Gln Pro Asp Leu Phe Glu Ser Ile Glu Lys Pro

290 295 300

Ser Ser Val Thr Val Asp Phe Trp Glu Met Val Lys Leu Glu Ala Ile

305 310 315 320

Met Lys Tyr Lys Glu Tyr Ile Pro Gly Tyr Thr Ser Glu His Phe Asp

325 330 335

Met Leu Asp Glu Glu Val Gln Ser Ser Phe Glu Ser Val Leu Ala Ser

340 345 350

Lys Ser Asp Lys Ser Glu Ile Phe Ser Ser Leu Gly Asp Met Glu Ala

355 360 365

Ser Pro Leu Glu Val Lys Ile Ala Phe Asn Ser Lys Gly Ile Ile Asn

370 375 380

Gln Gly Leu Ile Ser Val Lys Asp Ser Tyr Cys Ser Asn Leu Ile Val

385 390 395 400

Lys Gln Ile Glu Asn Arg Tyr Lys Ile Leu Asn Asn Ser Leu Asn Pro

405 410 415

Ala Ile Ser Glu Asp Asn Asp Phe Asn Thr Thr Thr Asn Ala Phe Ile

420 425 430

Asp Ser Ile Met Ala Glu Ala Asn Ala Asp Asn Gly Arg Phe Met Met

435 440 445

Glu Leu Gly Lys Tyr Leu Arg Val Gly Phe Phe Pro Asp Val Lys Thr

450 455 460

Thr Ile Asn Leu Ser Gly Pro Glu Ala Tyr Ala Ala Ala Tyr Gln Asp

465 470 475 480

Leu Leu Met Phe Lys Glu Gly Ser Met Asn Ile His Leu Ile Glu Ala

485 490 495

Asp Leu Arg Asn Phe Glu Ile Ser Lys Thr Asn Ile Ser Gln Ser Thr

500 505 510

Glu Gln Glu Met Ala Ser Leu Trp Ser Phe Asp Asp Ala Arg Ala Lys

515 520 525

Ala Gln Phe Glu Glu Tyr Lys Lys Asn Tyr Phe Glu Gly Ser Leu Gly

530 535 540

Glu Asp Asp Asn Leu Asp Phe Ser Gln Asn Thr Val Val Asp Lys Glu

545 550 555 560

Tyr Leu Leu Glu Lys Ile Ser Ser Leu Ala Arg Ser Ser Glu Arg Gly

565 570 575

Tyr Ile His Tyr Ile Val Gln Leu Gln Gly Asp Lys Ile Ser Tyr Glu

580 585 590

Ala Ala Cys Asn Leu Phe Ala Lys Thr Pro Tyr Asp Ser Val Leu Phe

595 600 605

Gln Lys Asn Ile Glu Asp Ser Glu Ile Ala Tyr Tyr Tyr Asn Pro Gly

610 615 620

Asp Gly Glu Ile Gln Glu Ile Asp Lys Tyr Lys Ile Pro Ser Ile Ile

625 630 635 640

Ser Asp Arg Pro Lys Ile Lys Leu Thr Phe Ile Gly His Gly Lys Asp

645 650 655

Glu Phe Asn Thr Asp Ile Phe Ala Gly Leu Asp Val Asp Ser Leu Ser

660 665 670

Thr Glu Ile Glu Thr Ala Ile Asp Leu Ala Lys Glu Asp Ile Ser Pro

675 680 685

Lys Ser Ile Glu Ile Asn Leu Leu Gly Cys Asn Met Phe Ser Tyr Ser

690 695 700

Val Asn Val Glu Glu Thr Tyr Pro Gly Lys Leu Leu Leu Arg Val Lys

705 710 715 720

Asp Lys Val Ser Glu Leu Met Pro Ser Ile Ser Gln Asp Ser Ile Ile

725 730 735

Val Ser Ala Asn Gln Tyr Glu Val Arg Ile Asn Ser Glu Gly Arg Arg

740 745 750

Glu Leu Leu Asp His Ser Gly Glu Trp Ile Asn Lys Glu Glu Ser Ile

755 760 765

Ile Lys Asp Ile Ser Ser Lys Glu Tyr Ile Ser Phe Asn Pro Lys Glu

770 775 780

Asn Lys Ile Ile Val Lys Ser Lys Asn Leu Pro Glu Leu Ser Thr Leu

785 790 795 800

Leu Gln Glu Ile Arg Asn Asn Ser Asn Ser Ser Asp Ile Glu Leu Glu

805 810 815

Glu Lys Val Met Leu Ala Glu Cys Glu Ile Asn Val Ile Ser Asn Ile

820 825 830

Asp Thr Gln Val Val Glu Gly Arg Ile Glu Glu Ala Lys Ser Leu Thr

835 840 845

Ser Asp Ser Ile Asn Tyr Ile Lys Asn Glu Phe Lys Leu Ile Glu Ser

850 855 860

Ile Ser Asp Ala Leu Tyr Asp Leu Lys Gln Gln Asn Glu Leu Glu Glu

865 870 875 880

Ser His Phe Ile Ser Phe Glu Asp Ile Leu Glu Thr Asp Glu Gly Phe

885 890 895

Ser Ile Arg Phe Ile Asp Lys Glu Thr Gly Glu Ser Ile Phe Val Glu

900 905 910

Thr Glu Lys Ala Ile Phe Ser Glu Tyr Ala Asn His Ile Thr Glu Glu

915 920 925

Ile Ser Lys Ile Lys Gly Thr Ile Phe Asp Thr Val Asn Gly Lys Leu

930 935 940

Val Lys Lys Val Asn Leu Asp Ala Thr His Glu Val Asn Thr Leu Asn

945 950 955 960

Ala Ala Phe Phe Ile Gln Ser Leu Ile Glu Tyr Asn Ser Ser Lys Glu

965 970 975

Ser Leu Ser Asn Leu Ser Val Ala Met Lys Val Gln Val Tyr Ala Gln

980 985 990

Leu Phe Ser Thr Gly Leu Asn Thr Ile Thr Asp Ala Ala Lys Val Val

995 1000 1005

Glu Leu Val Ser Thr Ala Leu Asp Glu Thr Ile Asp Leu Leu Pro

1010 1015 1020

Thr Leu Ser Glu Gly Leu Pro Val Ile Ala Thr Ile Ile Asp Gly

1025 1030 1035

Val Ser Leu Gly Ala Ala Ile Lys Glu Leu Ser Glu Thr Ser Asp

1040 1045 1050

Pro Leu Leu Arg Gln Glu Ile Glu Ala Lys Ile Gly Ile Met Ala

1055 1060 1065

Val Asn Leu Thr Ala Ala Thr Thr Ala Ile Ile Thr Ser Ser Leu

1070 1075 1080

Gly Ile Ala Ser Gly Phe Ser Ile Leu Leu Val Pro Leu Ala Gly

1085 1090 1095

Ile Ser Ala Gly Ile Pro Ser Leu Val Asn Asn Glu Leu Ile Leu

1100 1105 1110

Arg Asp Lys Ala Thr Lys Val Val Asp Tyr Phe Ser His Ile Ser

1115 1120 1125

Leu Ala Glu Ser Glu Gly Ala Phe Thr Ser Leu Asp Asp Lys Ile

1130 1135 1140

Met Met Pro Gln Asp Asp Leu Val Ile Ser Glu Ile Asp Phe Asn

1145 1150 1155

Asn Asn Ser Ile Thr Leu Gly Lys Cys Glu Ile Trp Arg Met Glu

1160 1165 1170

Gly Gly Ser Gly His Thr Val Thr Asp Asp Ile Asp His Phe Phe

1175 1180 1185

Ser Ala Pro Ser Ile Thr Tyr Arg Glu Pro His Leu Ser Ile Tyr

1190 1195 1200

Asp Val Leu Glu Val Gln Lys Glu Glu Leu Asp Leu Ser Lys Asp

1205 1210 1215

Leu Met Val Leu Pro Asn Ala Pro Asn Arg Val Phe Ala Trp Glu

1220 1225 1230

Thr Gly Trp Thr Pro Gly Leu Arg Ser Leu Glu Asn Asp Gly Thr

1235 1240 1245

Lys Leu Leu Asp Arg Ile Arg Asp Asn Tyr Glu Gly Glu Phe Tyr

1250 1255 1260

Trp Arg Tyr Phe Ala Phe Ile Ala Asp Ala Leu Ile Thr Thr Leu

1265 1270 1275

Lys Pro Arg Tyr Glu Asp Thr Asn Ile Arg Ile Asn Leu Asp Ser

1280 1285 1290

Asn Thr Arg Ser Phe Ile Val Pro Val Ile Thr Thr Glu Tyr Ile

1295 1300 1305

Arg Glu Lys Leu Ser Tyr Ser Phe Tyr Gly Ser Gly Gly Thr Tyr

1310 1315 1320

Ala Leu Ser Leu Ser Gln Tyr Asn Met Asn Ile Asn Ile Glu Leu

1325 1330 1335

Asn Glu Asn Asp Thr Trp Val Ile Asp Val Asp Asn Val Val Arg

1340 1345 1350

Asp Val Thr Ile Glu Ser Asp Lys Ile Lys Lys Gly Asp Leu Ile

1355 1360 1365

Glu Asn Ile Leu Ser Lys Leu Ser Ile Glu Asp Asn Lys Ile Ile

1370 1375 1380

Leu Asp Asn His Glu Ile Asn Phe Ser Gly Thr Leu Asn Gly Gly

1385 1390 1395

Asn Gly Phe Val Ser Leu Thr Phe Ser Ile Leu Glu Gly Ile Asn

1400 1405 1410

Ala Val Ile Glu Val Asp Leu Leu Ser Lys Ser Tyr Lys Val Leu

1415 1420 1425

Ile Ser Gly Glu Leu Lys Thr Leu Met Ala Asn Ser Asn Ser Val

1430 1435 1440

Gln Gln Lys Ile Asp Tyr Ile Gly Leu Asn Ser Glu Leu Gln Lys

1445 1450 1455

Asn Ile Pro Tyr Ser Phe Met Asp Asp Lys Gly Lys Glu Asn Gly

1460 1465 1470

Phe Ile Asn Cys Ser Thr Lys Glu Gly Leu Phe Val Ser Glu Leu

1475 1480 1485

Ser Asp Val Val Leu Ile Ser Lys Val Tyr Met Asp Asn Ser Lys

1490 1495 1500

Pro Leu Phe Gly Tyr Cys Ser Asn Asp Leu Lys Asp Val Lys Val

1505 1510 1515

Ile Thr Lys Asp Asp Val Ile Ile Leu Thr Gly Tyr Tyr Leu Lys

1520 1525 1530

Asp Asp Ile Lys Ile Ser Leu Ser Phe Thr Ile Gln Asp Glu Asn

1535 1540 1545

Thr Ile Lys Leu Asn Gly Val Tyr Leu Asp Glu Asn Gly Val Ala

1550 1555 1560

Glu Ile Leu Lys Phe Met Asn Lys Lys Gly Ser Thr Asn Thr Ser

1565 1570 1575

Asp Ser Leu Met Ser Phe Leu Glu Ser Met Asn Ile Lys Ser Ile

1580 1585 1590

Phe Ile Asn Ser Leu Gln Ser Asn Thr Lys Leu Ile Leu Asp Thr

1595 1600 1605

Asn Phe Ile Ile Ser Gly Thr Thr Ser Ile Gly Gln Phe Glu Phe

1610 1615 1620

Ile Cys Asp Lys Asp Asn Asn Ile Gln Pro Tyr Phe Ile Lys Phe

1625 1630 1635

Asn Thr Leu Glu Thr Lys Tyr Thr Leu Tyr Val Gly Asn Arg Gln

1640 1645 1650

Asn Met Ile Val Glu Pro Asn Tyr Asp Leu Asp Asp Ser Gly Asp

1655 1660 1665

Ile Ser Ser Thr Val Ile Asn Phe Ser Gln Lys Tyr Leu Tyr Gly

1670 1675 1680

Ile Asp Ser Cys Val Asn Lys Val Ile Ile Ser Pro Asn Ile Tyr

1685 1690 1695

Thr Asp Glu Ile Asn Ile Thr Pro Ile Tyr Glu Ala Asn Asn Thr

1700 1705 1710

Tyr Pro Glu Val Ile Val Leu Asp Thr Asn Tyr Ile Ser Glu Lys

1715 1720 1725

Ile Asn Ile Asn Ile Asn Asp Leu Ser Ile Arg Tyr Val Trp Ser

1730 1735 1740

Asn Asp Gly Ser Asp Phe Ile Leu Met Ser Thr Asp Glu Glu Asn

1745 1750 1755

Lys Val Ser Gln Val Lys Ile Arg Phe Thr Asn Val Phe Lys Gly

1760 1765 1770

Asn Thr Ile Ser Asp Lys Ile Ser Phe Asn Phe Ser Asp Lys Gln

1775 1780 1785

Asp Val Ser Ile Asn Lys Val Ile Ser Thr Phe Thr Pro Ser Tyr

1790 1795 1800

Tyr Val Glu Gly Leu Leu Asn Tyr Asp Leu Gly Leu Ile Ser Leu

1805 1810 1815

Tyr Asn Glu Lys Phe Tyr Ile Asn Asn Phe Gly Met Met Val Ser

1820 1825 1830

Gly Leu Val Tyr Ile Asn Asp Ser Leu Tyr Tyr Phe Lys Pro Pro

1835 1840 1845

Ile Lys Asn Leu Ile Thr Gly Phe Thr Thr Ile Gly Asp Asp Lys

1850 1855 1860

Tyr Tyr Phe Asn Pro Asp Asn Gly Gly Ala Ala Ser Val Gly Glu

1865 1870 1875

Thr Ile Ile Asp Gly Lys Asn Tyr Tyr Phe Ser Gln Asn Gly Val

1880 1885 1890

Leu Gln Thr Gly Val Phe Ser Thr Glu Asp Gly Phe Lys Tyr Phe

1895 1900 1905

Ala Pro Ala Asp Thr Leu Asp Glu Asn Leu Glu Gly Glu Ala Ile

1910 1915 1920

Asp Phe Thr Gly Lys Leu Thr Ile Asp Glu Asn Val Tyr Tyr Phe

1925 1930 1935

Gly Asp Asn Tyr Arg Ala Ala Ile Glu Trp Gln Thr Leu Asp Asp

1940 1945 1950

Glu Val Tyr Tyr Phe Ser Thr Asp Thr Gly Arg Ala Phe Lys Gly

1955 1960 1965

Leu Asn Gln Ile Gly Asp Asp Lys Phe Tyr Phe Asn Ser Asp Gly

1970 1975 1980

Ile Met Gln Lys Gly Phe Val Asn Ile Asn Asp Lys Thr Phe Tyr

1985 1990 1995

Phe Asp Asp Ser Gly Val Met Lys Ser Gly Tyr Thr Glu Ile Asp

2000 2005 2010

Gly Lys Tyr Phe Tyr Phe Ala Glu Asn Gly Glu Met Gln Ile Gly

2015 2020 2025

Val Phe Asn Thr Ala Asp Gly Phe Lys Tyr Phe Ala His His Asp

2030 2035 2040

Glu Asp Leu Gly Asn Glu Glu Gly Glu Ala Leu Ser Tyr Ser Gly

2045 2050 2055

Ile Leu Asn Phe Asn Asn Lys Ile Tyr Tyr Phe Asp Asp Ser Phe

2060 2065 2070

Thr Ala Val Val Gly Trp Lys Asp Leu Glu Asp Gly Ser Lys Tyr

2075 2080 2085

Tyr Phe Asp Glu Asp Thr Ala Glu Ala Tyr Ile Gly Ile Ser Ile

2090 2095 2100

Ile Asn Asp Gly Lys Tyr Tyr Phe Asn Asp Ser Gly Ile Met Gln

2105 2110 2115

Ile Gly Phe Val Thr Ile Asn Asn Glu Val Phe Tyr Phe Ser Asp

2120 2125 2130

Ser Gly Ile Val Glu Ser Gly Met Gln Asn Ile Asp Asp Asn Tyr

2135 2140 2145

Phe Tyr Ile Asp Glu Asn Gly Leu Val Gln Ile Gly Val Phe Asp

2150 2155 2160

Thr Ser Asp Gly Tyr Lys Tyr Phe Ala Pro Ala Asn Thr Val Asn

2165 2170 2175

Asp Asn Ile Tyr Gly Gln Ala Val Glu Tyr Ser Gly Leu Val Arg

2180 2185 2190

Val Gly Glu Asp Val Tyr Tyr Phe Gly Glu Thr Tyr Thr Ile Glu

2195 2200 2205

Thr Gly Trp Ile Tyr Asp Met Glu Asn Glu Ser Asp Lys Tyr Tyr

2210 2215 2220

Phe Asp Pro Glu Thr Lys Lys Ala Tyr Lys Gly Ile Asn Val Ile

2225 2230 2235

Asp Asp Ile Lys Tyr Tyr Phe Asp Glu Asn Gly Ile Met Arg Thr

2240 2245 2250

Gly Leu Ile Thr Phe Glu Asp Asn His Tyr Tyr Phe Asn Glu Asp

2255 2260 2265

Gly Ile Met Gln Tyr Gly Tyr Leu Asn Ile Glu Asp Lys Thr Phe

2270 2275 2280

Tyr Phe Ser Glu Asp Gly Ile Met Gln Ile Gly Val Phe Asn Thr

2285 2290 2295

Pro Asp Gly Phe Lys Tyr Phe Ala His Gln Asn Thr Leu Asp Glu

2300 2305 2310

Asn Phe Glu Gly Glu Ser Ile Asn Tyr Thr Gly Trp Leu Asp Leu

2315 2320 2325

Asp Glu Lys Arg Tyr Tyr Phe Thr Asp Glu Tyr Ile Ala Ala Thr

2330 2335 2340

Gly Ser Val Ile Ile Asp Gly Glu Glu Tyr Tyr Phe Asp Pro Asp

2345 2350 2355

Thr Ala Gln Leu Val Ile Ser Glu

2360 2365

<210> 8

<211> 2367

<212> PRT

<213> Artificial sequence

<220>

<223> TcdB3

<400> 8

Met Ser Leu Val Asn Arg Lys Gln Leu Glu Lys Met Ala Asn Val Arg

1 5 10 15

Phe Arg Val Gln Glu Asp Glu Tyr Val Ala Ile Leu Asp Ala Leu Glu

20 25 30

Glu Tyr His Asn Met Ser Glu Asn Thr Val Val Glu Lys Tyr Leu Lys

35 40 45

Leu Lys Asp Ile Asn Ser Leu Thr Asp Thr Tyr Ile Asp Thr Tyr Lys

50 55 60

Lys Ser Gly Arg Asn Lys Ala Leu Lys Lys Phe Lys Glu Tyr Leu Val

65 70 75 80

Ile Glu Ile Leu Glu Leu Lys Asn Ser Asn Leu Thr Pro Val Glu Lys

85 90 95

Asn Leu His Phe Ile Trp Ile Gly Gly Gln Ile Asn Asp Thr Ala Ile

100 105 110

Asn Tyr Ile Asn Gln Trp Lys Asp Val Asn Ser Asp Tyr Asn Val Asn

115 120 125

Val Phe Tyr Asp Ser Asn Ala Phe Leu Ile Asn Thr Leu Lys Lys Thr

130 135 140

Ile Ile Glu Ser Ala Ser Asn Asp Thr Leu Glu Ser Phe Arg Glu Asn

145 150 155 160

Leu Asn Asp Pro Glu Phe Asn His Thr Ala Phe Phe Arg Lys Arg Met

165 170 175

Gln Ile Ile Tyr Asp Lys Gln Gln Asn Phe Ile Asn Tyr Tyr Lys Ala

180 185 190

Gln Lys Glu Glu Asn Pro Asp Leu Ile Ile Asp Asp Ile Val Lys Thr

195 200 205

Tyr Leu Ser Asn Glu Tyr Ser Lys Asp Ile Asp Glu Leu Asn Ala Tyr

210 215 220

Ile Glu Glu Ser Leu Asn Lys Val Thr Glu Asn Ser Gly Asn Asp Val

225 230 235 240

Arg Asn Phe Glu Glu Phe Lys Thr Gly Glu Val Phe Asn Leu Tyr Glu

245 250 255

Gln Glu Leu Val Glu Arg Trp Asn Leu Ala Gly Ala Ser Asp Ile Leu

260 265 270

Arg Val Ala Ile Leu Lys Asn Ile Gly Gly Val Tyr Leu Asp Val Asp

275 280 285

Met Leu Pro Gly Ile His Pro Asp Leu Phe Lys Asp Ile Asn Lys Pro

290 295 300

Asp Ser Val Lys Thr Ala Val Asp Trp Glu Glu Met Gln Leu Glu Ala

305 310 315 320

Ile Met Lys His Lys Glu Tyr Ile Pro Glu Tyr Thr Ser Lys His Phe

325 330 335

Asp Thr Leu Asp Glu Glu Val Gln Ser Ser Phe Glu Ser Val Leu Ala

340 345 350

Ser Lys Ser Asp Lys Ser Glu Ile Phe Leu Pro Leu Gly Asp Ile Glu

355 360 365

Val Ser Pro Leu Glu Val Lys Ile Ala Phe Ala Lys Gly Ser Ile Ile

370 375 380

Asn Gln Ala Leu Ile Ser Ala Lys Asp Ser Tyr Cys Ser Asp Leu Leu

385 390 395 400

Ile Lys Gln Ile Gln Asn Arg Tyr Lys Ile Leu Asn Asp Thr Leu Gly

405 410 415

Pro Ile Ile Ser Gln Gly Asn Asp Phe Asn Thr Thr Met Asn Asn Phe

420 425 430

Gly Glu Ser Leu Gly Ala Ile Ala Asn Glu Glu Asn Ile Ser Phe Ile

435 440 445

Ala Lys Ile Gly Ser Tyr Leu Arg Val Gly Phe Tyr Pro Glu Ala Asn

450 455 460

Thr Thr Ile Thr Leu Ser Gly Pro Thr Ile Tyr Ala Gly Ala Tyr Lys

465 470 475 480

Asp Leu Leu Thr Phe Lys Glu Met Ser Ile Asp Thr Ser Ile Leu Ser

485 490 495

Ser Glu Leu Arg Asn Phe Glu Phe Pro Lys Val Asn Ile Ser Gln Ala

500 505 510

Thr Glu Gln Glu Lys Asn Ser Leu Trp Gln Phe Asn Glu Glu Arg Ala

515 520 525

Lys Ile Gln Phe Glu Glu Tyr Lys Lys Asn Tyr Phe Glu Gly Ala Leu

530 535 540

Gly Glu Asp Asp Asn Leu Asp Phe Ser Gln Asn Thr Val Thr Asp Lys

545 550 555 560

Glu Tyr Leu Leu Glu Lys Ile Ser Ser Ser Thr Lys Ser Ser Glu Arg

565 570 575

Gly Tyr Val His Tyr Ile Val Gln Leu Gln Gly Asp Lys Ile Ser Tyr

580 585 590

Glu Ala Ala Cys Asn Leu Phe Ala Lys Asn Pro Tyr Asp Ser Ile Leu

595 600 605

Phe Gln Lys Asn Ile Glu Asp Ser Glu Val Ala Tyr Tyr Tyr Asn Pro

610 615 620

Thr Asp Ser Glu Ile Gln Glu Ile Asp Lys Tyr Arg Ile Pro Asp Arg

625 630 635 640

Ile Ser Asp Arg Pro Lys Ile Lys Leu Thr Phe Ile Gly His Gly Lys

645 650 655

Ala Glu Phe Asn Thr Asp Ile Phe Ala Gly Leu Asp Val Asp Ser Leu

660 665 670

Ser Ser Glu Ile Glu Thr Ala Ile Gly Leu Ala Lys Glu Asp Ile Ser

675 680 685

Pro Lys Ser Ile Glu Ile Asn Leu Leu Gly Cys Asn Met Phe Ser Tyr

690 695 700

Ser Val Asn Val Glu Glu Thr Tyr Pro Gly Lys Leu Leu Leu Arg Val

705 710 715 720

Lys Asp Lys Val Ser Glu Leu Met Pro Ser Met Ser Gln Asp Ser Ile

725 730 735

Ile Val Ser Ala Asn Gln Tyr Glu Val Arg Ile Asn Ser Glu Gly Arg

740 745 750

Arg Glu Leu Leu Asp His Ser Gly Glu Trp Ile Asn Lys Glu Glu Ser

755 760 765

Ile Ile Lys Asp Ile Ser Ser Lys Glu Tyr Ile Ser Phe Asn Pro Lys

770 775 780

Glu Asn Lys Ile Ile Val Lys Ser Lys Asn Leu Pro Glu Leu Ser Thr

785 790 795 800

Leu Leu Gln Glu Ile Arg Asn Asn Ser Asn Ser Ser Asp Ile Glu Leu

805 810 815

Glu Glu Lys Val Met Leu Ala Glu Cys Glu Ile Asn Val Ile Ser Asn

820 825 830

Ile Glu Thr Gln Val Val Glu Glu Arg Ile Glu Glu Ala Lys Ser Leu

835 840 845

Thr Ser Asp Ser Ile Asn Tyr Ile Lys Asn Glu Phe Lys Leu Ile Glu

850 855 860

Ser Ile Ser Asp Ala Leu Cys Asp Leu Lys Gln Gln Asn Glu Leu Glu

865 870 875 880

Asp Ser His Phe Ile Ser Phe Glu Asp Ile Ser Glu Thr Asp Glu Gly

885 890 895

Phe Ser Ile Arg Phe Ile Asn Lys Glu Thr Gly Glu Ser Ile Phe Val

900 905 910

Glu Thr Glu Lys Thr Ile Phe Ser Glu Tyr Ala Asn His Ile Thr Glu

915 920 925

Glu Ile Ser Lys Ile Lys Gly Thr Ile Phe Asp Thr Val Asn Gly Lys

930 935 940

Leu Val Lys Lys Val Asn Leu Asp Thr Thr His Glu Val Asn Thr Leu

945 950 955 960

Asn Ala Ala Phe Phe Ile Gln Ser Leu Ile Glu Tyr Asn Ser Ser Lys

965 970 975

Glu Ser Leu Ser Asn Leu Ser Val Ala Met Lys Val Gln Val Tyr Ala

980 985 990

Gln Leu Phe Ser Thr Gly Leu Asn Thr Ile Thr Asp Ala Ala Lys Val

995 1000 1005

Val Glu Leu Val Ser Thr Ala Leu Asp Glu Thr Ile Asp Leu Leu

1010 1015 1020

Pro Thr Leu Ser Glu Gly Leu Pro Ile Ile Ala Thr Ile Ile Asp

1025 1030 1035

Gly Val Ser Leu Gly Ala Ala Ile Lys Glu Leu Ser Glu Thr Ser

1040 1045 1050

Asp Pro Leu Leu Arg Gln Glu Ile Glu Ala Lys Ile Gly Ile Met

1055 1060 1065

Ala Val Asn Leu Thr Thr Ala Thr Thr Ala Ile Ile Thr Ser Ser

1070 1075 1080

Leu Gly Ile Ala Ser Gly Phe Ser Ile Leu Leu Val Pro Leu Ala

1085 1090 1095

Gly Ile Ser Ala Gly Ile Pro Ser Leu Val Asn Asn Glu Leu Val

1100 1105 1110

Leu Arg Asp Lys Ala Thr Lys Val Val Asp Tyr Phe Lys His Val

1115 1120 1125

Ser Leu Val Glu Thr Glu Gly Val Phe Thr Leu Leu Asp Asp Lys

1130 1135 1140

Val Met Met Pro Gln Asp Asp Leu Val Ile Ser Glu Ile Asp Phe

1145 1150 1155

Asn Asn Asn Ser Ile Val Leu Gly Lys Cys Glu Ile Trp Arg Met

1160 1165 1170

Glu Gly Gly Ser Gly His Thr Val Thr Asp Asp Ile Asp His Phe

1175 1180 1185

Phe Ser Ala Pro Ser Ile Thr Tyr Arg Glu Pro His Leu Ser Ile

1190 1195 1200

Tyr Asp Val Leu Glu Val Gln Lys Glu Glu Leu Asp Leu Ser Lys

1205 1210 1215

Asp Leu Met Val Leu Pro Asn Ala Pro Asn Arg Val Phe Ala Trp

1220 1225 1230

Glu Thr Gly Trp Thr Pro Gly Leu Arg Ser Leu Glu Asn Asp Gly

1235 1240 1245

Thr Lys Leu Leu Asp Arg Ile Arg Asp Asn Tyr Glu Gly Glu Phe

1250 1255 1260

Tyr Trp Arg Tyr Phe Ala Phe Ile Ala Asp Ala Leu Ile Thr Thr

1265 1270 1275

Leu Lys Pro Arg Tyr Glu Asp Thr Asn Ile Arg Ile Asn Leu Asp

1280 1285 1290

Ser Asn Thr Arg Ser Phe Ile Val Pro Ile Ile Thr Thr Glu Tyr

1295 1300 1305

Ile Arg Glu Lys Leu Ser Tyr Ser Phe Tyr Gly Ser Gly Gly Thr

1310 1315 1320

Tyr Ala Leu Ser Leu Ser Gln Tyr Asn Met Gly Ile Asn Ile Glu

1325 1330 1335

Leu Ser Glu Ser Asp Val Trp Ile Ile Asp Val Asp Asn Val Val

1340 1345 1350

Arg Asp Val Thr Ile Glu Ser Asp Lys Ile Lys Lys Gly Asp Leu

1355 1360 1365

Ile Glu Gly Ile Leu Ser Thr Leu Ser Ile Glu Glu Asn Lys Ile

1370 1375 1380

Ile Leu Asn Ser His Glu Ile Asn Phe Ser Gly Glu Val Asn Gly

1385 1390 1395

Ser Asn Gly Phe Val Ser Leu Thr Phe Ser Ile Leu Glu Gly Ile

1400 1405 1410

Asn Ala Ile Ile Glu Val Asp Leu Leu Ser Lys Ser Tyr Lys Leu

1415 1420 1425

Leu Ile Ser Gly Glu Leu Lys Ile Leu Met Leu Asn Ser Asn His

1430 1435 1440

Ile Gln Gln Lys Ile Asp Tyr Ile Gly Phe Asn Ser Glu Leu Gln

1445 1450 1455

Lys Asn Ile Pro Tyr Ser Phe Val Asp Ser Glu Gly Lys Glu Asn

1460 1465 1470

Gly Phe Ile Asn Gly Ser Thr Lys Glu Gly Leu Phe Val Ser Glu

1475 1480 1485

Leu Pro Asp Val Val Leu Ile Ser Lys Val Tyr Met Asp Asp Ser

1490 1495 1500

Lys Pro Ser Phe Gly Tyr Tyr Ser Asn Asn Leu Lys Asp Val Lys

1505 1510 1515

Val Ile Thr Lys Asp Asn Val Asn Ile Leu Thr Gly Tyr Tyr Leu

1520 1525 1530

Lys Asp Asp Ile Lys Ile Ser Leu Ser Leu Thr Leu Gln Asp Glu

1535 1540 1545

Lys Thr Ile Lys Leu Asn Ser Val His Leu Asp Glu Ser Gly Val

1550 1555 1560

Ala Glu Ile Leu Lys Phe Met Asn Arg Lys Gly Ser Thr Asn Thr

1565 1570 1575

Ser Asp Ser Leu Met Ser Phe Leu Glu Ser Met Asn Ile Lys Ser

1580 1585 1590

Ile Phe Val Asn Phe Leu Gln Ser Asn Ile Lys Phe Ile Leu Asp

1595 1600 1605

Ala Asn Phe Ile Ile Ser Gly Thr Thr Ser Ile Gly Gln Phe Glu

1610 1615 1620

Phe Ile Cys Asp Glu Asn Asn Asn Ile Gln Pro Tyr Phe Ile Lys

1625 1630 1635

Phe Asn Thr Leu Glu Thr Asn Tyr Thr Leu Tyr Val Gly Asn Arg

1640 1645 1650

Gln Asn Met Ile Val Glu Pro Asn Tyr Asp Leu Asp Asp Ser Gly

1655 1660 1665

Asp Ile Ser Ser Thr Val Ile Asn Phe Ser Gln Lys Tyr Leu Tyr

1670 1675 1680

Gly Ile Asp Ser Cys Val Asn Lys Val Val Ile Ser Pro Asn Ile

1685 1690 1695

Tyr Thr Asp Glu Ile Asn Ile Thr Pro Val Tyr Glu Thr Asn Asn

1700 1705 1710

Thr Tyr Pro Glu Val Ile Val Leu Asp Ala Asn Tyr Ile Asn Glu

1715 1720 1725

Lys Ile Asn Val Asn Ile Asn Asp Leu Ser Ile Arg Tyr Val Trp

1730 1735 1740

Ser Asn Asp Gly Asn Asp Phe Ile Leu Met Ser Thr Ser Glu Glu

1745 1750 1755

Asn Lys Val Ser Gln Val Lys Ile Arg Phe Val Asn Val Phe Lys

1760 1765 1770

Asp Lys Thr Leu Ala Asn Lys Leu Ser Phe Asn Phe Ser Asp Lys

1775 1780 1785

Gln Asp Val Pro Val Ser Glu Ile Ile Leu Ser Phe Thr Pro Ser

1790 1795 1800

Tyr Tyr Glu Asp Gly Leu Ile Gly Tyr Asp Leu Gly Leu Val Ser

1805 1810 1815

Leu Tyr Asn Glu Lys Phe Tyr Ile Asn Asn Phe Gly Met Met Val

1820 1825 1830

Ser Gly Leu Ile Tyr Ile Asn Asp Ser Leu Tyr Tyr Phe Lys Pro

1835 1840 1845

Pro Val Asn Asn Leu Ile Thr Gly Phe Val Thr Val Gly Asp Asp

1850 1855 1860

Lys Tyr Tyr Phe Asn Pro Ile Asn Gly Gly Ala Ala Ser Ile Gly

1865 1870 1875

Glu Thr Ile Ile Asp Asp Lys Asn Tyr Tyr Phe Asn Gln Ser Gly

1880 1885 1890

Val Leu Gln Thr Gly Val Phe Ser Thr Glu Asp Gly Phe Lys Tyr

1895 1900 1905

Phe Ala Pro Ala Asn Thr Leu Asp Glu Asn Leu Glu Gly Glu Ala

1910 1915 1920

Ile Asp Phe Thr Gly Lys Leu Ile Ile Asp Glu Asn Ile Tyr Tyr

1925 1930 1935

Phe Glu Asp Asn Tyr Arg Gly Ala Val Glu Trp Lys Glu Leu Asp

1940 1945 1950

Gly Glu Met His Tyr Phe Ser Pro Glu Thr Gly Lys Ala Phe Lys

1955 1960 1965

Gly Leu Asn Gln Ile Gly Asp Asp Lys Tyr Tyr Phe Asn Ser Asp

1970 1975 1980

Gly Val Met Gln Lys Gly Phe Val Ser Ile Asn Asp Asn Lys His

1985 1990 1995

Tyr Phe Asp Asp Ser Gly Val Met Lys Val Gly Tyr Thr Glu Ile

2000 2005 2010

Asp Gly Lys His Phe Tyr Phe Ala Glu Asn Gly Glu Met Gln Ile

2015 2020 2025

Gly Val Phe Asn Thr Glu Asp Gly Phe Lys Tyr Phe Ala His His

2030 2035 2040

Asn Glu Asp Leu Gly Asn Glu Glu Gly Glu Glu Ile Ser Tyr Ser

2045 2050 2055

Gly Ile Leu Asn Phe Asn Asn Lys Ile Tyr Tyr Phe Asp Asp Ser

2060 2065 2070

Phe Thr Ala Val Val Gly Trp Lys Asp Leu Glu Asp Gly Ser Lys

2075 2080 2085

Tyr Tyr Phe Asp Glu Asp Thr Ala Glu Ala Tyr Ile Gly Leu Ser

2090 2095 2100

Leu Ile Asn Asp Gly Gln Tyr Tyr Phe Asn Asp Asp Gly Ile Met

2105 2110 2115

Gln Val Gly Phe Val Thr Ile Asn Asp Lys Val Phe Tyr Phe Ser

2120 2125 2130

Asp Ser Gly Ile Ile Glu Ser Gly Val Gln Asn Ile Asp Asp Asn

2135 2140 2145

Tyr Phe Tyr Ile Asp Asp Asn Gly Ile Val Gln Ile Gly Val Phe

2150 2155 2160

Asp Thr Ser Asp Gly Tyr Lys Tyr Phe Ala Pro Ala Asn Thr Val

2165 2170 2175

Asn Asp Asn Ile Tyr Gly Gln Ala Val Glu Tyr Ser Gly Leu Val

2180 2185 2190

Arg Val Gly Glu Asp Val Tyr Tyr Phe Gly Glu Thr Tyr Thr Ile

2195 2200 2205

Glu Thr Gly Trp Ile Tyr Asp Met Glu Asn Glu Ser Asp Lys Tyr

2210 2215 2220

Tyr Phe Asp Pro Glu Thr Lys Lys Ala Cys Lys Gly Ile Asn Leu

2225 2230 2235

Ile Asp Asp Ile Lys Tyr Tyr Phe Asp Glu Lys Gly Ile Met Arg

2240 2245 2250

Thr Gly Leu Ile Ser Phe Glu Asn Asn Asn Tyr Tyr Phe Asn Glu

2255 2260 2265

Asn Gly Glu Met Gln Phe Gly Tyr Ile Asn Ile Glu Asp Lys Met

2270 2275 2280

Phe Tyr Phe Gly Glu Asp Gly Val Met Gln Ile Gly Val Phe Asn

2285 2290 2295

Thr Pro Asp Gly Phe Lys Tyr Phe Ala His Gln Asn Thr Leu Asp

2300 2305 2310

Glu Asn Phe Glu Gly Glu Ser Ile Asn Tyr Thr Gly Trp Leu Asp

2315 2320 2325

Leu Asp Glu Lys Arg Tyr Tyr Phe Thr Asp Glu Tyr Ile Ala Ala

2330 2335 2340

Thr Gly Ser Val Ile Ile Asp Gly Glu Glu Tyr Tyr Phe Asp Pro

2345 2350 2355

Asp Thr Ala Gln Leu Val Ile Ser Glu

2360 2365

<210> 9

<211> 2367

<212> PRT

<213> Artificial sequence

<220>

<223> Full-length sequence of TcdB4 from Clostridium difficile strain 8864

<400> 9

Met Ser Leu Val Asn Arg Lys Gln Leu Glu Lys Met Ala Asn Val Arg

1 5 10 15

Phe Arg Val Gln Glu Asp Glu Tyr Val Ala Ile Leu Asp Ala Leu Glu

20 25 30

Glu Tyr His Asn Met Ser Glu Asn Thr Val Val Glu Lys Tyr Leu Lys

35 40 45

Leu Lys Asp Ile Asn Ser Leu Thr Asp Thr Tyr Ile Asp Thr Tyr Lys

50 55 60

Lys Ser Gly Arg Asn Lys Ala Leu Lys Lys Phe Lys Glu Tyr Leu Val

65 70 75 80

Thr Glu Ile Leu Glu Leu Lys Asn Ser Asn Leu Thr Pro Val Glu Lys

85 90 95

Asn Leu His Phe Ile Trp Ile Gly Gly Gln Ile Asn Asp Thr Ala Ile

100 105 110

Asn Tyr Ile Asn Gln Trp Lys Asp Val Asn Ser Asp Tyr Asn Val Asn

115 120 125

Val Phe Tyr Asp Ser Asn Ala Phe Leu Ile Asn Thr Leu Lys Lys Thr

130 135 140

Ile Ile Glu Ser Ala Ser Asn Asp Thr Leu Glu Ser Phe Arg Glu Asn

145 150 155 160

Leu Asn Asp Pro Glu Phe Asn His Thr Ala Phe Phe Arg Lys Arg Met

165 170 175

Gln Ile Ile Tyr Asp Lys Gln Gln Asn Phe Ile Asn Tyr Tyr Lys Ala

180 185 190

Gln Lys Glu Glu Asn Pro Asp Leu Ile Ile Asp Asp Ile Val Lys Thr

195 200 205

Tyr Leu Ser Asn Glu Tyr Ser Lys Asp Ile Asp Glu Leu Asn Ala Tyr

210 215 220

Ile Glu Glu Ser Leu Asn Lys Val Thr Glu Asn Ser Gly Asn Asp Val

225 230 235 240

Arg Asn Phe Glu Glu Phe Lys Thr Gly Glu Val Phe Asn Leu Tyr Glu

245 250 255

Gln Glu Leu Val Glu Arg Trp Asn Leu Ala Gly Ala Ser Asp Ile Leu

260 265 270

Arg Val Ala Ile Leu Lys Asn Ile Gly Gly Val Tyr Leu Asp Val Asp

275 280 285

Met Leu Pro Gly Ile His Pro Asp Leu Phe Lys Asp Ile Asn Lys Pro

290 295 300

Asp Ser Val Lys Thr Ala Val Asp Trp Glu Glu Met Gln Leu Glu Ala

305 310 315 320

Ile Met Lys Tyr Lys Glu Tyr Ile Pro Glu Tyr Thr Ser Lys His Phe

325 330 335

Asp Thr Leu Asp Glu Glu Val Gln Ser Ser Phe Glu Ser Val Leu Ala

340 345 350

Ser Lys Ser Asp Lys Ser Glu Ile Phe Leu Pro Leu Gly Gly Ile Glu

355 360 365

Val Ser Pro Leu Glu Val Lys Val Ala Phe Ala Lys Gly Ser Ile Ile

370 375 380

Asp Gln Ala Leu Ile Ser Ala Lys Asp Ser Tyr Cys Ser Asp Leu Leu

385 390 395 400

Ile Lys Gln Ile Gln Asn Arg Tyr Lys Ile Leu Asn Asp Thr Leu Gly

405 410 415

Pro Ile Ile Ser Gln Gly Asn Asp Phe Asn Thr Thr Met Asn Asn Phe

420 425 430

Gly Glu Ser Leu Gly Ala Ile Ala Asn Glu Glu Asn Ile Ser Phe Ile

435 440 445

Ala Lys Ile Gly Ser Tyr Leu Arg Val Gly Phe Tyr Pro Glu Ala Asn

450 455 460

Thr Thr Ile Thr Leu Ser Gly Pro Thr Ile Tyr Ala Gly Ala Tyr Lys

465 470 475 480

Asp Leu Leu Thr Phe Lys Glu Met Ser Ile Asp Thr Ser Ile Leu Ser

485 490 495

Ser Glu Leu Arg Asn Phe Glu Phe Pro Lys Val Asn Ile Ser Gln Ala

500 505 510

Thr Glu Gln Glu Lys Asn Ser Leu Trp Gln Phe Asn Glu Glu Arg Ala

515 520 525

Lys Ile Gln Phe Glu Glu Tyr Lys Lys Asn Tyr Phe Glu Gly Ala Leu

530 535 540

Gly Glu Asp Asp Asn Leu Asp Phe Ser Gln Asn Thr Val Thr Asp Lys

545 550 555 560

Glu Tyr Leu Leu Glu Lys Ile Ser Ser Ser Thr Lys Ser Ser Glu Arg

565 570 575

Gly Tyr Val His Tyr Ile Val Gln Leu Gln Gly Asp Lys Ile Ser Tyr

580 585 590

Glu Ala Ala Cys Asn Leu Phe Ala Lys Asn Pro Tyr Asp Ser Ile Leu

595 600 605

Phe Gln Lys Asn Ile Glu Asp Ser Glu Val Ala Tyr Tyr Tyr Asn Pro

610 615 620

Thr Asp Ser Glu Ile Gln Glu Ile Asp Lys Tyr Arg Ile Pro Asp Arg

625 630 635 640

Ile Ser Asp Arg Pro Lys Ile Lys Leu Thr Leu Ile Gly His Gly Lys

645 650 655

Ala Glu Phe Asn Thr Asp Ile Phe Ala Gly Leu Asp Val Asp Ser Leu

660 665 670

Ser Ser Glu Ile Glu Thr Ile Leu Asp Leu Ala Lys Ala Asp Ile Ser

675 680 685

Pro Lys Ser Ile Glu Ile Asn Leu Leu Gly Cys Asn Met Phe Ser Tyr

690 695 700

Ser Val Asn Val Glu Glu Thr Tyr Pro Gly Lys Leu Leu Leu Arg Val

705 710 715 720

Lys Asp Lys Val Ser Glu Leu Met Pro Ser Ile Ser Gln Asp Ser Ile

725 730 735

Ile Val Ser Ala Asn Gln Tyr Glu Val Arg Ile Asn Ser Glu Gly Arg

740 745 750

Arg Glu Leu Leu Asp His Ser Gly Glu Trp Ile Asn Lys Glu Glu Ser

755 760 765

Ile Ile Lys Asp Ile Ser Ser Lys Glu Tyr Ile Ser Phe Asn Pro Lys

770 775 780

Glu Asn Lys Ile Ile Val Lys Ser Lys Asn Leu Pro Glu Leu Ser Thr

785 790 795 800

Leu Leu Gln Glu Ile Arg Asn Asn Ser Asn Ser Ser Asp Ile Glu Leu

805 810 815

Glu Glu Lys Val Met Leu Ala Glu Cys Glu Ile Asn Val Ile Ser Asn

820 825 830

Ile Glu Thr Gln Val Val Glu Glu Arg Ile Glu Glu Ala Lys Ser Leu

835 840 845

Thr Ser Asp Ser Ile Asn Tyr Ile Lys Asn Glu Phe Lys Leu Ile Glu

850 855 860

Ser Ile Ser Asp Ala Leu Tyr Asp Leu Lys Gln Gln Asn Glu Leu Glu

865 870 875 880

Glu Ser His Phe Ile Ser Phe Glu Asp Ile Ser Lys Thr Asp Glu Gly

885 890 895

Phe Ser Ile Arg Phe Ile Asp Lys Glu Thr Gly Glu Ser Ile Phe Val

900 905 910

Glu Thr Glu Lys Ala Ile Phe Ser Glu Tyr Ala Asn His Ile Thr Glu

915 920 925

Glu Ile Ser Lys Leu Lys Asp Thr Ile Phe Asp Thr Val Asn Gly Lys

930 935 940

Leu Val Lys Lys Val Thr Leu Asp Ala Thr His Glu Val Asn Thr Leu

945 950 955 960

Asn Ala Ala Phe Phe Ile Gln Ser Leu Ile Gly Tyr Asn Ser Ser Lys

965 970 975

Glu Ser Leu Ser Asn Leu Ser Val Ala Met Lys Val Gln Val Tyr Ala

980 985 990

Gln Leu Phe Ser Thr Gly Leu Asn Thr Ile Thr Asp Ala Ala Lys Val

995 1000 1005

Val Glu Leu Val Ser Thr Ala Leu Asp Glu Thr Ile Asp Leu Leu

1010 1015 1020

Pro Thr Leu Ser Glu Gly Leu Pro Val Ile Ala Thr Ile Ile Asp

1025 1030 1035

Gly Val Ser Leu Gly Ala Ser Ile Lys Glu Leu Ser Glu Thr Ser

1040 1045 1050

Asp Pro Leu Leu Arg Gln Glu Ile Glu Ala Lys Ile Gly Ile Met

1055 1060 1065

Ala Val Asn Leu Thr Ala Ala Thr Thr Ala Ile Ile Thr Ser Ser

1070 1075 1080

Leu Gly Ile Ala Ser Gly Phe Ser Ile Leu Leu Val Pro Leu Ala

1085 1090 1095

Gly Ile Ser Ala Gly Ile Pro Ser Leu Val Asn Asn Glu Leu Ile

1100 1105 1110

Leu Arg Ala Glu Ala Lys Asn Val Val Asp Tyr Phe Gly His Ile

1115 1120 1125

Ser Leu Ala Glu Ser Glu Gly Ala Phe Thr Leu Leu Asp Asp Lys

1130 1135 1140

Ile Met Met Pro Gln Asp Asp Leu Val Ile Ser Glu Ile Asp Phe

1145 1150 1155

Asn Asn Asn Ser Ile Thr Leu Gly Lys Cys Glu Ile Trp Arg Met

1160 1165 1170

Glu Gly Gly Ser Gly His Thr Val Thr Asp Asp Ile Asp His Phe

1175 1180 1185

Phe Ser Ala Pro Ser Thr Thr Tyr Arg Glu Pro Tyr Leu Ser Ile

1190 1195 1200

Tyr Asp Val Leu Asp Val Lys Glu Glu Glu Leu Asp Leu Ser Lys

1205 1210 1215

Asp Leu Met Val Leu Pro Asn Ala Pro Asp Arg Ile Phe Gly Trp

1220 1225 1230

Glu Arg Gly Trp Thr Pro Gly Leu Arg Ser Leu Glu Asn Asp Gly

1235 1240 1245

Thr Lys Leu Leu Asp Arg Ile Arg Asp His Tyr Glu Gly Gln Phe

1250 1255 1260

Tyr Trp Arg Phe Phe Ala Phe Ile Ala Asp Ser Val Ile Thr Lys

1265 1270 1275

Leu Lys Pro Arg Tyr Glu Asp Thr Asn Ile Arg Ile Ser Leu Asp

1280 1285 1290

Ser Asn Thr Arg Ser Phe Ile Val Pro Val Ile Thr Thr Glu Tyr

1295 1300 1305

Ile Arg Glu Lys Leu Ser Tyr Ser Phe Tyr Gly Ser Gly Gly Thr

1310 1315 1320

Tyr Ala Leu Ser Leu Ser Gln Tyr Asn Met Asn Ile Asn Ile Glu

1325 1330 1335

Leu Asn Glu Asn Asp Thr Trp Val Ile Asp Val Asp Asn Val Val

1340 1345 1350

Arg Asp Val Thr Ile Glu Ser Asp Lys Ile Lys Lys Gly Asp Leu

1355 1360 1365

Ile Glu Asn Ile Leu Ser Lys Leu Ser Ile Glu Asp Asn Lys Ile

1370 1375 1380

Ile Leu Asp Asn His Glu Ile Asn Phe Ser Gly Thr Leu Asn Gly

1385 1390 1395

Gly Asn Gly Phe Val Ser Leu Thr Phe Ser Ile Leu Glu Gly Ile

1400 1405 1410

Asn Ala Val Ile Glu Val Asp Leu Leu Ser Lys Ser Tyr Lys Val

1415 1420 1425

Leu Ile Ser Gly Glu Leu Lys Thr Leu Met Ala Asn Ser Asn Ser

1430 1435 1440

Val Gln Gln Lys Ile Asp Tyr Ile Gly Leu Asn Ser Glu Leu Gln

1445 1450 1455

Lys Asn Ile Pro Tyr Ser Phe Met Asp Asp Glu Gly Lys Glu Asn

1460 1465 1470

Gly Phe Ile Asn Cys Phe Thr Lys Glu Gly Leu Phe Val Ser Glu

1475 1480 1485

Leu Ser Asp Val Val Leu Ile Ile Lys Val Tyr Met Asp Asn Ser

1490 1495 1500

Lys Pro Pro Phe Gly Tyr Tyr Ser Asn Asp Leu Lys Asp Val Lys

1505 1510 1515

Val Ile Thr Lys Asp Asp Val Ile Ile Ile Thr Gly Tyr Tyr Leu

1520 1525 1530

Lys Asp Asp Ile Lys Ile Ser Leu Ser Phe Thr Ile Gln Asp Lys

1535 1540 1545

Asn Thr Ile Lys Leu Asn Gly Val Tyr Leu Asp Glu Asn Gly Val

1550 1555 1560

Ala Glu Ile Leu Lys Phe Met Asn Lys Lys Gly Ser Thr Asn Thr

1565 1570 1575

Ser Asp Ser Leu Met Ser Phe Leu Glu Ser Met Asn Ile Lys Ser

1580 1585 1590

Ile Phe Ile Lys Ser Leu Lys Ser Asn Ala Lys Leu Ile Leu Asp

1595 1600 1605

Thr Asn Phe Ile Ile Ser Gly Thr Thr Phe Ile Gly Gln Phe Glu

1610 1615 1620

Phe Ile Cys Asp Lys Asp Asn Asn Ile Gln Pro Tyr Phe Ile Lys

1625 1630 1635

Phe Asn Thr Leu Glu Thr Lys Tyr Thr Leu Tyr Val Gly Asn Arg

1640 1645 1650

Gln Asn Met Ile Val Glu Pro Asn Tyr Asn Leu Asp Asp Ser Gly

1655 1660 1665

Asp Ile Ser Ser Thr Val Ile Asn Phe Ser Gln Lys Tyr Leu Tyr

1670 1675 1680

Gly Ile Asp Ser Cys Val Asn Lys Val Val Ile Ser Pro Gly Ile

1685 1690 1695

Tyr Thr Asp Glu Ile Asn Ile Thr Pro Val His Glu Ala Asn Asn

1700 1705 1710

Thr Tyr Pro Glu Val Ile Val Leu Asp Thr Asn Tyr Ile Ser Glu

1715 1720 1725

Lys Ile Asn Ile Asn Ile Asn Asp Leu Ser Ile Arg Tyr Val Trp

1730 1735 1740

Ser Asn Asp Gly Ser Asp Phe Ile Leu Met Ser Thr Asp Glu Glu

1745 1750 1755

Asn Lys Val Ser Gln Val Lys Ile Arg Phe Thr Asn Val Phe Lys

1760 1765 1770

Gly Asn Thr Ile Ser Asp Lys Ile Ser Phe Asn Phe Ser Asp Lys

1775 1780 1785

Gln Asp Ile Ser Ile Asn Lys Ile Ile Ser Thr Phe Thr Pro Ser

1790 1795 1800

Tyr Tyr Val Glu Gly Leu Leu Asn Tyr Asp Leu Gly Leu Ile Ser

1805 1810 1815

Leu Tyr Asn Glu Lys Phe Tyr Ile Asn Asn Leu Gly Met Met Val

1820 1825 1830

Ser Gly Leu Val Tyr Ile Asn Asp Ser Leu Tyr Tyr Phe Lys Pro

1835 1840 1845

Pro Ile Lys Asn Leu Ile Thr Gly Phe Thr Thr Ile Gly Asp Asp

1850 1855 1860

Lys Tyr Tyr Phe Asn Pro Asp Asn Gly Gly Pro Ala Ser Val Gly

1865 1870 1875

Glu Thr Ile Ile Asp Gly Lys Asn Tyr Tyr Phe Ser Gln Asn Gly

1880 1885 1890

Val Leu Gln Thr Gly Val Phe Ser Thr Glu Asp Gly Phe Lys Tyr

1895 1900 1905

Phe Ala Pro Ala Asp Thr Leu Asp Glu Asn Leu Glu Gly Glu Ala

1910 1915 1920

Ile Asp Phe Thr Gly Lys Leu Ile Ile Asp Glu Asn Val Tyr Tyr

1925 1930 1935

Phe Gly Asp Asn Tyr Arg Ala Ala Ile Glu Trp Gln Thr Leu Asp

1940 1945 1950

Asp Glu Val Tyr Tyr Phe Ser Thr Asp Thr Gly Arg Ala Phe Lys

1955 1960 1965

Gly Leu Asn Gln Ile Gly Asp Asp Lys Phe Tyr Phe Asn Ser Asp

1970 1975 1980

Gly Ile Met Gln Lys Gly Phe Val Asn Ile Asn Asp Lys Thr Phe

1985 1990 1995

Tyr Phe Asp Asp Ser Gly Val Met Lys Ser Gly Tyr Thr Glu Ile

2000 2005 2010

Asp Gly Arg Tyr Phe Tyr Phe Ala Glu Asn Gly Glu Met Gln Ile

2015 2020 2025

Gly Val Phe Asn Thr Ala Asp Gly Phe Lys Tyr Phe Ala His His

2030 2035 2040

Asp Glu Asp Leu Gly Asn Glu Glu Gly Glu Ala Leu Ser Tyr Ser

2045 2050 2055

Gly Ile Leu Asn Phe Asn Asn Lys Ile Tyr Tyr Phe Asp Asp Ser

2060 2065 2070

Phe Thr Ala Val Val Gly Trp Lys Asp Leu Glu Asp Gly Ser Lys

2075 2080 2085

Tyr Tyr Phe Asp Glu Asn Thr Ala Glu Ala Ser Ile Gly Ile Ser

2090 2095 2100

Ile Ile Asn Asp Gly Lys Tyr Tyr Phe Asn Asp Ser Gly Ile Met

2105 2110 2115

Gln Ile Gly Phe Val Thr Ile Asn Asn Glu Val Phe Tyr Phe Ser

2120 2125 2130

Asp Ser Gly Ile Val Glu Ser Gly Met Gln Asn Ile Asp Asp Asn

2135 2140 2145

Tyr Phe Tyr Ile Ser Glu Asn Gly Leu Val Gln Ile Gly Val Phe

2150 2155 2160

Asp Thr Ser Asp Gly Tyr Lys Tyr Phe Ala Pro Ala Asn Thr Val

2165 2170 2175

Asn Asp Asn Ile Tyr Gly Gln Ala Val Glu Tyr Ser Gly Leu Val

2180 2185 2190

Arg Val Asn Glu Asp Val Tyr Ser Phe Gly Glu Ser Tyr Thr Ile

2195 2200 2205

Glu Thr Gly Trp Ile Tyr Asp Ser Glu Asn Glu Ser Asp Lys Tyr

2210 2215 2220

Tyr Phe Asp Pro Glu Ala Lys Lys Ala Tyr Lys Gly Ile Asn Val

2225 2230 2235

Ile Asp Asp Ile Lys Tyr Tyr Phe Asp Glu Asn Gly Ile Met Arg

2240 2245 2250

Thr Gly Leu Ile Thr Phe Glu Asp Asn His Tyr Tyr Phe Asn Glu

2255 2260 2265

Asp Gly Glu Met Gln Tyr Gly Tyr Leu Asn Ile Glu Asp Lys Met

2270 2275 2280

Phe Tyr Phe Ser Glu Asp Gly Ile Met Gln Ile Gly Val Phe Asn

2285 2290 2295

Thr Pro Asp Gly Phe Lys Tyr Phe Ala His Gln Asn Thr Leu Asp

2300 2305 2310

Glu Asn Phe Glu Gly Glu Ser Ile Asn Tyr Thr Gly Trp Leu Asp

2315 2320 2325

Leu Asp Glu Lys Arg Tyr Tyr Phe Thr Asp Glu Tyr Ile Ala Ala

2330 2335 2340

Thr Gly Ser Val Ile Ile Asp Gly Glu Glu Tyr Tyr Phe Asp Pro

2345 2350 2355

Asp Thr Ala Gln Leu Val Ile Ser Glu

2360 2365

<210> 10

<211> 2367

<212> PRT

<213> Artificial sequence

<220>

<223> TcdB5

<400> 10

Met Ser Leu Val Asn Arg Lys Gln Leu Glu Lys Met Ala Asn Val Arg

1 5 10 15

Phe Arg Thr Gln Glu Asp Glu Tyr Val Ala Ile Leu Asp Ala Leu Glu

20 25 30

Glu Tyr His Asn Met Ser Glu Asn Thr Val Val Glu Lys Tyr Leu Lys

35 40 45

Leu Lys Asp Ile Asn Ser Leu Thr Asp Ile Tyr Ile Asp Thr Tyr Lys

50 55 60

Lys Ser Gly Arg Asn Lys Ala Leu Lys Lys Phe Lys Glu Tyr Leu Val

65 70 75 80

Thr Glu Val Leu Glu Leu Lys Asn Asn Asn Leu Thr Pro Val Glu Lys

85 90 95

Asn Leu His Phe Val Trp Ile Gly Gly Gln Ile Asn Asp Thr Ala Ile

100 105 110

Asn Tyr Ile Asn Gln Trp Lys Asp Val Asn Ser Asp Tyr Asn Val Asn

115 120 125

Val Phe Tyr Asp Ser Asn Ala Phe Leu Ile Asn Thr Leu Lys Lys Thr

130 135 140

Ile Val Glu Ser Ala Thr Asn Asp Thr Leu Glu Ser Phe Arg Glu Asn

145 150 155 160

Leu Asn Asp Pro Glu Phe Asn His Thr Ala Phe Phe Arg Lys Arg Met

165 170 175

Gln Ile Ile Tyr Asp Lys Gln Gln Asn Phe Ile Asn Tyr Tyr Lys Ala

180 185 190

Gln Lys Glu Glu Asn Pro Asp Leu Ile Ile Asp Asp Thr Val Lys Ser

195 200 205

Tyr Leu Ser Asp Glu Tyr Ser Lys Asp Ile Asp Glu Leu Asn Ala Tyr

210 215 220

Ile Glu Glu Ser Leu Asn Lys Ile Ala Glu Asn Ser Gly Asn Asp Val

225 230 235 240

Arg Asn Phe Glu Glu Phe Lys Asp Gly Glu Val Phe Asn Leu Tyr Glu

245 250 255

Gln Glu Leu Val Glu Arg Trp Asn Leu Ala Ala Ala Ser Asp Ile Leu

260 265 270

Arg Val Ala Ile Leu Lys Asn Ile Gly Gly Val Tyr Leu Asp Val Asp

275 280 285

Met Leu Pro Gly Ile His Pro Asp Leu Phe Lys Asn Ile Asn Lys Pro

290 295 300

Asp Ser Val Lys Thr Ala Val Asp Trp Glu Glu Met Lys Leu Glu Ala

305 310 315 320

Ile Met Lys Tyr Lys Glu Tyr Ile Pro Glu Tyr Thr Ser Lys His Phe

325 330 335

Asp Thr Leu Asp Glu Glu Val Gln Ser Ser Phe Glu Ser Val Leu Ala

340 345 350

Ser Lys Ser Asp Lys Ser Glu Ile Phe Leu Pro Leu Gly Asp Ile Glu

355 360 365

Val Ser Pro Leu Glu Val Lys Ile Ala Phe Ala Lys Gly Ser Ile Ile

370 375 380

Asn Gln Ala Leu Ile Ser Val Lys Asp Ser Tyr Cys Ser Asp Leu Leu

385 390 395 400

Ile Lys Gln Ile Gln Asn Arg Tyr Lys Ile Leu Asn Asp Thr Leu Gly

405 410 415

Pro Ile Ile Ser Gln Gly Asn Asp Phe Asn Thr Thr Met Asn Ser Phe

420 425 430

Gly Glu Ser Leu Gly Ala Ile Ser Ser Glu Asp Asn Ile Ser Phe Ile

435 440 445

Ala Lys Ile Gly Ser Tyr Leu Arg Val Gly Phe Tyr Pro Glu Ala Asn

450 455 460

Thr Thr Ile Thr Leu Ser Gly Pro Thr Val Tyr Ala Gly Ala Tyr Lys

465 470 475 480

Asp Leu Leu Thr Phe Lys Glu Ile Ser Leu Asp Thr Ser Ile Leu Thr

485 490 495

Ser Glu Leu Arg Asn Phe Glu Phe Pro Lys Asp Asn Ile Ser Gln Ala

500 505 510

Thr Glu Gln Glu Lys Asn Ser Leu Trp Gln Phe Asn Glu Glu Arg Ala

515 520 525

Lys Ile Gln Phe Glu Glu Tyr Lys Arg Ala Tyr Phe Glu Gly Ala Leu

530 535 540

Gly Glu Asp Asp Asn Leu Asp Phe Ser Gln Asn Thr Val Thr Asp Lys

545 550 555 560

Glu Tyr Leu Leu Glu Lys Ile Ser Ser Ser Thr Lys Ser Ser Glu Arg

565 570 575

Gly Tyr Val His Tyr Ile Val Gln Leu Gln Gly Asp Lys Ile Ser Tyr

580 585 590

Glu Ala Ala Cys Asn Leu Phe Ala Lys Asn Pro Tyr Asp Ser Ile Leu

595 600 605

Phe Gln Lys Asn Ile Glu Asp Ser Glu Ile Ala Tyr Tyr Tyr Asn Pro

610 615 620

Ala Asp Gly Glu Ile Gln Glu Ile Asp Lys Tyr Arg Ile Pro Asp Arg

625 630 635 640

Ile Ser Asp Arg Pro Lys Ile Lys Leu Thr Phe Ile Gly His Gly Lys

645 650 655

Asp Glu Phe Asn Thr Asp Ile Phe Ala Gly Leu Asp Val Asp Ser Leu

660 665 670

Ser Thr Glu Ile Glu Thr Ala Ile Asp Leu Ala Lys Glu Asp Ile Ser

675 680 685

Ser Lys Ser Ile Glu Ile Asn Leu Leu Gly Cys Asn Met Phe Ser Tyr

690 695 700

Ser Ile Asn Val Glu Glu Thr Tyr Pro Gly Lys Leu Leu Leu Lys Val

705 710 715 720

Lys Asp Lys Ile Ser Glu Leu Met Pro Ser Ile Ser Gln Asp Ser Ile

725 730 735

Ile Val Ser Ala Asn Gln Tyr Glu Val Arg Ile Asn Ser Glu Gly Arg

740 745 750

Arg Glu Leu Leu Asp His Ser Gly Glu Trp Ile Asn Lys Glu Glu Ser

755 760 765

Ile Ile Lys Asp Ile Ser Ser Lys Glu Tyr Ile Ser Phe Asn Pro Lys

770 775 780

Glu Asn Lys Ile Thr Val Lys Ser Lys Asn Leu Pro Glu Leu Ser Thr

785 790 795 800

Leu Leu Gln Glu Ile Arg Asn Asn Ser Asn Leu Ser Asp Ile Glu Leu

805 810 815

Glu Glu Lys Val Met Leu Ala Glu Cys Glu Ile Asn Val Ile Ser Asn

820 825 830

Ile Asp Thr Gln Ile Val Glu Glu Arg Ile Glu Glu Ala Lys Asn Leu

835 840 845

Thr Ser Asp Ser Ile Asn Tyr Ile Lys Asn Glu Phe Lys Leu Ile Glu

850 855 860

Ser Ile Ser Asp Ser Leu Tyr Asp Leu Lys Gln Gln Asn Glu Leu Asp

865 870 875 880

Asp Ser His Phe Ile Ser Phe Glu Asp Ile Ser Lys Thr Glu Asp Gly

885 890 895

Phe Ser Ile Arg Phe Ile Asn Lys Glu Thr Gly Glu Ser Ile Phe Val

900 905 910

Glu Thr Glu Lys Glu Ile Phe Ser Glu Tyr Ala Asn His Ile Glu Arg

915 920 925

Glu Ile Ser Asn Ile Lys Asp Thr Ile Phe Asp Thr Val Asn Gly Lys

930 935 940

Leu Val Lys Lys Val Asn Leu Asp Ala Ile His Glu Val Asn Thr Leu

945 950 955 960

Asn Ala Ala Phe Phe Ile Gln Ser Leu Ile Gly Tyr Ser Ser Ser Lys

965 970 975

Glu Ser Leu Ser Asn Leu Ser Val Ala Met Lys Val Gln Val Tyr Ala

980 985 990

Gln Leu Phe Ser Thr Gly Leu Asn Thr Ile Thr Asp Ala Ala Lys Val

995 1000 1005

Val Glu Leu Val Ser Thr Ala Leu Asp Glu Thr Ile Asp Leu Leu

1010 1015 1020

Pro Thr Leu Ser Glu Gly Leu Pro Val Ile Ala Thr Ile Ile Asp

1025 1030 1035

Gly Val Ser Leu Gly Ala Ala Ile Lys Glu Leu Ser Glu Thr Ser

1040 1045 1050

Asp Pro Leu Leu Arg Gln Glu Ile Glu Ala Lys Ile Gly Ile Met

1055 1060 1065

Ala Val Asn Leu Thr Ala Ala Thr Thr Ala Ile Ile Thr Ser Ser

1070 1075 1080

Leu Gly Ile Ala Ser Gly Phe Ser Ile Leu Leu Val Pro Leu Ala

1085 1090 1095

Gly Ile Ser Ala Gly Ile Pro Ser Leu Val Asn Asn Glu Leu Val

1100 1105 1110

Leu Arg Asp Lys Ala Thr Lys Val Val Asp Tyr Phe Lys His Ile

1115 1120 1125

Ser Leu Val Glu Thr Glu Gly Ala Phe Thr Leu Leu Asp Asp Lys

1130 1135 1140

Ile Met Ile Pro Gln Asp Asp Leu Val Ile Ser Glu Ile Asp Phe

1145 1150 1155

Asn Asn Asn Ser Ile Val Leu Gly Lys Cys Glu Ile Trp Arg Met

1160 1165 1170

Glu Gly Gly Ser Gly His Thr Val Thr Asn Asp Ile Asp His Phe

1175 1180 1185

Phe Ser Ser Pro Thr Ile Thr Tyr Ile Lys Pro His Leu Ser Ile

1190 1195 1200

Tyr Asp Val Leu Glu Val Gln Lys Glu Glu Leu Asp Leu Ser Lys

1205 1210 1215

Asp Leu Met Val Leu Pro Asn Ala Pro Asn Arg Val Phe Ala Trp

1220 1225 1230

Glu Thr Gly Trp Thr Pro Gly Leu Arg Ser Leu Glu Asn Glu Gly

1235 1240 1245

Thr Lys Leu Leu Asp Arg Ile Arg Asp His Tyr Lys Gly Glu Phe

1250 1255 1260

Tyr Trp Arg Tyr Phe Ala Phe Ile Ala Asp Ala Leu Ile Thr Thr

1265 1270 1275

Leu Lys Pro Arg Tyr Glu Asp Thr Asn Ile Arg Ile Asn Leu Asp

1280 1285 1290

Ser Asn Asn Arg Ser Phe Ile Val Pro Ile Ile Thr Thr Glu His

1295 1300 1305

Ile Arg Glu Lys Leu Ser Tyr Ser Phe His Gly Ser Gly Gly Thr

1310 1315 1320

Tyr Ala Leu Ser Leu Ser Gln Tyr Asn Met Gly Ile Asn Ile Glu

1325 1330 1335

Leu Ser Glu Ser Asp Val Trp Ile Ile Asp Val Asp Asn Val Val

1340 1345 1350

Arg Asp Val Thr Ile Asp Ser Asp Lys Ile Lys Lys Gly Asp Leu

1355 1360 1365

Ile Glu Gly Ile Leu Ser Thr Leu Ser Ile Glu Asp Asn Lys Ile

1370 1375 1380

Ile Leu Asn His His Glu Ile Asn Phe Ser Gly Asp Val Asn Gly

1385 1390 1395

Ser Asn Gly Phe Ile Ser Leu Thr Phe Ser Ile Leu Glu Gly Ile

1400 1405 1410

Asn Ala Ile Ile Glu Val Asp Leu Leu Ser Lys Ser Tyr Lys Leu

1415 1420 1425

Leu Ile Ser Gly Glu Leu Lys Ile Leu Met Leu Asn Ser Asn His

1430 1435 1440

Ile Gln Gln Lys Ile Asp Tyr Ile Gly Phe Asn Ser Glu Leu Gln

1445 1450 1455

Lys Asn Ile Pro Tyr Ser Phe Val Asp Ser Glu Gly Lys Glu Asn

1460 1465 1470

Gly Phe Ile Asn Gly Ser Thr Lys Glu Gly Leu Phe Val Ser Glu

1475 1480 1485

Leu Pro Asp Val Val Leu Ile Ser Lys Val Tyr Met Asp Asp Ser

1490 1495 1500

Lys Pro Ser Phe Gly Tyr Tyr Ser Asn Asn Leu Lys Asp Val Lys

1505 1510 1515

Val Ile Thr Lys Asp Asn Val Asn Ile Leu Thr Gly Tyr Tyr Leu

1520 1525 1530

Lys Asp Asp Ile Lys Ile Ser Leu Ser Phe Thr Leu Gln Asp Glu

1535 1540 1545

Lys Thr Ile Lys Leu Asn Gly Val His Leu Asp Glu Ser Gly Val

1550 1555 1560

Ala Glu Ile Leu Lys Phe Met Asn Lys Lys Gly Ser Thr Asn Thr

1565 1570 1575

Ser Asp Ser Leu Met Ser Phe Leu Glu Ser Val Asn Ile Lys Ser

1580 1585 1590

Ile Phe Val Asn Phe Leu Gln Ser Lys Ile Asn Phe Ile Leu Asp

1595 1600 1605

Ala Asn Phe Ile Ile Ser Gly Thr Thr Ser Ile Gly Gln Phe Glu

1610 1615 1620

Phe Ile Cys Asp Glu Asn Asp Asn Ile Gln Pro Tyr Phe Ile Lys

1625 1630 1635

Phe Asn Thr Leu Glu Thr Thr Tyr Thr Leu Tyr Val Gly Asn Arg

1640 1645 1650

Gln Asn Met Ile Val Glu Pro Asn Tyr Asp Leu Asp Asp Ser Gly

1655 1660 1665

Asp Ile Ser Ser Thr Val Ile Asn Phe Ser Gln Lys Tyr Leu Tyr

1670 1675 1680

Gly Ile Asp Ser Cys Val Asn Lys Val Val Ile Ser Pro Asn Ile

1685 1690 1695

Tyr Thr Asp Glu Ile Asn Ile Thr Pro Val Tyr Glu Thr Asn Asn

1700 1705 1710

Asn Tyr Pro Glu Val Ile Val Leu Asp Ala Asn Tyr Ile Asn Glu

1715 1720 1725

Lys Ile Asn Val Asn Ile Asn Asp Leu Ser Ile Arg Tyr Val Trp

1730 1735 1740

Ser Asn Asp Gly Asn Asp Phe Ile Leu Met Ser Thr Ser Glu Glu

1745 1750 1755

Asn Lys Val Ser Gln Val Lys Ile Arg Phe Val Asn Val Phe Lys

1760 1765 1770

Asp Lys Thr Leu Ala Asn Lys Leu Ser Phe Asn Phe Ser Asp Lys

1775 1780 1785

Gln Asp Val Pro Val Ser Glu Ile Ile Ser Ala Phe Thr Pro Ser

1790 1795 1800

Tyr Tyr Glu Asp Gly Leu Ile Gly Tyr Asp Leu Gly Leu Val Ser

1805 1810 1815

Leu Tyr Asn Glu Lys Phe Tyr Ile Asn Asn Phe Gly Met Met Val

1820 1825 1830

Ser Gly Leu Ile Tyr Ile Asn Asp Ser Leu Tyr Tyr Phe Lys Pro

1835 1840 1845

Pro Val Asn Asn Leu Ile Thr Gly Phe Val Thr Val Gly Asp Asp

1850 1855 1860

Lys Tyr Tyr Phe Asn Pro Thr Asn Gly Gly Ala Ala Ser Ile Gly

1865 1870 1875

Glu Thr Ile Ile Asp Asp Lys Asn Tyr Tyr Phe Asn Gln Ser Gly

1880 1885 1890

Ile Leu Gln Thr Gly Val Phe Ser Thr Glu Asp Gly Leu Lys Tyr

1895 1900 1905

Phe Ala Pro Ala Asn Thr Leu Asp Glu Asn Leu Glu Gly Glu Ala

1910 1915 1920

Ile Asp Phe Thr Gly Lys Leu Ile Ile Asp Glu Asn Ile Tyr Tyr

1925 1930 1935

Phe Glu Asp Asn Tyr Arg Gly Ala Val Glu Trp Lys Glu Leu Asp

1940 1945 1950

Gly Glu Met Tyr Tyr Phe Ser Pro Glu Thr Gly Lys Ala Phe Lys

1955 1960 1965

Gly Leu Asn Gln Ile Gly Asp Asp Lys Tyr Tyr Phe Asn Ser Asp

1970 1975 1980

Gly Ile Met Lys Lys Gly Phe Val Ser Ile Asn Asp Lys Lys Tyr

1985 1990 1995

Tyr Phe Asp Asp Ser Gly Val Met Lys Val Gly Tyr Ile Glu Ile

2000 2005 2010

Asp Gly Lys Tyr Phe Tyr Phe Ala Glu Asn Gly Glu Met Gln Ile

2015 2020 2025

Gly Val Phe Asn Thr Ser Asp Gly Phe Lys Tyr Phe Ala His His

2030 2035 2040

Asn Glu Asp Leu Gly Asn Glu Glu Gly Glu Ala Ile Ser Tyr Ser

2045 2050 2055

Gly Ile Leu Asn Phe Asn Asn Lys Ile Tyr Tyr Phe Asp Tyr Ser

2060 2065 2070

Phe Thr Ala Val Val Gly Trp Lys Asp Leu Glu Asp Gly Ser Lys

2075 2080 2085

Tyr Tyr Phe Asp Glu Asp Thr Ala Glu Ala Tyr Val Gly Leu Ser

2090 2095 2100

Leu Ile Asn Asp Gly Gln Tyr Tyr Phe Asn Asp Asp Gly Ile Met

2105 2110 2115

Gln Val Gly Phe Val Thr Ile Asn Asn Lys Val Phe Tyr Phe Ser

2120 2125 2130

Asp Ser Gly Ile Ile Glu Ser Gly Val Gln Asn Ile Asp Asp Asn

2135 2140 2145

Tyr Phe Tyr Ile Asp Glu Lys Gly Ile Val Gln Ile Gly Val Phe

2150 2155 2160

Asp Thr Ser Asp Gly Tyr Lys Tyr Phe Ala Pro Ala Asn Thr Val

2165 2170 2175

Asn Asp Asn Ile Tyr Gly Gln Ala Val Asp Tyr Ser Gly Leu Val

2180 2185 2190

Arg Val Gly Glu Asp Ile Tyr Tyr Phe Gly Glu Thr Tyr Thr Ile

2195 2200 2205

Glu Thr Gly Trp Ile Tyr Asp Met Glu Asn Glu Ser Asp Lys Tyr

2210 2215 2220

Tyr Phe Asn Pro Glu Thr Lys Lys Ala Cys Lys Gly Ile Asn Leu

2225 2230 2235

Ile Asp Asp Ile Lys Tyr Tyr Phe Asp Glu Asn Gly Ile Met Arg

2240 2245 2250

Thr Gly Leu Ile Ser Phe Glu Asn Asn Asp Tyr Tyr Phe Asn Glu

2255 2260 2265

Asn Gly Glu Met His Phe Gly Tyr Ile Asn Ile Glu Asp Lys Met

2270 2275 2280

Phe Tyr Phe Gly Glu Asp Gly Val Met Gln Ile Gly Val Phe Asn

2285 2290 2295

Thr Pro Asp Gly Phe Lys Tyr Phe Ala His Gln Asn Thr Leu Asp

2300 2305 2310

Glu Asn Phe Glu Gly Glu Ser Ile Asn Tyr Thr Gly Trp Leu Asp

2315 2320 2325

Leu Glu Gly Lys Arg Tyr Tyr Phe Thr Asp Glu Tyr Ile Ala Ala

2330 2335 2340

Thr Gly Thr Val Thr Ile Asp Gly Glu Glu Tyr Tyr Phe Asp Pro

2345 2350 2355

Asp Thr Ala Glu Leu Val Val Ser Glu

2360 2365

<210> 11

<211> 2366

<212> PRT

<213> Artificial sequence

<220>

<223> TcdB6

<400> 11

Met Ser Leu Ile Asn Arg Lys Gln Leu Glu Lys Met Ala Asn Val Lys

1 5 10 15

Phe Arg Val Gln Glu Asp Glu Tyr Ile Ala Ile Leu Asp Ala Leu Glu

20 25 30

Glu Tyr His Asn Met Ser Glu Asn Thr Val Val Glu Lys Tyr Leu Lys

35 40 45

Leu Lys Asp Ile Asn Ser Leu Thr Glu Thr Tyr Ile Asp Thr Tyr Lys

50 55 60

Lys Ser Gly Arg Asn Lys Ala Leu Lys Lys Phe Lys Glu Tyr Leu Val

65 70 75 80

Thr Glu Val Leu Glu Leu Lys Asn Ser Asn Val Ala Pro Val Glu Lys

85 90 95

Asn Leu His Phe Val Trp Ile Gly Gly Lys Ile Asn Asp Thr Ala Ile

100 105 110

Asn Tyr Ile Asn Gln Trp Lys Asp Val Asn Ser Asp Tyr Asn Val Asn

115 120 125

Val Phe Tyr Asp Ser Asn Ala Phe Leu Ile Asn Thr Leu Lys Lys Thr

130 135 140

Ile Val Asp Ser Ala Thr Asn Glu Thr Leu Glu Ser Phe Arg Glu Asn

145 150 155 160

Leu Asp Asp Pro Arg Phe Asp Tyr Asn Lys Phe Tyr Arg Lys Arg Met

165 170 175

Glu Ile Ile Tyr Asp Lys Gln Lys Asn Phe Ile Asn Tyr Tyr Lys Ala

180 185 190

Gln Arg Glu Glu Asn Pro Asp Leu Ile Ile Asp Asp Ile Val Lys Thr

195 200 205

Tyr Leu Ser Asn Glu Tyr Ser Lys Asp Ile Asp Glu Leu Asn Ala Tyr

210 215 220

Ile Glu Glu Ser Leu Ser Lys Val Thr Glu Asn Ser Gly Asn Asp Val

225 230 235 240

Arg Asn Phe Glu Glu Phe Lys Gly Gly Glu Ser Phe Asn Leu Tyr Glu

245 250 255

Gln Glu Leu Val Glu Arg Trp Asn Leu Ala Ala Ala Ser Asp Ile Leu

260 265 270

Arg Val Ser Ala Leu Lys Glu Val Gly Gly Val Tyr Leu Asp Val Asp

275 280 285

Met Leu Pro Gly Ile Gln Pro Asp Leu Phe Glu Ser Ile Glu Lys Pro

290 295 300

Ser Ser Val Thr Val Asp Phe Trp Glu Met Val Lys Leu Glu Ala Ile

305 310 315 320

Met Lys Tyr Lys Glu Tyr Ile Pro Gly Tyr Thr Ser Glu His Phe Asp

325 330 335

Met Leu Asp Glu Glu Val Gln Ser Ser Phe Glu Ser Val Leu Ala Ser

340 345 350

Lys Ser Asp Lys Ser Glu Ile Phe Ser Ser Leu Gly Asp Val Glu Ser

355 360 365

Ser Pro Leu Glu Val Lys Ile Ala Phe Asn Ser Lys Gly Ile Ile Asn

370 375 380

Gln Gly Leu Ile Ser Val Lys Asp Ser Tyr Cys Ser Asn Leu Ile Val

385 390 395 400

Lys Gln Ile Glu Asn Arg Tyr Lys Ile Leu Asn Asn Ser Leu Asn Pro

405 410 415

Ala Ile Ser Glu Asp Asn Asp Phe Asn Thr Thr Thr Asn Thr Phe Ile

420 425 430

Asp Ser Ile Met Ala Glu Ala Asn Ala Asp Asn Ser Arg Phe Met Met

435 440 445

Glu Leu Gly Lys Tyr Leu Arg Val Gly Phe Phe Pro Asp Val Lys Thr

450 455 460

Thr Ile Asn Leu Ser Gly Pro Glu Ala Tyr Ala Ala Ala Tyr Gln Asp

465 470 475 480

Leu Leu Met Phe Lys Glu Tyr Ser Met Asn Ile His Leu Leu Glu Ser

485 490 495

Asp Leu Arg Asn Phe Glu Ile Ser Lys Thr Asn Ile Ser Gln Ser Thr

500 505 510

Glu Gln Glu Met Ala Ser Leu Trp Ser Phe Asp Asp Ala Arg Ala Lys

515 520 525

Ala Gln Phe Gln Glu Tyr Lys Lys Asn Tyr Phe Glu Gly Ala Leu Gly

530 535 540

Glu Asp Asp Asn Leu Asp Phe Ser Glu Asn Thr Val Leu Asp Lys Asp

545 550 555 560

Tyr Ile Leu Glu Lys Ile Ser Ser Ser Thr Arg Ser Ser Glu Arg Gly

565 570 575

Tyr Val His Tyr Ile Val Gln Leu Gln Gly Asp Lys Ile Ser Tyr Glu

580 585 590

Ala Ala Cys Asn Leu Phe Ala Lys Asn Pro Tyr Asp Ser Ile Leu Phe

595 600 605

Gln Asn Asn Ile Glu Asp Ser Glu Ile Ala Tyr Tyr Tyr Asn Pro Ala

610 615 620

Asp Gly Glu Ile Gln Glu Ile Asp Lys Tyr Arg Ile Pro Asp Ile Ile

625 630 635 640

Ser Asp Arg Pro Lys Val Lys Leu Thr Phe Ile Gly His Gly Lys Ser

645 650 655

Glu Phe Asn Thr Asp Ile Phe Ala Asn Leu Asp Val Asp Ser Leu Ser

660 665 670

Ser Glu Ile Glu Thr Val Ile Asp Leu Ala Lys Thr Asp Ile Ser Pro

675 680 685

Lys Ala Ile Glu Ile Asn Leu Leu Gly Cys Asn Met Phe Ser Tyr Ser

690 695 700

Ile Asn Val Glu Asp Thr Tyr Pro Gly Lys Leu Leu Leu Lys Val Lys

705 710 715 720

Asp Lys Val Ser Glu Leu Leu Pro Ser Ile Asn Gln Asp Ser Ile Ile

725 730 735

Val Ser Ala Asn Gln Tyr Glu Val Arg Ile Asn Ser Glu Gly Arg Arg

740 745 750

Glu Leu Leu Asp His Ser Gly Glu Trp Ile Asn Lys Glu Glu Ser Ile

755 760 765

Ile Lys Asp Ile Ser Ser Lys Glu Tyr Ile Ser Phe Asn Pro Gln Glu

770 775 780

Asn Lys Ile Ile Val Lys Ser Lys Asn Leu Pro Glu Leu Ser Thr Leu

785 790 795 800

Val Gln Glu Ile Arg Asn Asn Ser Asn Ser Gly Asp Ile Glu Leu Glu

805 810 815

Glu Lys Val Met Leu Ala Glu Cys Glu Ile Ser Val Val Ser Asp Ile

820 825 830

Asp Thr Gln Val Val Glu Glu Arg Ile Glu Glu Ala Lys Asn Leu Thr

835 840 845

Ser Asp Ser Ile Asn Tyr Ile Lys Asn Glu Phe Lys Leu Ile Glu Ser

850 855 860

Ile Ser Asp Ala Leu Tyr Asp Leu Lys Gln Gln Asn Glu Leu Glu Asp

865 870 875 880

Ser His Phe Ile Ser Phe Glu Asp Ile Ser Glu Thr Asp Glu Gly Phe

885 890 895

Ser Ile Arg Phe Ile Asp Lys Glu Thr Gly Glu Ser Ile Phe Val Glu

900 905 910

Thr Glu Lys Thr Ile Phe Ser Glu Tyr Ala Asn His Ile Thr Glu Glu

915 920 925

Ile Thr Lys Val Lys Asp Thr Ile Phe Asp Thr Val Asn Gly Lys Leu

930 935 940

Val Lys Lys Val Asn Leu Asp Ala Thr His Glu Val Asn Thr Leu Asn

945 950 955 960

Ala Ala Phe Phe Ile Gln Ser Leu Ile Gly Tyr Asn Ser Ser Lys Glu

965 970 975

Ser Leu Ser Asn Leu Ser Val Ala Met Lys Val Gln Val Tyr Ala Gln

980 985 990

Leu Phe Ser Thr Gly Leu Asn Thr Ile Thr Asp Ala Ala Lys Val Val

995 1000 1005

Glu Leu Val Ser Thr Ala Leu Asp Glu Thr Ile Asp Leu Leu Pro

1010 1015 1020

Thr Leu Ser Glu Gly Leu Pro Ile Ile Ala Thr Ile Ile Asp Gly

1025 1030 1035

Val Ser Leu Gly Ala Ser Ile Lys Glu Leu Ser Glu Thr Ser Asp

1040 1045 1050

Pro Leu Leu Arg Gln Glu Ile Glu Ala Lys Ile Gly Ile Met Ala

1055 1060 1065

Val Asn Leu Thr Ala Ala Thr Thr Ala Ile Ile Thr Ser Ala Leu

1070 1075 1080

Gly Ile Ala Ser Gly Phe Ser Ile Leu Leu Val Pro Leu Ala Gly

1085 1090 1095

Ile Ser Ala Gly Val Pro Ser Leu Val Asn Asn Glu Leu Val Leu

1100 1105 1110

Arg Asp Lys Ala Thr Lys Val Val Asp Tyr Phe Ser His Ile Ser

1115 1120 1125

Leu Ala Glu Ser Glu Gly Ala Phe Thr Leu Leu Asp Asp Lys Ile

1130 1135 1140

Met Met Leu Gln Asp Asp Leu Val Ile Ser Glu Ile Asp Phe Asn

1145 1150 1155

Asn Asn Ser Ile Thr Leu Gly Lys Cys Glu Ile Trp Arg Met Glu

1160 1165 1170

Gly Gly Ser Gly His Thr Val Val Asp Asp Ile Asp His Phe Phe

1175 1180 1185

Ser Ser Pro Pro Ile Thr Tyr Arg Glu Pro His Leu Ser Ile Tyr

1190 1195 1200

Asp Val Leu Glu Val Lys Lys Glu Glu Leu Asp Leu Ser Lys Asp

1205 1210 1215

Leu Met Val Leu Pro Asn Ala Pro Asn Arg Val Phe Gly Trp Glu

1220 1225 1230

Thr Gly Trp Thr Pro Gly Leu Arg Gly Leu Glu Asn Asp Gly Thr

1235 1240 1245

Lys Leu Leu Asp Arg Ile Arg Asp Tyr Tyr Glu Gly Gln Phe Tyr

1250 1255 1260

Trp Arg Phe Tyr Ala Phe Val Ala Asp Ala Leu Ile Thr Thr Leu

1265 1270 1275

Lys Pro Arg Tyr Glu Asp Thr Asn Val Arg Ile Ser Leu Asp Ser

1280 1285 1290

Asn Thr Arg Ser Phe Ile Val Pro Val Ile Thr Thr Glu Tyr Ile

1295 1300 1305

Arg Glu Lys Leu Ser Tyr Ser Phe Tyr Gly Ser Gly Gly Thr Tyr

1310 1315 1320

Ala Leu Ser Leu Ser Gln Tyr Asn Met Asn Ile Asn Ile Glu Leu

1325 1330 1335

Asn Glu Asn Asp Thr Trp Val Ile Asp Val Asp Asn Val Val Arg

1340 1345 1350

Asp Val Thr Ile Glu Ser Asp Lys Ile Lys Lys Gly Asp Leu Ile

1355 1360 1365

Glu Asn Ile Leu Ser Lys Leu Ser Ile Glu Glu Asn Lys Ile Ile

1370 1375 1380

Leu Asp Asn His Glu Ile Asn Phe Ser Gly Thr Leu Asn Gly Gly

1385 1390 1395

Asn Gly Phe Val Ser Leu Thr Phe Ser Ile Leu Glu Gly Ile Asn

1400 1405 1410

Ala Val Ile Glu Val Asp Leu Leu Ser Lys Ser Tyr Lys Val Leu

1415 1420 1425

Val Ser Gly Glu Leu Lys Thr Leu Met Leu Asn Ser Asn Ser Val

1430 1435 1440

Gln Gln Lys Ile Asp Tyr Ile Gly Leu Asn Ser Glu Val Gln Lys

1445 1450 1455

Asn Ile Pro Tyr Ser Phe Thr Asp Asp Lys Gly Lys Glu Asn Gly

1460 1465 1470

Phe Ile Asn Cys Ser Thr Lys Glu Gly Leu Phe Ile Ser Glu Leu

1475 1480 1485

Ser Asp Val Val Leu Ile Ser Lys Val Tyr Met Asp Asp Ser Lys

1490 1495 1500

Pro Ser Phe Gly Tyr Tyr Ser Asp Asp Leu Lys Asp Val Lys Val

1505 1510 1515

Ile Thr Lys Asp Asp Val Ile Leu Leu Thr Gly Tyr Tyr Leu Lys

1520 1525 1530

Asp Asp Ile Lys Ile Ser Leu Ser Phe Thr Ile Gln Asp Glu Asn

1535 1540 1545

Thr Ile Lys Leu Asn Gly Val Tyr Leu Asp Glu Asn Gly Val Ala

1550 1555 1560

Glu Ile Leu Lys Phe Met Asn Lys Lys Gly Ser Thr Asn Thr Ser

1565 1570 1575

Asp Ser Leu Met Ser Phe Leu Glu Ser Met Asn Ile Lys Ser Ile

1580 1585 1590

Phe Met Asn Phe Val Gln Ser Asn Val Lys Leu Ile Leu Asp Thr

1595 1600 1605

Asn Phe Ile Ile Asn Gly Thr Thr Ser Ile Gly Gln Phe Glu Phe

1610 1615 1620

Ile Cys Asp Lys Asp Asn Asn Ile Gln Pro Tyr Phe Ile Lys Phe

1625 1630 1635

Asn Thr Leu Glu Thr Lys Tyr Thr Leu Tyr Val Gly Asn Arg Glu

1640 1645 1650

Asn Met Ile Val Glu Pro Asn Tyr Asn Leu Asp Asp Ser Gly Asp

1655 1660 1665

Ile Ser Ser Thr Val Ile Asn Phe Ser Gln Lys Tyr Leu Tyr Gly

1670 1675 1680

Ile Asp Ser Cys Ile Asn Lys Val Ile Ile Ser Pro Asn Ile Tyr

1685 1690 1695

Thr Asp Glu Ile Asn Ile Thr Pro Val Tyr Glu Ala Asn Asn Thr

1700 1705 1710

Tyr Pro Glu Val Ile Val Leu Asp Ala Asn Tyr Ile Ser Glu Lys

1715 1720 1725

Ile Asn Ile Asn Ile Asn Asp Leu Ser Ile Arg Tyr Val Trp Ser

1730 1735 1740

Asn Asp Gly Ser Asp Phe Ile Leu Met Ser Thr Asn Glu Glu Asp

1745 1750 1755

Lys Val Ser Gln Ile Lys Ile Arg Phe Thr Asn Val Phe Lys Gly

1760 1765 1770

Asn Thr Met Ser Asp Lys Leu Ser Phe Asn Phe Ser Asp Lys Gln

1775 1780 1785

Asp Val Ser Ile Ser Lys Ile Ile Ser Thr Phe Thr Pro Ser Tyr

1790 1795 1800

Tyr Arg Glu Asn Leu Leu Asn Tyr Asp Leu Gly Met Ile Ser Leu

1805 1810 1815

Tyr Asn Glu Lys Phe Tyr Ile Asn Asn Phe Gly Met Met Val Ser

1820 1825 1830

Gly Leu Val Tyr Ile Asn Asp Ser Leu Tyr Tyr Phe Lys Pro Pro

1835 1840 1845

Leu Lys Asn Leu Ile Thr Gly Phe Thr Thr Ile Gly Asp Asp Lys

1850 1855 1860

Tyr Tyr Phe Asn Pro Asp Asn Gly Gly Ala Ala Ser Val Gly Glu

1865 1870 1875

Thr Ile Ile Asp Gly Lys Asn Tyr Tyr Phe Ser Gln Asn Gly Val

1880 1885 1890

Leu Gln Thr Gly Val Phe Ser Thr Glu Asp Gly Phe Lys Tyr Phe

1895 1900 1905

Ala Pro Ala Asn Thr Leu Asp Glu Asn Leu Glu Gly Glu Ala Ile

1910 1915 1920

Asp Phe Thr Gly Lys Leu Thr Ile Asp Glu Asn Val Tyr Tyr Phe

1925 1930 1935

Gly Asp Asn Tyr Arg Ala Ala Ile Glu Trp Gln Thr Leu Asp Asp

1940 1945 1950

Glu Met Tyr Tyr Phe Ser Thr Glu Thr Gly Arg Ala Phe Lys Gly

1955 1960 1965

Leu Asn Gln Ile Gly Asp Asp Lys Phe Tyr Phe Asn Ser Ser Gly

1970 1975 1980

Ile Met Gln Lys Gly Phe Val Asn Ile Asn Asp Lys Thr Phe Tyr

1985 1990 1995

Phe Asp Asp Ser Gly Val Met Lys Ser Gly Tyr Ile Glu Ile Asp

2000 2005 2010

Gly Lys Tyr Phe Tyr Phe Ala Glu Asn Gly Glu Met Gln Ile Gly

2015 2020 2025

Val Phe Asn Thr Thr Asp Gly Phe Lys Tyr Phe Ala His Gln Asp

2030 2035 2040

Glu Asp Leu Gly Asn Glu Glu Gly Glu Ala Leu Ser Tyr Ser Gly

2045 2050 2055

Ile Leu Asn Phe Asn Asn Lys Ile Tyr Tyr Phe Asp Asp Ser Phe

2060 2065 2070

Thr Ala Val Val Gly Trp Lys Asp Leu Glu Asp Gly Ser Lys Tyr

2075 2080 2085

Tyr Phe Asp Glu Asp Thr Ala Glu Ala Tyr Ile Gly Ile Ser Thr

2090 2095 2100

Ile Asn Asp Gly Gln Tyr Tyr Phe Asn Asp Ser Gly Ile Met Gln

2105 2110 2115

Ile Gly Phe Val Thr Ile Asn Asp Lys Val Phe Tyr Phe Ser Asp

2120 2125 2130

Ser Gly Ile Val Glu Ser Gly Met Gln Asn Ile Asp Asp Asn Tyr

2135 2140 2145

Phe Tyr Ile Asp Asp Asn Gly Leu Val Gln Ile Gly Val Phe Asp

2150 2155 2160

Thr Ser Asp Gly Tyr Lys Tyr Phe Ala Pro Ala Asn Thr Val Asn

2165 2170 2175

Asp Asn Ile Tyr Gly Gln Ala Val Glu Cys Ser Gly Leu Val Arg

2180 2185 2190

Val Gly Glu Asp Val Tyr Cys Phe Gly Glu Ser Tyr Thr Ile Glu

2195 2200 2205

Thr Gly Trp Ile Tyr Asp Met Glu Asn Glu Ser Asp Lys Tyr Tyr

2210 2215 2220

Phe Asp Ser Glu Thr Lys Lys Ala Tyr Lys Gly Ile Asn Val Ile

2225 2230 2235

Asp Asp Ile Lys Tyr Tyr Phe Asp Glu Asn Gly Ile Met Arg Thr

2240 2245 2250

Gly Leu Ile Ser Phe Glu Asn Asn His Tyr Tyr Phe Asn Ala Asp

2255 2260 2265

Gly Glu Met Gln Tyr Gly Tyr Leu Asn Ile Glu Asp Lys Met Phe

2270 2275 2280

Tyr Phe Ser Glu Asp Gly Phe Met Gln Ile Gly Val Phe Asn Thr

2285 2290 2295

Pro Asp Gly Phe Lys Tyr Phe Ala His Gln Ser Thr Leu Asp Glu

2300 2305 2310

Asn Phe Glu Gly Glu Ser Ile Asn Tyr Thr Gly Trp Leu Asp Leu

2315 2320 2325

Asp Asp Lys Lys Tyr Tyr Phe Thr Asp Glu Tyr Ile Ala Ala Thr

2330 2335 2340

Gly Ser Val Ile Ile Asp Asp Glu Glu Tyr Tyr Phe Asp Pro Glu

2345 2350 2355

Thr Ala Glu Leu Val Val Ser Glu

2360 2365

<210> 12

<211> 2366

<212> PRT

<213> Artificial sequence

<220>

<223> TcdB7

<400> 12

Met Ser Leu Val Asn Arg Lys Gln Leu Glu Lys Met Ala Asn Val Arg

1 5 10 15

Phe Arg Val Gln Glu Asp Glu Tyr Val Ala Ile Leu Asp Ala Leu Glu

20 25 30

Glu Tyr His Asn Met Ser Glu Asn Thr Val Val Glu Lys Tyr Leu Lys

35 40 45

Leu Lys Asp Ile Asn Ser Leu Thr Asp Thr Tyr Ile Asp Thr Tyr Lys

50 55 60

Lys Ser Gly Arg Asn Lys Ala Leu Lys Lys Phe Lys Glu Tyr Leu Val

65 70 75 80

Thr Glu Val Leu Glu Leu Lys Ser Ser Asn Val Val Pro Val Glu Lys

85 90 95

Asn Leu His Phe Val Trp Ile Gly Gly Lys Ile Asn Asp Thr Ala Ile

100 105 110

Asn Tyr Ile Asn Gln Trp Lys Asp Val Asn Ser Asp Tyr Asn Val Asn

115 120 125

Val Phe Tyr Asp Ser Asn Ala Phe Leu Ile Asn Thr Leu Lys Lys Thr

130 135 140

Ile Val Asp Ser Ala Thr Asn Glu Thr Leu Glu Ser Phe Arg Glu Asn

145 150 155 160

Leu Asp Asp Pro Arg Phe Asp Tyr Asn Lys Phe Tyr Arg Lys Arg Met

165 170 175

Glu Ile Ile Tyr Asp Lys Gln Lys Asn Phe Ile Asn Tyr Tyr Lys Ala

180 185 190

Gln Arg Glu Glu Asn Pro Asp Phe Ile Ile Asp Asp Ile Val Lys Ser

195 200 205

Tyr Leu Ser Asn Glu Tyr Ser Lys Asp Ile Asp Glu Leu Asn Ala Tyr

210 215 220

Ile Glu Glu Ser Leu Asn Lys Val Lys Glu Asn Ser Gly Asn Asp Ile

225 230 235 240

Arg Asp Phe Glu Glu Phe Lys Gly Gly Asn Ser Phe Asn Leu Tyr Glu

245 250 255

Gln Glu Leu Val Glu Arg Trp Asn Leu Ala Ala Ala Ser Asp Ile Leu

260 265 270

Arg Ile Ser Ala Leu Lys Glu Val Gly Gly Val Tyr Leu Asp Val Asp

275 280 285

Met Leu Pro Gly Ile Gln Pro Asp Leu Phe Glu Ser Ile Glu Lys Pro

290 295 300

Ser Ser Val Thr Val Asp Phe Trp Glu Met Val Lys Leu Glu Ala Ile

305 310 315 320

Met Lys Tyr Lys Glu Tyr Ile Pro Gly Tyr Thr Ser Glu His Phe Asp

325 330 335

Ile Leu Asp Glu Glu Val Gln Ser Ser Phe Glu Ser Val Leu Ala Ser

340 345 350

Lys Ser Asp Lys Ser Glu Ile Phe Ser Ser Leu Gly Asp Ile Glu Ala

355 360 365

Ser Pro Leu Glu Val Lys Ile Ala Phe Asn Ser Lys Gly Ile Ile Asn

370 375 380

Gln Gly Leu Ile Ser Val Lys Asp Ser Tyr Cys Ser Asn Leu Ile Val

385 390 395 400

Lys Gln Ile Glu Asn Arg Tyr Lys Ile Leu Asn Asp Ser Leu Asn Pro

405 410 415

Ala Ile Ser Glu Asp Asn Asp Phe Asn Thr Thr Thr Asn Thr Phe Ile

420 425 430

Asp Ser Ile Met Ala Glu Ala Asn Ala Asp Asn Gly Arg Phe Met Met

435 440 445

Glu Leu Gly Lys Tyr Leu Arg Val Gly Phe Phe Pro Asp Val Lys Thr

450 455 460

Thr Ile Asn Leu Ser Gly Pro Glu Ala Tyr Ala Ala Ala Tyr Gln Asp

465 470 475 480

Leu Leu Met Phe Lys Glu Tyr Ser Ile Asn Ile His Leu Leu Glu Ser

485 490 495

Asp Leu Arg Asn Phe Glu Ile Ser Lys Thr Asn Ile Ser Gln Ser Thr

500 505 510

Glu Gln Glu Met Ala Ser Leu Trp Ser Phe Asp Asp Ala Arg Ala Lys

515 520 525

Ala Gln Phe Gln Glu Tyr Lys Arg Asn Tyr Phe Glu Gly Ala Leu Gly

530 535 540

Glu Asp Asp Asn Leu Asp Phe Ser Gln Asn Thr Ile Thr Asp Lys Glu

545 550 555 560

Tyr Leu Ile Glu Lys Ile Ser Ser Ser Ala Lys Asn Ser Glu Arg Gly

565 570 575

Tyr Val His Tyr Ile Ile Gln Leu Gln Gly Asp Asn Ile Ser Tyr Glu

580 585 590

Ala Ala Cys Asn Leu Phe Ala Lys Asn Pro Tyr Asp Ser Ile Leu Phe

595 600 605

Gln Lys Asn Ile Glu Asp Ser Thr Ile Ala Tyr Tyr Tyr Asn Pro Ala

610 615 620

Asp Gly Glu Ile Gln Glu Ile Asp Lys Tyr Arg Ile Pro Asp Arg Ile

625 630 635 640

Ser Asp Arg Pro Lys Ile Lys Leu Thr Phe Ile Gly His Gly Lys Ser

645 650 655

Glu Phe Asn Thr Asp Ile Phe Ala Asn Leu Asn Val Asp Ser Leu Ser

660 665 670

Ser Glu Ile Glu Thr Ala Ile Asp Leu Ala Lys Thr Asp Ile Ser Pro

675 680 685

Lys Ala Ile Glu Ile Asn Leu Leu Gly Cys Asn Met Phe Ser Tyr Ser

690 695 700

Val Asn Val Glu Glu Thr Tyr Pro Gly Lys Leu Leu Leu Lys Val Lys

705 710 715 720

Asp Lys Val Ser Glu Leu Met Pro Ser Ile Ser Gln Asp Ser Ile Ile

725 730 735

Val Ser Ala Asn Gln Tyr Glu Val Arg Ile Asn Ser Glu Gly Arg Arg

740 745 750

Glu Leu Leu Asp His Ser Gly Glu Trp Ile Asn Lys Glu Glu Ser Ile

755 760 765

Ile Lys Asp Ile Ser Ser Lys Glu Tyr Ile Ser Phe Asn Ser Lys Glu

770 775 780

Asn Lys Ile Ile Val Lys Ser Lys Asn Leu Pro Glu Leu Ser Thr Leu

785 790 795 800

Leu Gln Glu Ile Arg Asn Asn Ser Asn Leu Ser Asp Ile Glu Leu Glu

805 810 815

Glu Lys Val Met Leu Ala Glu Cys Glu Ile Ser Val Val Ser Asp Ile

820 825 830

Asp Thr Gln Val Val Glu Glu Arg Ile Glu Glu Ala Lys Asn Leu Thr

835 840 845

Ser Asp Ser Ile Asn Tyr Ile Lys Asn Glu Phe Lys Leu Ile Glu Ser

850 855 860

Ile Ser Asp Ala Leu Tyr Asp Leu Lys Gln Gln Asn Glu Leu Glu Asp

865 870 875 880

Ser His Phe Ile Ser Phe Glu Asp Ile Ser Glu Thr Asp Glu Gly Phe

885 890 895

Ser Ile Arg Phe Ile Asp Lys Glu Thr Gly Glu Ser Ile Phe Val Glu

900 905 910

Thr Glu Lys Thr Ile Phe Ser Glu Tyr Ala Asn His Ile Thr Glu Glu

915 920 925

Ile Ser Lys Val Lys Asp Thr Ile Phe Asp Thr Val Asn Gly Lys Leu

930 935 940

Val Lys Lys Val Asn Leu Asp Ala Thr His Glu Val Asn Thr Leu Asn

945 950 955 960

Ala Ala Phe Phe Ile Gln Ser Leu Ile Gly Tyr Asn Ser Ser Lys Glu

965 970 975

Ser Leu Ser Asn Leu Ser Val Ala Met Lys Val Gln Val Tyr Ala Gln

980 985 990

Leu Phe Ser Thr Gly Leu Asn Thr Ile Thr Asp Ala Ala Lys Val Val

995 1000 1005

Glu Leu Val Ser Thr Ala Leu Asp Glu Thr Ile Asp Leu Leu Pro

1010 1015 1020

Thr Leu Ser Glu Gly Leu Pro Ile Ile Ala Thr Ile Ile Asp Gly

1025 1030 1035

Val Ser Leu Gly Ala Ser Ile Lys Glu Leu Ser Glu Thr Ser Asp

1040 1045 1050

Pro Leu Leu Arg Gln Glu Ile Glu Ala Lys Ile Gly Ile Met Ala

1055 1060 1065

Val Asn Leu Thr Ala Ala Thr Thr Ala Ile Ile Thr Ser Ser Leu

1070 1075 1080

Gly Ile Ala Ser Gly Phe Ser Ile Leu Leu Val Pro Leu Ala Gly

1085 1090 1095

Ile Ser Ala Gly Ile Pro Ser Leu Val Asn Asn Glu Leu Ile Leu

1100 1105 1110

Arg Ala Glu Ala Lys Asn Val Val Asp Tyr Phe Ser His Ile Ser

1115 1120 1125

Leu Ala Glu Ser Glu Gly Ala Phe Thr Leu Leu Asp Asp Lys Ile

1130 1135 1140

Met Met Pro Gln Asp Asp Leu Val Ile Ser Glu Ile Asp Phe Asn

1145 1150 1155

Ser Asn Ser Ile Thr Leu Gly Lys Cys Glu Ile Trp Arg Met Glu

1160 1165 1170

Gly Gly Ser Gly His Thr Val Thr Asn Asp Ile Asp His Phe Phe

1175 1180 1185

Ser Ala Pro Ser Thr Thr Tyr Arg Glu Pro Tyr Leu Ser Ile Tyr

1190 1195 1200

Asp Val Leu Asp Val Lys Lys Glu Glu Leu Asp Leu Ser Lys Asp

1205 1210 1215

Leu Met Val Leu Pro Asn Ala Pro Asp Arg Ile Phe Gly Trp Glu

1220 1225 1230

Arg Gly Trp Thr Pro Gly Leu Arg Gly Leu Glu Asn Asp Gly Thr

1235 1240 1245

Lys Leu Leu Asp Arg Ile Arg Asp His Tyr Glu Gly Gln Phe Tyr

1250 1255 1260

Trp Arg Phe Phe Ala Phe Ile Ala Asp Ser Val Ile Thr Lys Leu

1265 1270 1275

Lys Pro Arg Tyr Glu Asp Thr Asn Ile Arg Ile Ser Leu Asp Ser

1280 1285 1290

Asn Thr Arg Ser Phe Ile Val Pro Val Ile Thr Thr Glu Tyr Ile

1295 1300 1305

Arg Glu Lys Leu Ser Tyr Ser Phe Tyr Gly Ser Gly Gly Thr Tyr

1310 1315 1320

Ala Leu Ser Leu Ser Gln Tyr Asn Met Asn Ile Asn Ile Glu Leu

1325 1330 1335

Asn Glu Ser Asp Thr Trp Val Ile Asp Ile Asp Asn Val Val Arg

1340 1345 1350

Asp Val Thr Ile Glu Ser Asp Lys Ile Lys Lys Gly Asp Leu Ile

1355 1360 1365

Glu Asn Ile Leu Ser Lys Leu Ser Ile Glu Glu Asn Lys Ile Ile

1370 1375 1380

Leu Asp Asn His Glu Ile Asn Phe Ser Gly Thr Leu Asn Gly Gly

1385 1390 1395

Asn Gly Phe Val Ser Leu Thr Phe Ser Ile Leu Glu Gly Ile Asn

1400 1405 1410

Ala Val Ile Glu Val Asp Leu Leu Ser Lys Ser Tyr Lys Val Leu

1415 1420 1425

Ile Ser Gly Glu Leu Lys Thr Leu Met Glu Asn Ser Asn Ser Val

1430 1435 1440

Gln Gln Lys Met Asp Tyr Ile Gly Leu Asn Ser Glu Val Gln Lys

1445 1450 1455

Asn Ile Pro Tyr Ser Phe Thr Asp Asp Lys Gly Lys Glu Asn Gly

1460 1465 1470

Phe Ile Asn Cys Ser Thr Lys Glu Gly Leu Phe Val Ser Glu Leu

1475 1480 1485

Ser Asp Val Val Leu Ile Ser Lys Val Tyr Met Asp Asp Ser Lys

1490 1495 1500

Pro Ser Ser Gly Tyr Tyr Ser Tyr Asp Leu Lys Asp Val Lys Val

1505 1510 1515

Ile Thr Lys Asp Asp Val Ile Ile Leu Thr Gly Tyr Tyr Leu Lys

1520 1525 1530

Asp Asp Ile Lys Ile Ser Leu Ser Phe Thr Ile Gln Asp Glu Asn

1535 1540 1545

Thr Ile Lys Leu Asn Gly Val Tyr Leu Asp Glu Asn Gly Val Ala

1550 1555 1560

Glu Ile Leu Lys Phe Met Asn Lys Lys Gly Ser Thr Asn Thr Ser

1565 1570 1575

Asp Ser Leu Met Ser Phe Leu Glu Ser Met Asn Ile Lys Ser Ile

1580 1585 1590

Phe Ile Asn Ser Leu Gln Ser Asn Thr Lys Leu Ile Leu Asp Thr

1595 1600 1605

Asn Phe Ile Ile Ser Gly Ala Thr Ser Ile Gly Gln Phe Glu Phe

1610 1615 1620

Ile Cys Asp Lys Asp Asn Asn Ile Gln Pro Tyr Phe Ile Lys Phe

1625 1630 1635

Asn Thr Leu Glu Thr Lys Tyr Thr Leu Tyr Val Gly Asn Arg Gln

1640 1645 1650

Asn Met Ile Val Glu Pro Asn Tyr Asn Leu Asp Asp Ser Gly Asp

1655 1660 1665

Ile Ser Ser Thr Val Ile Asn Phe Ser Gln Lys Tyr Leu Tyr Gly

1670 1675 1680

Ile Asp Ser Cys Val Asn Lys Val Ile Ile Ser Pro Asn Ile Tyr

1685 1690 1695

Thr Asp Glu Ile Asn Ile Thr Pro Val Tyr Glu Ala Asn Asn Thr

1700 1705 1710

Tyr Pro Glu Val Ile Val Leu Asp Thr Asn Tyr Ile Ser Glu Lys

1715 1720 1725

Ile Asn Ile Asn Ile Asn Asp Leu Ser Ile Arg Tyr Val Trp Ser

1730 1735 1740

Asn Asp Gly Ser Asp Phe Ile Leu Met Ser Thr Asp Glu Glu Asn

1745 1750 1755

Lys Ile Ser Gln Val Lys Ile Arg Phe Thr Asn Val Phe Lys Gly

1760 1765 1770

Asn Thr Ile Ala Asp Lys Ile Ser Phe Asn Phe Ser Asp Lys Gln

1775 1780 1785

Asp Val Ser Ile Asn Lys Ile Ile Ser Thr Phe Thr Pro Ser Tyr

1790 1795 1800

Tyr Val Glu Gln Leu Leu Asn Tyr Asp Leu Gly Leu Ile Ser Leu

1805 1810 1815

Tyr Asn Glu Lys Phe Tyr Ile Asn Asn Phe Gly Met Met Val Ser

1820 1825 1830

Gly Leu Val Tyr Ile Asn Asp Ser Leu Tyr Tyr Phe Lys Pro Pro

1835 1840 1845

Ile Lys Asn Leu Ile Thr Gly Phe Thr Thr Ile Gly Asp Asp Lys

1850 1855 1860

Tyr Tyr Phe Asn Pro Asp Asn Gly Gly Ala Ala Ser Val Gly Glu

1865 1870 1875

Thr Ile Ile Asp Gly Lys Asn Tyr Tyr Phe Ser Gln Asn Gly Val

1880 1885 1890

Leu Gln Thr Gly Val Phe Ser Thr Glu Asp Gly Phe Lys Tyr Phe

1895 1900 1905

Ala Pro Ala Asp Thr Leu Asp Glu Asn Leu Glu Gly Glu Ala Ile

1910 1915 1920

Asn Phe Thr Gly Lys Leu Ile Ile Asp Glu Asn Ile Tyr Tyr Phe

1925 1930 1935

Gly Asp Asn Tyr Arg Ala Ala Glu Glu Trp Gln Thr Leu Asp Asp

1940 1945 1950

Glu Val Tyr Tyr Phe Ser Thr Asp Thr Gly Lys Ala Phe Lys Gly

1955 1960 1965

Leu Asn Gln Ile Gly Asp Asp Lys Phe Tyr Phe Asn Ser Asp Gly

1970 1975 1980

Ile Met Gln Lys Gly Phe Val Asn Ile Asn Asp Lys Thr Phe Tyr

1985 1990 1995

Phe Asp Asp Ser Gly Val Met Lys Ser Gly Tyr Leu Glu Ile Tyr

2000 2005 2010

Gly Lys Tyr Phe Tyr Phe Ala Glu Asn Gly Glu Met Gln Ile Gly

2015 2020 2025

Val Phe Asn Thr Thr Asp Gly Phe Lys Tyr Phe Ala His Gln Asp

2030 2035 2040

Glu Asp Leu Gly Asn Glu Glu Gly Glu Ala Leu Ser Tyr Ser Gly

2045 2050 2055

Ile Leu Asn Phe Asn Asn Lys Ile Tyr Tyr Phe Asp Asp Ser Phe

2060 2065 2070

Thr Ala Ile Val Gly Trp Lys Asp Leu Glu Asp Gly Ser Lys Tyr

2075 2080 2085

Tyr Phe Asp Glu Asn Thr Ala Glu Ala Ser Ile Gly Ile Ser Ile

2090 2095 2100

Ile Asn Asp Gly Lys Tyr Tyr Phe Asn Asp Ser Gly Ile Met Gln

2105 2110 2115

Ile Gly Phe Val Thr Ile Asn Asp Lys Val Phe Tyr Phe Ser Asp

2120 2125 2130

Ser Gly Ile Val Glu Ser Gly Met Gln Asn Ile Asp Asp Asn Tyr

2135 2140 2145

Phe Tyr Ile Ser Glu Asn Gly Leu Val Gln Ile Gly Val Phe Asp

2150 2155 2160

Thr Ser Asp Gly Tyr Lys Tyr Phe Ala Pro Ala Asn Thr Val Asn

2165 2170 2175

Asp Asn Ile Tyr Gly Gln Ala Val Glu Tyr Ser Gly Leu Val Lys

2180 2185 2190

Val Asn Glu Asp Val Tyr Ser Phe Gly Glu Ser Tyr Thr Ile Glu

2195 2200 2205

Thr Gly Trp Ile Tyr Asp Ser Glu Asn Glu Ser Asp Lys Tyr Tyr

2210 2215 2220

Phe Asp Pro Glu Thr Lys Lys Ala Tyr Lys Gly Ile Asn Thr Ile

2225 2230 2235

Asp Asp Ile Lys Tyr Tyr Phe Asp Glu Asn Gly Ile Met Arg Thr

2240 2245 2250

Gly Leu Ile Thr Phe Glu Asp Asn His Tyr Tyr Phe Asn Glu Asp

2255 2260 2265

Gly Val Met Gln Tyr Gly Tyr Leu Asn Ile Glu Asp Lys Met Phe

2270 2275 2280

Tyr Phe Asn Glu Asp Gly Val Met Gln Ile Gly Val Phe Asn Thr

2285 2290 2295

Pro Asp Gly Phe Lys Tyr Phe Ala His Gln Asn Thr Leu Asp Glu

2300 2305 2310

Asn Phe Glu Gly Glu Ser Ile Asn Tyr Thr Gly Trp Leu Asp Leu

2315 2320 2325

Asp Gly Lys Lys Tyr Tyr Phe Thr Glu Glu Tyr Ile Ala Ala Thr

2330 2335 2340

Gly Ser Val Thr Ile Asp Asp Glu Glu Tyr Tyr Phe Asp Pro Asp

2345 2350 2355

Thr Ala Glu Leu Val Val Ser Glu

2360 2365

<210> 13

<211> 2366

<212> PRT

<213> Artificial sequence

<220>

<223> TcdB8

<400> 13

Met Ser Leu Val Asn Arg Lys Gln Leu Glu Lys Met Ala Asn Val Lys

1 5 10 15

Phe Arg Thr Gln Glu Asp Glu Tyr Val Ala Ile Leu Asp Ala Leu Glu

20 25 30

Glu Tyr His Asn Met Ser Glu Asn Thr Val Val Glu Lys Tyr Leu Lys

35 40 45

Leu Lys Asp Ile Asn Ser Leu Thr Asp Ala Tyr Ile Asp Thr Tyr Lys

50 55 60

Lys Ser Gly Arg Asn Lys Ala Leu Lys Lys Phe Lys Glu Tyr Leu Thr

65 70 75 80

Thr Glu Val Ile Glu Leu Lys Asn Ser Asn Leu Thr Pro Val Glu Lys

85 90 95

Asn Leu His Phe Val Trp Ile Gly Gly Gln Ile Ser Asp Thr Ala Ile

100 105 110

Asn Tyr Ile Asn Gln Trp Lys Asp Val Asn Ser Asp Tyr Asn Thr Asn

115 120 125

Val Phe Tyr Asp Ser Asn Ala Phe Leu Ile Asn Thr Leu Lys Lys Thr

130 135 140

Ile Val Glu Ala Thr Thr Asn Asp Thr Leu Glu Ser Phe Ser Glu Asn

145 150 155 160

Leu Asn Asp Pro Arg Phe Asp His Asn Asn Phe Tyr Arg Lys Arg Met

165 170 175

Glu Met Ile Tyr Asp Lys Gln Lys Asn Phe Ile Asn Tyr Tyr Lys Ala

180 185 190

Gln Arg Glu Glu Asn Pro Glu Leu Ile Ile Asp Asp Ile Val Lys Thr

195 200 205

Tyr Leu Ser Asn Glu Tyr Ser Lys Glu Ile Asp Glu Leu Asn Ala Tyr

210 215 220

Ile Glu Glu Ser Leu Asn Lys Ile Thr Gln Asn Ser Gly Asn Asp Val

225 230 235 240

Arg Asn Phe Glu Glu Phe Lys Asn Gly Glu Ser Phe Lys Leu Tyr Glu

245 250 255

Gln Glu Leu Val Glu Arg Trp Asn Leu Ala Ala Ala Ser Asp Ile Leu

260 265 270

Arg Ile Ser Ala Leu Lys Glu Ile Gly Gly Val Tyr Leu Asp Val Asp

275 280 285

Met Leu Pro Gly Ile Gln Pro Asp Leu Phe Glu Ser Ile Glu Lys Pro

290 295 300

Ser Ser Val Thr Val Asp Phe Trp Glu Met Thr Lys Leu Glu Ala Ile

305 310 315 320

Met Lys Tyr Lys Glu Tyr Ile Pro Gly Tyr Thr Ser Glu His Phe Asp

325 330 335

Met Leu Asp Glu Glu Val Gln Ser Ser Phe Glu Ser Ala Leu Ala Ser

340 345 350

Lys Ser Asp Lys Ser Glu Ile Phe Ser Ser Leu Gly Asp Met Glu Ala

355 360 365

Ser Pro Leu Glu Val Lys Ile Ala Phe Asn Ser Lys Gly Ile Ile Asn

370 375 380

Gln Gly Leu Ile Ser Val Lys Asp Ser Tyr Cys Ser Asn Leu Ile Val

385 390 395 400

Lys Gln Ile Glu Asn Arg Tyr Lys Ile Leu Asn Asn Ser Leu Asn Pro

405 410 415

Ala Ile Ser Glu Asp Asn Asp Phe Asn Thr Thr Thr Asn Thr Phe Ile

420 425 430

Asp Ser Ile Met Ala Glu Ala Asn Ala Asp Asn Gly Arg Phe Met Met

435 440 445

Glu Leu Gly Lys Tyr Leu Arg Val Gly Phe Phe Pro Asp Val Lys Thr

450 455 460

Thr Ile Asn Leu Ser Gly Pro Glu Ala Tyr Ala Ala Ala Tyr Gln Asp

465 470 475 480

Leu Leu Met Phe Lys Glu Asp Ser Met Asn Ile His Leu Ile Glu Ala

485 490 495

Asp Leu Arg Asn Phe Glu Ile Ser Lys Thr Asn Ile Ser Gln Ser Thr

500 505 510

Glu Gln Glu Met Ala Ser Leu Trp Ser Phe Asp Asp Ala Arg Ala Lys

515 520 525

Ala Gln Phe Glu Glu Tyr Lys Arg Asn Tyr Phe Glu Gly Ala Leu Gly

530 535 540

Glu Asp Asp Asn Leu Asp Phe Ser Gln Asn Thr Val Val Asp Lys Glu

545 550 555 560

Tyr Leu Leu Glu Lys Ile Ser Ser Leu Ala Arg Ser Ser Glu Arg Gly

565 570 575

Tyr Ile His Tyr Ile Val Gln Leu Gln Gly Asp Lys Ile Ser Tyr Glu

580 585 590

Ala Ala Cys Asn Leu Phe Ala Lys Thr Pro Tyr Asp Ser Ile Leu Phe

595 600 605

Gln Lys Asn Ile Glu Asp Ser Glu Ile Ala Tyr Tyr Tyr Asn Pro Gly

610 615 620

Asp Asp Glu Ile Gln Glu Ile Asp Lys Tyr Arg Ile Pro Ser Ile Ile

625 630 635 640

Ser Asp Arg Pro Lys Ile Lys Leu Thr Phe Ile Gly His Gly Lys Asp

645 650 655

Glu Phe Asn Thr Asp Ile Phe Ala Gly Leu Asp Val Asp Ser Leu Ser

660 665 670

Thr Glu Ile Glu Thr Val Ile Asp Leu Ala Lys Glu Asp Ile Ser Pro

675 680 685

Lys Ala Ile Glu Ile Asn Leu Leu Gly Cys Asn Met Phe Ser Tyr Ser

690 695 700

Ile Asn Ile Glu Glu Thr Tyr Pro Gly Lys Leu Leu Leu Lys Val Lys

705 710 715 720

Asp Lys Ile Ser Glu Leu Met Pro Ser Ile Ser Gln Asp Ser Ile Ile

725 730 735

Val Ser Ala Asn Gln Tyr Glu Val Arg Ile Asn Ser Glu Gly Arg Arg

740 745 750

Glu Leu Leu Asp His Ser Gly Glu Trp Ile Asn Lys Glu Glu Ser Ile

755 760 765

Ile Lys Asp Ile Ser Ser Lys Glu Tyr Ile Ser Phe Asn Ser Lys Glu

770 775 780

Asn Lys Ile Ile Val Lys Ser Lys Asn Leu Pro Glu Leu Ser Thr Leu

785 790 795 800

Leu Gln Glu Ile Arg Asn Asn Ser Asn Ser Ser Asp Ile Glu Leu Glu

805 810 815

Glu Lys Val Met Leu Ala Glu Cys Glu Ile Asn Val Ile Ser Asn Ile

820 825 830

Asp Thr Gln Ile Val Glu Glu Arg Ile Glu Glu Ala Lys Asn Leu Thr

835 840 845

Ser Asp Ser Ile Asn Tyr Ile Lys Asn Glu Phe Lys Leu Ile Glu Ser

850 855 860

Ile Ser Asp Ala Leu Cys Asp Leu Lys Gln Gln Asn Glu Leu Glu Asp

865 870 875 880

Ser His Phe Ile Ser Phe Glu Asp Ile Ser Glu Thr Asp Glu Gly Phe

885 890 895

Ser Ile Arg Phe Ile Asn Lys Glu Thr Gly Glu Ser Ile Phe Val Glu

900 905 910

Thr Glu Lys Thr Ile Phe Ser Glu Tyr Ala Asn His Ile Thr Glu Glu

915 920 925

Ile Ser Lys Val Lys Asp Thr Ile Phe Asp Thr Val Asn Gly Lys Leu

930 935 940

Val Lys Lys Val Asn Leu Asp Thr Thr His Glu Val Asn Thr Leu Asn

945 950 955 960

Ala Ala Phe Phe Ile Gln Ser Leu Ile Glu Tyr Asn Ser Ser Lys Glu

965 970 975

Ser Leu Ser Asn Leu Ser Val Ala Met Lys Val Gln Val Tyr Ala Gln

980 985 990

Leu Phe Ser Thr Gly Leu Asn Thr Ile Thr Asp Ala Ala Lys Val Val

995 1000 1005

Glu Leu Val Ser Thr Ala Leu Asp Glu Thr Ile Asp Leu Leu Pro

1010 1015 1020

Thr Leu Ser Glu Gly Leu Pro Val Ile Ala Thr Ile Ile Asp Gly

1025 1030 1035

Val Ser Leu Gly Ser Ala Ile Lys Glu Leu Ser Glu Thr Ser Asp

1040 1045 1050

Pro Leu Leu Arg Gln Glu Ile Glu Ala Lys Ile Gly Ile Met Ala

1055 1060 1065

Val Asn Leu Thr Ala Ala Thr Thr Ala Ile Ile Thr Ser Ala Leu

1070 1075 1080

Gly Ile Ala Ser Gly Phe Ser Ile Leu Leu Val Pro Leu Ala Gly

1085 1090 1095

Ile Ser Ala Gly Ile Pro Ser Leu Val Asn Asn Glu Leu Ile Leu

1100 1105 1110

Arg Asp Lys Ala Thr Lys Val Val Asp Tyr Phe Lys His Ile Ser

1115 1120 1125

Leu Ala Glu Thr Glu Gly Ala Phe Thr Leu Leu Asp Asp Lys Ile

1130 1135 1140

Ile Met Pro Gln Asp Asp Leu Val Ile Ser Glu Ile Asp Phe Asn

1145 1150 1155

Asn Asn Ser Ile Thr Leu Gly Lys Cys Glu Ile Trp Arg Met Glu

1160 1165 1170

Gly Gly Ser Gly His Thr Val Thr Asp Asp Ile Asp His Phe Phe

1175 1180 1185

Ser Ser Pro Ser Ile Thr Tyr Arg Glu Pro Tyr Leu Ser Ile Tyr

1190 1195 1200

Asp Val Leu Glu Val Gln Lys Glu Glu Leu Asp Leu Ser Lys Asp

1205 1210 1215

Leu Met Val Leu Pro Asn Ala Pro Asn Arg Val Phe Ala Trp Glu

1220 1225 1230

Thr Gly Trp Thr Pro Gly Leu Arg Ser Leu Glu Asn Asp Gly Thr

1235 1240 1245

Lys Leu Leu Asp Arg Ile Arg Asn His Tyr Glu Gly Glu Phe Tyr

1250 1255 1260

Trp Arg Tyr Phe Ala Phe Ile Ala Asp Ala Leu Ile Thr Thr Leu

1265 1270 1275

Lys Pro Arg Tyr Glu Asp Thr Asn Ile Arg Ile Asn Leu Asp Ser

1280 1285 1290

Asn Thr Arg Ser Phe Val Val Pro Val Ile Thr Thr Glu Tyr Ile

1295 1300 1305

Arg Glu Asn Leu Ser Tyr Ser Phe Tyr Gly Ser Gly Gly Thr Tyr

1310 1315 1320

Ala Leu Ser Leu Ser Gln Tyr Asn Met Gly Ile Asn Ile Glu Leu

1325 1330 1335

Ser Glu Ser Asp Val Trp Ile Ile Asp Val Asp Asn Val Val Arg

1340 1345 1350

Asp Val Thr Ile Glu Ser Asp Lys Ile Lys Lys Gly Asp Leu Ile

1355 1360 1365

Glu Gly Ile Leu Ser Thr Leu Ser Ile Glu Asp Asn Lys Ile Ile

1370 1375 1380

Leu Asn Ser His Glu Leu Asn Phe Ser Gly Asp Val Asn Gly Ser

1385 1390 1395

Asn Gly Phe Val Ser Leu Thr Phe Ser Ile Leu Glu Gly Ile Asn

1400 1405 1410

Ala Ile Ile Glu Val Asp Leu Leu Ser Lys Ser Tyr Lys Leu Leu

1415 1420 1425

Leu Ser Gly Glu Leu Lys Thr Leu Met Ser Asn Ser Asn Tyr Ile

1430 1435 1440

Gln Gln Lys Ile Asp Tyr Ile Gly Phe Asn Ser Glu Leu Gln Lys

1445 1450 1455

Asn Ile Pro Tyr Ser Phe Val Asp Ala Glu Gly Lys Lys Asn Gly

1460 1465 1470

Phe Ile Asn Val Ser Thr Lys Glu Gly Leu Phe Ala Ser Glu Leu

1475 1480 1485

Ser Asp Val Val Leu Ile Ser Lys Val Tyr Met Asp Asn Ser Lys

1490 1495 1500

Pro Ser Phe Gly His Tyr Ser Asp Ile Leu Lys Asp Val Lys Val

1505 1510 1515

Ile Thr Lys Asp Asp Ile Asn Ile Leu Thr Gly Tyr Tyr Leu Lys

1520 1525 1530

Asp Asp Ile Lys Ile Ser Leu Ser Phe Thr Leu Gln Asp Glu His

1535 1540 1545

Thr Ile Lys Leu Asn Gly Val His Leu Asp Glu Lys Gly Val Ala

1550 1555 1560

Glu Ile Leu Thr Phe Met Asn Lys Lys Val Gly Thr Asn Thr Ser

1565 1570 1575

Asp Ser Leu Met Ser Phe Leu Lys Ser Met Asn Ile Asn Asn Val

1580 1585 1590

Phe Ser His Ser Leu Gln Asp Lys Val Asn Leu Val Leu Glu Thr

1595 1600 1605

Asn Phe Ile Ile Ser Gly Met Thr Ser Ile Gly Gln Phe Glu Phe

1610 1615 1620

Ile Cys Asp Glu Asn Asp Asn Ile Gln Pro Tyr Phe Ile Lys Phe

1625 1630 1635

Asn Ala Leu Asp Thr Lys Tyr Thr Leu Tyr Leu Gly Asn Arg Gln

1640 1645 1650

Asn Met Ile Val Glu Pro Asn Tyr Asp Leu Asp Asp Ser Gly Asn

1655 1660 1665

Ile Ser Ser Thr Val Ile Asn Phe Ser Gln Lys His Leu Tyr Gly

1670 1675 1680

Ile Asp Ser Phe Ile Asn Lys Val Ile Ile Ser Pro Asn Leu Tyr

1685 1690 1695

Thr Asp Glu Ile Asn Ile Thr Pro Val His Glu Thr Asn Asn Thr

1700 1705 1710

Tyr Pro Glu Val Ile Val Leu Asp Ala Asn Tyr Ile Ser Glu Lys

1715 1720 1725

Ile Lys Val Asn Ile Asn Asp Leu Ser Ile Arg Tyr Ile Trp Ser

1730 1735 1740

Asn Asp Gly Asn Asp Phe Ile Leu Met Ser Thr Ile Gly Glu Asp

1745 1750 1755

Lys Ala Ser Gln Val Lys Ile Arg Phe Ala Asn Val Phe Lys Gly

1760 1765 1770

Asn Thr Leu Ala Asn Lys Leu Ser Phe Asn Phe Ser Asp Lys Gln

1775 1780 1785

Asp Val Ser Leu Ser Glu Ile Ile Ser Ala Phe Thr Pro Ser Lys

1790 1795 1800

Tyr Glu Asp Gly Phe Ser Ser Tyr Lys Leu Gly Leu Ile Ser Phe

1805 1810 1815

Tyr Asn Glu Lys Phe Tyr Ile Asn Asn Phe Gly Met Lys Val Ser

1820 1825 1830

Gly Leu Ile Tyr Ile Asn Asp Ser Leu Tyr Tyr Phe Lys Pro Pro

1835 1840 1845

Val Asn Asn Leu Ile Thr Gly Phe Thr Thr Val Gly Asp Asp Lys

1850 1855 1860

Tyr Tyr Phe Asn Pro Thr Asn Gly Gly Ala Ala Ser Ile Gly Asp

1865 1870 1875

Thr Ile Ile Asp Asp Lys Asn Tyr Tyr Phe Asn Gln Ile Gly Val

1880 1885 1890

Leu Gln Thr Gly Val Phe Ser Thr Glu Asp Gly Phe Lys Tyr Phe

1895 1900 1905

Ala Pro Ala Asn Thr Leu Asp Glu Asn Leu Glu Gly Glu Ala Ile

1910 1915 1920

Asp Phe Thr Gly Lys Leu Ile Ile Asp Glu Asn Ile Tyr Tyr Phe

1925 1930 1935

Glu Asp Asn Tyr Arg Gly Ala Val Glu Trp Lys Glu Leu Asp Gly

1940 1945 1950

Glu Met Tyr Tyr Phe Ser Pro Glu Thr Gly Lys Ala Phe Lys Gly

1955 1960 1965

Leu Asn Gln Ile Gly Asp Asp Lys Tyr Tyr Phe Asn Ser Asp Gly

1970 1975 1980

Ile Met Gln Lys Gly Phe Val Ser Ile Asn Asp Lys Lys His Tyr

1985 1990 1995

Phe Asp Asp Ser Gly Val Met Lys Val Gly Tyr Thr Glu Ile Asp

2000 2005 2010

Gly Lys Tyr Phe Tyr Phe Ala Glu Asn Gly Glu Met Gln Ile Gly

2015 2020 2025

Val Phe Asn Thr Ser Asp Gly Phe Lys Tyr Phe Ala His Tyr Asn

2030 2035 2040

Glu Asp Leu Gly Asn Glu Glu Gly Glu Ala Leu Ser Tyr Ser Gly

2045 2050 2055

Ile Leu Asn Phe Asn Asn Lys Ile Tyr Tyr Phe Asp Asp Ser Phe

2060 2065 2070

Thr Ala Val Val Gly Trp Arg Asn Leu Asp Asp Gly Ser Lys Tyr

2075 2080 2085

Tyr Phe Asp Glu Asn Thr Ala Glu Ala Phe Ile Gly Phe Ser Leu

2090 2095 2100

Ile Asn Asp Glu Gln Tyr Tyr Phe Asn Glu Asp Gly Ile Met Gln

2105 2110 2115

Val Gly Phe Val Thr Ile Asn Asp Arg Val Phe Tyr Phe Ser Asp

2120 2125 2130

Ser Gly Ile Ile Glu Ser Gly Val Gln Asn Ile Asp Asp Asn Tyr

2135 2140 2145

Phe Tyr Ile Asp Glu Lys Gly Ile Val Gln Ile Gly Val Phe Asp

2150 2155 2160

Thr Ser Asp Gly Tyr Lys Tyr Phe Ala Pro Pro Asn Thr Val Asn

2165 2170 2175

Glu Asn Ile Tyr Gly Gln Ala Val Glu Tyr Ser Gly Leu Val Lys

2180 2185 2190

Val Asn Glu Asp Val Tyr Tyr Phe Gly Glu Thr Tyr Leu Ile Glu

2195 2200 2205

Thr Gly Trp Ile Tyr Asp Met Glu Asn Glu Ser Asp Lys Tyr Tyr

2210 2215 2220

Phe Asp Pro Glu Thr Lys Lys Ala Tyr Lys Gly Ile Asn Val Ile

2225 2230 2235

Asn Asp Thr Lys Tyr Tyr Phe Asp Glu Asn Gly Ile Met Arg Thr

2240 2245 2250

Gly Leu Ile Ser Phe Glu Asn Asn His Tyr Tyr Phe Asn Glu Asp

2255 2260 2265

Gly Val Met Gln Ser Gly Tyr Ile Asn Ile Glu Asp Lys Met Phe

2270 2275 2280

Tyr Phe Ser Glu Asp Gly Ile Met Gln Ile Gly Val Phe Asn Thr

2285 2290 2295

Pro Asp Gly Phe Lys Tyr Phe Ala His Gln Asn Thr Leu Asp Asp

2300 2305 2310

Asn Phe Glu Gly Glu Ser Ile Asn Tyr Thr Gly Trp Leu Asp Phe

2315 2320 2325

Asn Glu Lys Arg Tyr Tyr Phe Thr Asp Glu Tyr Ile Ala Ala Thr

2330 2335 2340

Gly Ser Val Thr Ile Asp Asp Glu Glu Tyr Tyr Phe Asp Pro Asp

2345 2350 2355

Thr Ala Glu Leu Val Leu Ser Glu

2360 2365

<210> 14

<211> 992

<212> PRT

<213> Artificial sequence

<220>

<223> Receptor binding domain of TcdB4 (DRBD)

<400> 14

Arg Ile Glu Glu Ala Lys Ser Leu Thr Ser Asp Ser Ile Asn Tyr Ile

1 5 10 15

Lys Asn Glu Phe Lys Leu Ile Glu Ser Ile Ser Asp Ala Leu Tyr Asp

20 25 30

Leu Lys Gln Gln Asn Glu Leu Glu Glu Ser His Phe Ile Ser Phe Glu

35 40 45

Asp Ile Ser Lys Thr Asp Glu Gly Phe Ser Ile Arg Phe Ile Asp Lys

50 55 60

Glu Thr Gly Glu Ser Ile Phe Val Glu Thr Glu Lys Ala Ile Phe Ser

65 70 75 80

Glu Tyr Ala Asn His Ile Thr Glu Glu Ile Ser Lys Leu Lys Asp Thr

85 90 95

Ile Phe Asp Thr Val Asn Gly Lys Leu Val Lys Lys Val Thr Leu Asp

100 105 110

Ala Thr His Glu Val Asn Thr Leu Asn Ala Ala Phe Phe Ile Gln Ser

115 120 125

Leu Ile Gly Tyr Asn Ser Ser Lys Glu Ser Leu Ser Asn Leu Ser Val

130 135 140

Ala Met Lys Val Gln Val Tyr Ala Gln Leu Phe Ser Thr Gly Leu Asn

145 150 155 160

Thr Ile Thr Asp Ala Ala Lys Val Val Glu Leu Val Ser Thr Ala Leu

165 170 175

Asp Glu Thr Ile Asp Leu Leu Pro Thr Leu Ser Glu Gly Leu Pro Val

180 185 190

Ile Ala Thr Ile Ile Asp Gly Val Ser Leu Gly Ala Ser Ile Lys Glu

195 200 205

Leu Ser Glu Thr Ser Asp Pro Leu Leu Arg Gln Glu Ile Glu Ala Lys

210 215 220

Ile Gly Ile Met Ala Val Asn Leu Thr Ala Ala Thr Thr Ala Ile Ile

225 230 235 240

Thr Ser Ser Leu Gly Ile Ala Ser Gly Phe Ser Ile Leu Leu Val Pro

245 250 255

Leu Ala Gly Ile Ser Ala Gly Ile Pro Ser Leu Val Asn Asn Glu Leu

260 265 270

Ile Leu Arg Ala Glu Ala Lys Asn Val Val Asp Tyr Phe Gly His Ile

275 280 285

Ser Leu Ala Glu Ser Glu Gly Ala Phe Thr Leu Leu Asp Asp Lys Ile

290 295 300

Met Met Pro Gln Asp Asp Leu Val Ile Ser Glu Ile Asp Phe Asn Asn

305 310 315 320

Asn Ser Ile Thr Leu Gly Lys Cys Glu Ile Trp Arg Met Glu Gly Gly

325 330 335

Ser Gly His Thr Val Thr Asp Asp Ile Asp His Phe Phe Ser Ala Pro

340 345 350

Ser Thr Thr Tyr Arg Glu Pro Tyr Leu Ser Ile Tyr Asp Val Leu Asp

355 360 365

Val Lys Glu Glu Glu Leu Asp Leu Ser Lys Asp Leu Met Val Leu Pro

370 375 380

Asn Ala Pro Asp Arg Ile Phe Gly Trp Glu Arg Gly Trp Thr Pro Gly

385 390 395 400

Leu Arg Ser Leu Glu Asn Asp Gly Thr Lys Leu Leu Asp Arg Ile Arg

405 410 415

Asp His Tyr Glu Gly Gln Phe Tyr Trp Arg Phe Phe Ala Phe Ile Ala

420 425 430

Asp Ser Val Ile Thr Lys Leu Lys Pro Arg Tyr Glu Asp Thr Asn Ile

435 440 445

Arg Ile Ser Leu Asp Ser Asn Thr Arg Ser Phe Ile Val Pro Val Ile

450 455 460

Thr Thr Glu Tyr Ile Arg Glu Lys Leu Ser Tyr Ser Phe Tyr Gly Ser

465 470 475 480

Gly Gly Thr Tyr Ala Leu Ser Leu Ser Gln Tyr Asn Met Asn Ile Asn

485 490 495

Ile Glu Leu Asn Glu Asn Asp Thr Trp Val Ile Asp Val Asp Asn Val

500 505 510

Val Arg Asp Val Thr Ile Glu Ser Asp Lys Ile Lys Lys Gly Asp Leu

515 520 525

Ile Glu Asn Ile Leu Ser Lys Leu Ser Ile Glu Asp Asn Lys Ile Ile

530 535 540

Leu Asp Asn His Glu Ile Asn Phe Ser Gly Thr Leu Asn Gly Gly Asn

545 550 555 560

Gly Phe Val Ser Leu Thr Phe Ser Ile Leu Glu Gly Ile Asn Ala Val

565 570 575

Ile Glu Val Asp Leu Leu Ser Lys Ser Tyr Lys Val Leu Ile Ser Gly

580 585 590

Glu Leu Lys Thr Leu Met Ala Asn Ser Asn Ser Val Gln Gln Lys Ile

595 600 605

Asp Tyr Ile Gly Leu Asn Ser Glu Leu Gln Lys Asn Ile Pro Tyr Ser

610 615 620

Phe Met Asp Asp Glu Gly Lys Glu Asn Gly Phe Ile Asn Cys Phe Thr

625 630 635 640

Lys Glu Gly Leu Phe Val Ser Glu Leu Ser Asp Val Val Leu Ile Ile

645 650 655

Lys Val Tyr Met Asp Asn Ser Lys Pro Pro Phe Gly Tyr Tyr Ser Asn

660 665 670

Asp Leu Lys Asp Val Lys Val Ile Thr Lys Asp Asp Val Ile Ile Ile

675 680 685

Thr Gly Tyr Tyr Leu Lys Asp Asp Ile Lys Ile Ser Leu Ser Phe Thr

690 695 700

Ile Gln Asp Lys Asn Thr Ile Lys Leu Asn Gly Val Tyr Leu Asp Glu

705 710 715 720

Asn Gly Val Ala Glu Ile Leu Lys Phe Met Asn Lys Lys Gly Ser Thr

725 730 735

Asn Thr Ser Asp Ser Leu Met Ser Phe Leu Glu Ser Met Asn Ile Lys

740 745 750

Ser Ile Phe Ile Lys Ser Leu Lys Ser Asn Ala Lys Leu Ile Leu Asp

755 760 765

Thr Asn Phe Ile Ile Ser Gly Thr Thr Phe Ile Gly Gln Phe Glu Phe

770 775 780

Ile Cys Asp Lys Asp Asn Asn Ile Gln Pro Tyr Phe Ile Lys Phe Asn

785 790 795 800

Thr Leu Glu Thr Lys Tyr Thr Leu Tyr Val Gly Asn Arg Gln Asn Met

805 810 815

Ile Val Glu Pro Asn Tyr Asn Leu Asp Asp Ser Gly Asp Ile Ser Ser

820 825 830

Thr Val Ile Asn Phe Ser Gln Lys Tyr Leu Tyr Gly Ile Asp Ser Cys

835 840 845

Val Asn Lys Val Val Ile Ser Pro Gly Ile Tyr Thr Asp Glu Ile Asn

850 855 860

Ile Thr Pro Val His Glu Ala Asn Asn Thr Tyr Pro Glu Val Ile Val

865 870 875 880

Leu Asp Thr Asn Tyr Ile Ser Glu Lys Ile Asn Ile Asn Ile Asn Asp

885 890 895

Leu Ser Ile Arg Tyr Val Trp Ser Asn Asp Gly Ser Asp Phe Ile Leu

900 905 910

Met Ser Thr Asp Glu Glu Asn Lys Val Ser Gln Val Lys Ile Arg Phe

915 920 925

Thr Asn Val Phe Lys Gly Asn Thr Ile Ser Asp Lys Ile Ser Phe Asn

930 935 940

Phe Ser Asp Lys Gln Asp Ile Ser Ile Asn Lys Ile Ile Ser Thr Phe

945 950 955 960

Thr Pro Ser Tyr Tyr Val Glu Gly Leu Leu Asn Tyr Asp Leu Gly Leu

965 970 975

Ile Ser Leu Tyr Asn Glu Lys Phe Tyr Ile Asn Asn Leu Gly Met Met

980 985 990

<210> 15

<211> 991

<212> PRT

<213> Artificial sequence

<220>

<223> Fragments of 842 to 1834 bits of TcdB4

<400> 15

Ile Glu Glu Ala Lys Ser Leu Thr Ser Asp Ser Ile Asn Tyr Ile Lys

1 5 10 15

Asn Glu Phe Lys Leu Ile Glu Ser Ile Ser Asp Ala Leu Tyr Asp Leu

20 25 30

Lys Gln Gln Asn Glu Leu Glu Glu Ser His Phe Ile Ser Phe Glu Asp

35 40 45

Ile Ser Lys Thr Asp Glu Gly Phe Ser Ile Arg Phe Ile Asp Lys Glu

50 55 60

Thr Gly Glu Ser Ile Phe Val Glu Thr Glu Lys Ala Ile Phe Ser Glu

65 70 75 80

Tyr Ala Asn His Ile Thr Glu Glu Ile Ser Lys Leu Lys Asp Thr Ile

85 90 95

Phe Asp Thr Val Asn Gly Lys Leu Val Lys Lys Val Thr Leu Asp Ala

100 105 110

Thr His Glu Val Asn Thr Leu Asn Ala Ala Phe Phe Ile Gln Ser Leu

115 120 125

Ile Gly Tyr Asn Ser Ser Lys Glu Ser Leu Ser Asn Leu Ser Val Ala

130 135 140

Met Lys Val Gln Val Tyr Ala Gln Leu Phe Ser Thr Gly Leu Asn Thr

145 150 155 160

Ile Thr Asp Ala Ala Lys Val Val Glu Leu Val Ser Thr Ala Leu Asp

165 170 175

Glu Thr Ile Asp Leu Leu Pro Thr Leu Ser Glu Gly Leu Pro Val Ile

180 185 190

Ala Thr Ile Ile Asp Gly Val Ser Leu Gly Ala Ser Ile Lys Glu Leu

195 200 205

Ser Glu Thr Ser Asp Pro Leu Leu Arg Gln Glu Ile Glu Ala Lys Ile

210 215 220

Gly Ile Met Ala Val Asn Leu Thr Ala Ala Thr Thr Ala Ile Ile Thr

225 230 235 240

Ser Ser Leu Gly Ile Ala Ser Gly Phe Ser Ile Leu Leu Val Pro Leu

245 250 255

Ala Gly Ile Ser Ala Gly Ile Pro Ser Leu Val Asn Asn Glu Leu Ile

260 265 270

Leu Arg Ala Glu Ala Lys Asn Val Val Asp Tyr Phe Gly His Ile Ser

275 280 285

Leu Ala Glu Ser Glu Gly Ala Phe Thr Leu Leu Asp Asp Lys Ile Met

290 295 300

Met Pro Gln Asp Asp Leu Val Ile Ser Glu Ile Asp Phe Asn Asn Asn

305 310 315 320

Ser Ile Thr Leu Gly Lys Cys Glu Ile Trp Arg Met Glu Gly Gly Ser

325 330 335

Gly His Thr Val Thr Asp Asp Ile Asp His Phe Phe Ser Ala Pro Ser

340 345 350

Thr Thr Tyr Arg Glu Pro Tyr Leu Ser Ile Tyr Asp Val Leu Asp Val

355 360 365

Lys Glu Glu Glu Leu Asp Leu Ser Lys Asp Leu Met Val Leu Pro Asn

370 375 380

Ala Pro Asp Arg Ile Phe Gly Trp Glu Arg Gly Trp Thr Pro Gly Leu

385 390 395 400

Arg Ser Leu Glu Asn Asp Gly Thr Lys Leu Leu Asp Arg Ile Arg Asp

405 410 415

His Tyr Glu Gly Gln Phe Tyr Trp Arg Phe Phe Ala Phe Ile Ala Asp

420 425 430

Ser Val Ile Thr Lys Leu Lys Pro Arg Tyr Glu Asp Thr Asn Ile Arg

435 440 445

Ile Ser Leu Asp Ser Asn Thr Arg Ser Phe Ile Val Pro Val Ile Thr

450 455 460

Thr Glu Tyr Ile Arg Glu Lys Leu Ser Tyr Ser Phe Tyr Gly Ser Gly

465 470 475 480

Gly Thr Tyr Ala Leu Ser Leu Ser Gln Tyr Asn Met Asn Ile Asn Ile

485 490 495

Glu Leu Asn Glu Asn Asp Thr Trp Val Ile Asp Val Asp Asn Val Val

500 505 510

Arg Asp Val Thr Ile Glu Ser Asp Lys Ile Lys Lys Gly Asp Leu Ile

515 520 525

Glu Asn Ile Leu Ser Lys Leu Ser Ile Glu Asp Asn Lys Ile Ile Leu

530 535 540

Asp Asn His Glu Ile Asn Phe Ser Gly Thr Leu Asn Gly Gly Asn Gly

545 550 555 560

Phe Val Ser Leu Thr Phe Ser Ile Leu Glu Gly Ile Asn Ala Val Ile

565 570 575

Glu Val Asp Leu Leu Ser Lys Ser Tyr Lys Val Leu Ile Ser Gly Glu

580 585 590

Leu Lys Thr Leu Met Ala Asn Ser Asn Ser Val Gln Gln Lys Ile Asp

595 600 605

Tyr Ile Gly Leu Asn Ser Glu Leu Gln Lys Asn Ile Pro Tyr Ser Phe

610 615 620

Met Asp Asp Glu Gly Lys Glu Asn Gly Phe Ile Asn Cys Phe Thr Lys

625 630 635 640

Glu Gly Leu Phe Val Ser Glu Leu Ser Asp Val Val Leu Ile Ile Lys

645 650 655

Val Tyr Met Asp Asn Ser Lys Pro Pro Phe Gly Tyr Tyr Ser Asn Asp

660 665 670

Leu Lys Asp Val Lys Val Ile Thr Lys Asp Asp Val Ile Ile Ile Thr

675 680 685

Gly Tyr Tyr Leu Lys Asp Asp Ile Lys Ile Ser Leu Ser Phe Thr Ile

690 695 700

Gln Asp Lys Asn Thr Ile Lys Leu Asn Gly Val Tyr Leu Asp Glu Asn

705 710 715 720

Gly Val Ala Glu Ile Leu Lys Phe Met Asn Lys Lys Gly Ser Thr Asn

725 730 735

Thr Ser Asp Ser Leu Met Ser Phe Leu Glu Ser Met Asn Ile Lys Ser

740 745 750

Ile Phe Ile Lys Ser Leu Lys Ser Asn Ala Lys Leu Ile Leu Asp Thr

755 760 765

Asn Phe Ile Ile Ser Gly Thr Thr Phe Ile Gly Gln Phe Glu Phe Ile

770 775 780

Cys Asp Lys Asp Asn Asn Ile Gln Pro Tyr Phe Ile Lys Phe Asn Thr

785 790 795 800

Leu Glu Thr Lys Tyr Thr Leu Tyr Val Gly Asn Arg Gln Asn Met Ile

805 810 815

Val Glu Pro Asn Tyr Asn Leu Asp Asp Ser Gly Asp Ile Ser Ser Thr

820 825 830

Val Ile Asn Phe Ser Gln Lys Tyr Leu Tyr Gly Ile Asp Ser Cys Val

835 840 845

Asn Lys Val Val Ile Ser Pro Gly Ile Tyr Thr Asp Glu Ile Asn Ile

850 855 860

Thr Pro Val His Glu Ala Asn Asn Thr Tyr Pro Glu Val Ile Val Leu

865 870 875 880

Asp Thr Asn Tyr Ile Ser Glu Lys Ile Asn Ile Asn Ile Asn Asp Leu

885 890 895

Ser Ile Arg Tyr Val Trp Ser Asn Asp Gly Ser Asp Phe Ile Leu Met

900 905 910

Ser Thr Asp Glu Glu Asn Lys Val Ser Gln Val Lys Ile Arg Phe Thr

915 920 925

Asn Val Phe Lys Gly Asn Thr Ile Ser Asp Lys Ile Ser Phe Asn Phe

930 935 940

Ser Asp Lys Gln Asp Ile Ser Ile Asn Lys Ile Ile Ser Thr Phe Thr

945 950 955 960

Pro Ser Tyr Tyr Val Glu Gly Leu Leu Asn Tyr Asp Leu Gly Leu Ile

965 970 975

Ser Leu Tyr Asn Glu Lys Phe Tyr Ile Asn Asn Leu Gly Met Met

980 985 990

<210> 16

<211> 550

<212> PRT

<213> Artificial sequence

<220>

<223> Fragments of 1285 to 1834 bits of TcdB4

<400> 16

Asp Thr Asn Ile Arg Ile Ser Leu Asp Ser Asn Thr Arg Ser Phe Ile

1 5 10 15

Val Pro Val Ile Thr Thr Glu Tyr Ile Arg Glu Lys Leu Ser Tyr Ser

20 25 30

Phe Tyr Gly Ser Gly Gly Thr Tyr Ala Leu Ser Leu Ser Gln Tyr Asn

35 40 45

Met Asn Ile Asn Ile Glu Leu Asn Glu Asn Asp Thr Trp Val Ile Asp

50 55 60

Val Asp Asn Val Val Arg Asp Val Thr Ile Glu Ser Asp Lys Ile Lys

65 70 75 80

Lys Gly Asp Leu Ile Glu Asn Ile Leu Ser Lys Leu Ser Ile Glu Asp

85 90 95

Asn Lys Ile Ile Leu Asp Asn His Glu Ile Asn Phe Ser Gly Thr Leu

100 105 110

Asn Gly Gly Asn Gly Phe Val Ser Leu Thr Phe Ser Ile Leu Glu Gly

115 120 125

Ile Asn Ala Val Ile Glu Val Asp Leu Leu Ser Lys Ser Tyr Lys Val

130 135 140

Leu Ile Ser Gly Glu Leu Lys Thr Leu Met Ala Asn Ser Asn Ser Val

145 150 155 160

Gln Gln Lys Ile Asp Tyr Ile Gly Leu Asn Ser Glu Leu Gln Lys Asn

165 170 175

Ile Pro Tyr Ser Phe Met Asp Asp Glu Gly Lys Glu Asn Gly Phe Ile

180 185 190

Asn Cys Phe Thr Lys Glu Gly Leu Phe Val Ser Glu Leu Ser Asp Val

195 200 205

Val Leu Ile Ile Lys Val Tyr Met Asp Asn Ser Lys Pro Pro Phe Gly

210 215 220

Tyr Tyr Ser Asn Asp Leu Lys Asp Val Lys Val Ile Thr Lys Asp Asp

225 230 235 240

Val Ile Ile Ile Thr Gly Tyr Tyr Leu Lys Asp Asp Ile Lys Ile Ser

245 250 255

Leu Ser Phe Thr Ile Gln Asp Lys Asn Thr Ile Lys Leu Asn Gly Val

260 265 270

Tyr Leu Asp Glu Asn Gly Val Ala Glu Ile Leu Lys Phe Met Asn Lys

275 280 285

Lys Gly Ser Thr Asn Thr Ser Asp Ser Leu Met Ser Phe Leu Glu Ser

290 295 300

Met Asn Ile Lys Ser Ile Phe Ile Lys Ser Leu Lys Ser Asn Ala Lys

305 310 315 320

Leu Ile Leu Asp Thr Asn Phe Ile Ile Ser Gly Thr Thr Phe Ile Gly

325 330 335

Gln Phe Glu Phe Ile Cys Asp Lys Asp Asn Asn Ile Gln Pro Tyr Phe

340 345 350

Ile Lys Phe Asn Thr Leu Glu Thr Lys Tyr Thr Leu Tyr Val Gly Asn

355 360 365

Arg Gln Asn Met Ile Val Glu Pro Asn Tyr Asn Leu Asp Asp Ser Gly

370 375 380

Asp Ile Ser Ser Thr Val Ile Asn Phe Ser Gln Lys Tyr Leu Tyr Gly

385 390 395 400

Ile Asp Ser Cys Val Asn Lys Val Val Ile Ser Pro Gly Ile Tyr Thr

405 410 415

Asp Glu Ile Asn Ile Thr Pro Val His Glu Ala Asn Asn Thr Tyr Pro

420 425 430

Glu Val Ile Val Leu Asp Thr Asn Tyr Ile Ser Glu Lys Ile Asn Ile

435 440 445

Asn Ile Asn Asp Leu Ser Ile Arg Tyr Val Trp Ser Asn Asp Gly Ser

450 455 460

Asp Phe Ile Leu Met Ser Thr Asp Glu Glu Asn Lys Val Ser Gln Val

465 470 475 480

Lys Ile Arg Phe Thr Asn Val Phe Lys Gly Asn Thr Ile Ser Asp Lys

485 490 495

Ile Ser Phe Asn Phe Ser Asp Lys Gln Asp Ile Ser Ile Asn Lys Ile

500 505 510

Ile Ser Thr Phe Thr Pro Ser Tyr Tyr Val Glu Gly Leu Leu Asn Tyr

515 520 525

Asp Leu Gly Leu Ile Ser Leu Tyr Asn Glu Lys Phe Tyr Ile Asn Asn

530 535 540

Leu Gly Met Met Val Ser

545 550

<210> 17

<211> 181

<212> PRT

<213> Artificial sequence

<220>

<223> TFPIK1+K2

<400> 17

Asp Ser Glu Glu Asp Glu Glu His Thr Ile Ile Thr Asp Thr Glu Leu

1 5 10 15

Pro Pro Leu Lys Leu Met His Ser Phe Cys Ala Phe Lys Ala Asp Asp

20 25 30

Gly Pro Cys Lys Ala Ile Met Lys Arg Phe Phe Phe Asn Ile Phe Thr

35 40 45

Arg Gln Cys Glu Glu Phe Ile Tyr Gly Gly Cys Glu Gly Asn Gln Asn

50 55 60

Arg Phe Glu Ser Leu Glu Glu Cys Lys Lys Met Cys Thr Arg Asp Asn

65 70 75 80

Ala Asn Arg Ile Ile Lys Thr Thr Leu Gln Gln Glu Lys Pro Asp Phe

85 90 95

Cys Phe Leu Glu Glu Asp Pro Gly Ile Cys Arg Gly Tyr Ile Thr Arg

100 105 110

Tyr Phe Tyr Asn Asn Gln Thr Lys Gln Cys Glu Arg Phe Lys Tyr Gly

115 120 125

Gly Cys Leu Gly Asn Met Asn Asn Phe Glu Thr Leu Glu Glu Cys Lys

130 135 140

Asn Ile Cys Glu Asp Gly Pro Asn Gly Phe Gln Val Asp Asn Tyr Gly

145 150 155 160

Thr Gln Leu Asn Ala Val Asn Asn Ser Leu Thr Pro Gln Ser Thr Lys

165 170 175

Val Pro Ser Leu Phe

180

<210> 18

<211> 51

<212> PRT

<213> Artificial sequence

<220>

<223> TFPIK1

<400> 18

Cys Ala Phe Lys Ala Asp Asp Gly Pro Cys Lys Ala Ile Met Lys Arg

1 5 10 15

Phe Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu Phe Ile Tyr Gly

20 25 30

Gly Cys Glu Gly Asn Gln Asn Arg Phe Glu Ser Leu Glu Glu Cys Lys

35 40 45

Lys Met Cys

50

<210> 19

<211> 51

<212> PRT

<213> Artificial sequence

<220>

<223> TfpiK1

<400> 19

Cys Ala Met Lys Ala Asp Asp Gly Pro Cys Lys Ala Met Ile Arg Ser

1 5 10 15

Tyr Phe Phe Asn Met Tyr Thr His Gln Cys Glu Glu Phe Ile Tyr Gly

20 25 30

Gly Cys Glu Gly Asn Glu Asn Arg Phe Asp Thr Leu Glu Glu Cys Lys

35 40 45

Lys Thr Cys

50

<210> 20

<211> 142

<212> PRT

<213> Artificial sequence

<220>

<223> CSPG4R1

<400> 20

Glu Leu Pro Glu Pro Cys Val Pro Glu Pro Gly Leu Pro Pro Val Phe

1 5 10 15

Ala Asn Phe Thr Gln Leu Leu Thr Ile Ser Pro Leu Val Val Ala Glu

20 25 30

Gly Gly Thr Ala Trp Leu Glu Trp Arg His Val Gln Pro Thr Leu Asp

35 40 45

Leu Met Glu Ala Glu Leu Arg Lys Ser Gln Val Leu Phe Ser Val Thr

50 55 60

Arg Gly Ala Arg His Gly Glu Leu Glu Leu Asp Ile Pro Gly Ala Gln

65 70 75 80

Ala Arg Lys Met Phe Thr Leu Leu Asp Val Val Asn Arg Lys Ala Arg

85 90 95

Phe Ile His Asp Gly Ser Glu Asp Thr Ser Asp Gln Leu Val Leu Glu

100 105 110

Val Ser Val Thr Ala Arg Val Pro Met Pro Ser Cys Leu Arg Arg Gly

115 120 125

Gln Thr Tyr Leu Leu Pro Ile Gln Val Asn Pro Val Asn Asp

130 135 140

<210> 21

<211> 208

<212> PRT

<213> Artificial sequence

<220>

<223> TFPIK2-CSPG4R1

<400> 21

Cys Phe Leu Glu Glu Asp Pro Gly Ile Cys Arg Gly Tyr Ile Thr Arg

1 5 10 15

Tyr Phe Tyr Asn Asn Gln Thr Lys Gln Cys Glu Arg Phe Lys Tyr Gly

20 25 30

Gly Cys Leu Gly Asn Met Asn Asn Phe Glu Thr Leu Glu Glu Cys Lys

35 40 45

Asn Ile Cys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

50 55 60

Gly Ser Glu Leu Pro Glu Pro Cys Val Pro Glu Pro Gly Leu Pro Pro

65 70 75 80

Val Phe Ala Asn Phe Thr Gln Leu Leu Thr Ile Ser Pro Leu Val Val

85 90 95

Ala Glu Gly Gly Thr Ala Trp Leu Glu Trp Arg His Val Gln Pro Thr

100 105 110

Leu Asp Leu Met Glu Ala Glu Leu Arg Lys Ser Gln Val Leu Phe Ser

115 120 125

Val Thr Arg Gly Ala Arg His Gly Glu Leu Glu Leu Asp Ile Pro Gly

130 135 140

Ala Gln Ala Arg Lys Met Phe Thr Leu Leu Asp Val Val Asn Arg Lys

145 150 155 160

Ala Arg Phe Ile His Asp Gly Ser Glu Asp Thr Ser Asp Gln Leu Val

165 170 175

Leu Glu Val Ser Val Thr Ala Arg Val Pro Met Pro Ser Cys Leu Arg

180 185 190

Arg Gly Gln Thr Tyr Leu Leu Pro Ile Gln Val Asn Pro Val Asn Asp

195 200 205

<210> 22

<211> 304

<212> PRT

<213> Artificial sequence

<220>

<223> TFPI alpha containing Signal peptide

<400> 22

Met Ile Tyr Thr Met Lys Lys Val His Ala Leu Trp Ala Ser Val Cys

1 5 10 15

Leu Leu Leu Asn Leu Ala Pro Ala Pro Leu Asn Ala Asp Ser Glu Glu

20 25 30

Asp Glu Glu His Thr Ile Ile Thr Asp Thr Glu Leu Pro Pro Leu Lys

35 40 45

Leu Met His Ser Phe Cys Ala Phe Lys Ala Asp Asp Gly Pro Cys Lys

50 55 60

Ala Ile Met Lys Arg Phe Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu

65 70 75 80

Glu Phe Ile Tyr Gly Gly Cys Glu Gly Asn Gln Asn Arg Phe Glu Ser

85 90 95

Leu Glu Glu Cys Lys Lys Met Cys Thr Arg Asp Asn Ala Asn Arg Ile

100 105 110

Ile Lys Thr Thr Leu Gln Gln Glu Lys Pro Asp Phe Cys Phe Leu Glu

115 120 125

Glu Asp Pro Gly Ile Cys Arg Gly Tyr Ile Thr Arg Tyr Phe Tyr Asn

130 135 140

Asn Gln Thr Lys Gln Cys Glu Arg Phe Lys Tyr Gly Gly Cys Leu Gly

145 150 155 160

Asn Met Asn Asn Phe Glu Thr Leu Glu Glu Cys Lys Asn Ile Cys Glu

165 170 175

Asp Gly Pro Asn Gly Phe Gln Val Asp Asn Tyr Gly Thr Gln Leu Asn

180 185 190

Ala Val Asn Asn Ser Leu Thr Pro Gln Ser Thr Lys Val Pro Ser Leu

195 200 205

Phe Glu Phe His Gly Pro Ser Trp Cys Leu Thr Pro Ala Asp Arg Gly

210 215 220

Leu Cys Arg Ala Asn Glu Asn Arg Phe Tyr Tyr Asn Ser Val Ile Gly

225 230 235 240

Lys Cys Arg Pro Phe Lys Tyr Ser Gly Cys Gly Gly Asn Glu Asn Asn

245 250 255

Phe Thr Ser Lys Gln Glu Cys Leu Arg Ala Cys Lys Lys Gly Phe Ile

260 265 270

Gln Arg Ile Ser Lys Gly Gly Leu Ile Lys Thr Lys Arg Lys Arg Lys

275 280 285

Lys Gln Arg Val Lys Ile Ala Tyr Glu Glu Ile Phe Val Lys Asn Met

290 295 300

<210> 23

<211> 251

<212> PRT

<213> Artificial sequence

<220>

<223> TFPI beta containing Signal peptide

<400> 23

Met Ile Tyr Thr Met Lys Lys Val His Ala Leu Trp Ala Ser Val Cys

1 5 10 15

Leu Leu Leu Asn Leu Ala Pro Ala Pro Leu Asn Ala Asp Ser Glu Glu

20 25 30

Asp Glu Glu His Thr Ile Ile Thr Asp Thr Glu Leu Pro Pro Leu Lys

35 40 45

Leu Met His Ser Phe Cys Ala Phe Lys Ala Asp Asp Gly Pro Cys Lys

50 55 60

Ala Ile Met Lys Arg Phe Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu

65 70 75 80

Glu Phe Ile Tyr Gly Gly Cys Glu Gly Asn Gln Asn Arg Phe Glu Ser

85 90 95

Leu Glu Glu Cys Lys Lys Met Cys Thr Arg Asp Asn Ala Asn Arg Ile

100 105 110

Ile Lys Thr Thr Leu Gln Gln Glu Lys Pro Asp Phe Cys Phe Leu Glu

115 120 125

Glu Asp Pro Gly Ile Cys Arg Gly Tyr Ile Thr Arg Tyr Phe Tyr Asn

130 135 140

Asn Gln Thr Lys Gln Cys Glu Arg Phe Lys Tyr Gly Gly Cys Leu Gly

145 150 155 160

Asn Met Asn Asn Phe Glu Thr Leu Glu Glu Cys Lys Asn Ile Cys Glu

165 170 175

Asp Gly Pro Asn Gly Phe Gln Val Asp Asn Tyr Gly Thr Gln Leu Asn

180 185 190

Ala Val Asn Asn Ser Leu Thr Pro Gln Ser Thr Lys Val Pro Ser Leu

195 200 205

Phe Val Thr Lys Glu Gly Thr Asn Asp Gly Trp Lys Asn Ala Ala His

210 215 220

Ile Tyr Gln Val Phe Leu Asn Ala Phe Cys Ile His Ala Ser Met Phe

225 230 235 240

Phe Leu Gly Leu Asp Ser Ile Ser Cys Leu Cys

245 250

<210> 24

<211> 238

<212> PRT

<213> Artificial sequence

<220>

<223> Fc of human IgG1

<400> 24

Gly Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

1 5 10 15

Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro

20 25 30

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

35 40 45

Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val

50 55 60

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

65 70 75 80

Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln

85 90 95

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala

100 105 110

Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

115 120 125

Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr

130 135 140

Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser

145 150 155 160

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

165 170 175

Lys Ala Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr

180 185 190

Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe

195 200 205

Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys

210 215 220

Ser Leu Ser Leu Ser Pro Gly Lys His His His His His His

225 230 235

<210> 25

<211> 356

<212> PRT

<213> Artificial sequence

<220>

<223> TFPIK1-Fc

<400> 25

Met Gly Ile Leu Pro Ser Pro Gly Met Pro Ala Leu Leu Ser Leu Val

1 5 10 15

Ser Leu Leu Ser Val Leu Leu Met Gly Cys Val Ala Glu Thr Gly Asp

20 25 30

Thr Glu Leu Pro Pro Leu Lys Leu Met His Ser Phe Cys Ala Phe Lys

35 40 45

Ala Asp Asp Gly Pro Cys Lys Ala Ile Met Lys Arg Phe Phe Phe Asn

50 55 60

Ile Phe Thr Arg Gln Cys Glu Glu Phe Ile Tyr Gly Gly Cys Glu Gly

65 70 75 80

Asn Gln Asn Arg Phe Glu Ser Leu Glu Glu Cys Lys Lys Met Cys Thr

85 90 95

Arg Asp Asn Ala Asn Arg Ile Ile Lys Thr Thr Leu Gln Gln Gly Thr

100 105 110

Leu Glu Val Leu Phe Gln Gly Pro Lys Ser Cys Asp Lys Thr His Thr

115 120 125

Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe

130 135 140

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro

145 150 155 160

Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val

165 170 175

Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr

180 185 190

Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val

195 200 205

Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys

210 215 220

Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser

225 230 235 240

Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro

245 250 255

Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val

260 265 270

Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly

275 280 285

Gln Pro Glu Asn Asn Tyr Lys Ala Thr Pro Pro Val Leu Asp Ser Asp

290 295 300

Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp

305 310 315 320

Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

325 330 335

Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys His His

340 345 350

His His His His

355

<210> 26

<211> 367

<212> PRT

<213> Artificial sequence

<220>

<223> TFPIK2-Fc

<400> 26

Met Gly Ile Leu Pro Ser Pro Gly Met Pro Ala Leu Leu Ser Leu Val

1 5 10 15

Ser Leu Leu Ser Val Leu Leu Met Gly Cys Val Ala Glu Thr Gly Glu

20 25 30

Lys Pro Asp Phe Cys Phe Leu Glu Glu Asp Pro Gly Ile Cys Arg Gly

35 40 45

Tyr Ile Thr Arg Tyr Phe Tyr Asn Asn Gln Thr Lys Gln Cys Glu Arg

50 55 60

Phe Lys Tyr Gly Gly Cys Leu Gly Asn Met Asn Asn Phe Glu Thr Leu

65 70 75 80

Glu Glu Cys Lys Asn Ile Cys Glu Asp Gly Pro Asn Gly Phe Gln Val

85 90 95

Asp Asn Tyr Gly Thr Gln Leu Asn Ala Val Asn Asn Ser Leu Thr Pro

100 105 110

Gln Ser Thr Lys Val Pro Ser Leu Phe Gly Thr Leu Glu Val Leu Phe

115 120 125

Gln Gly Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro

130 135 140

Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys

145 150 155 160

Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val

165 170 175

Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr

180 185 190

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

195 200 205

Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His

210 215 220

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

225 230 235 240

Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln

245 250 255

Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu

260 265 270

Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro

275 280 285

Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn

290 295 300

Tyr Lys Ala Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu

305 310 315 320

Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val

325 330 335

Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln

340 345 350

Lys Ser Leu Ser Leu Ser Pro Gly Lys His His His His His His

355 360 365

<210> 27

<211> 351

<212> PRT

<213> Artificial sequence

<220>

<223> TfpiK2-Fc

<400> 27

Met Gly Ile Leu Pro Ser Pro Gly Met Pro Ala Leu Leu Ser Leu Val

1 5 10 15

Ser Leu Leu Ser Val Leu Leu Met Gly Cys Val Ala Glu Thr Gly Tyr

20 25 30

Glu Lys Thr Ala Val Lys Ala Ala Ser Gly Ala Glu Arg Pro Asp Phe

35 40 45

Cys Phe Leu Glu Glu Asp Pro Gly Leu Cys Arg Gly Tyr Met Lys Arg

50 55 60

Tyr Leu Tyr Asn Asn Gln Thr Lys Gln Cys Glu Arg Phe Val Tyr Gly

65 70 75 80

Gly Cys Leu Gly Asn Arg Asn Asn Phe Glu Thr Leu Asp Glu Cys Lys

85 90 95

Lys Ile Cys Glu Asn Pro Val His Ser Gly Thr Leu Glu Val Leu Phe

100 105 110

Gln Gly Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro

115 120 125

Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys

130 135 140

Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val

145 150 155 160

Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr

165 170 175

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

180 185 190

Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His

195 200 205

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

210 215 220

Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln

225 230 235 240

Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu

245 250 255

Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro

260 265 270

Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn

275 280 285

Tyr Lys Ala Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu

290 295 300

Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val

305 310 315 320

Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln

325 330 335

Lys Ser Leu Ser Leu Ser Pro Gly Lys His His His His His His

340 345 350

<210> 28

<211> 994

<212> PRT

<213> Artificial sequence

<220>

<223> Receptor binding domain of TcdB2 (DRBD)

<400> 28

Arg Ile Glu Glu Ala Lys Ser Leu Thr Ser Asp Ser Ile Asn Tyr Ile

1 5 10 15

Lys Asn Glu Phe Lys Leu Ile Glu Ser Ile Ser Asp Ala Leu Tyr Asp

20 25 30

Leu Lys Gln Gln Asn Glu Leu Glu Glu Ser His Phe Ile Ser Phe Glu

35 40 45

Asp Ile Leu Glu Thr Asp Glu Gly Phe Ser Ile Arg Phe Ile Asp Lys

50 55 60

Glu Thr Gly Glu Ser Ile Phe Val Glu Thr Glu Lys Ala Ile Phe Ser

65 70 75 80

Glu Tyr Ala Asn His Ile Thr Glu Glu Ile Ser Lys Ile Lys Gly Thr

85 90 95

Ile Phe Asp Thr Val Asn Gly Lys Leu Val Lys Lys Val Asn Leu Asp

100 105 110

Ala Thr His Glu Val Asn Thr Leu Asn Ala Ala Phe Phe Ile Gln Ser

115 120 125

Leu Ile Glu Tyr Asn Ser Ser Lys Glu Ser Leu Ser Asn Leu Ser Val

130 135 140

Ala Met Lys Val Gln Val Tyr Ala Gln Leu Phe Ser Thr Gly Leu Asn

145 150 155 160

Thr Ile Thr Asp Ala Ala Lys Val Val Glu Leu Val Ser Thr Ala Leu

165 170 175

Asp Glu Thr Ile Asp Leu Leu Pro Thr Leu Ser Glu Gly Leu Pro Val

180 185 190

Ile Ala Thr Ile Ile Asp Gly Val Ser Leu Gly Ala Ala Ile Lys Glu

195 200 205

Leu Ser Glu Thr Ser Asp Pro Leu Leu Arg Gln Glu Ile Glu Ala Lys

210 215 220

Ile Gly Ile Met Ala Val Asn Leu Thr Ala Ala Thr Thr Ala Ile Ile

225 230 235 240

Thr Ser Ser Leu Gly Ile Ala Ser Gly Phe Ser Ile Leu Leu Val Pro

245 250 255

Leu Ala Gly Ile Ser Ala Gly Ile Pro Ser Leu Val Asn Asn Glu Leu

260 265 270

Ile Leu Arg Asp Lys Ala Thr Lys Val Val Asp Tyr Phe Ser His Ile

275 280 285

Ser Leu Ala Glu Ser Glu Gly Ala Phe Thr Ser Leu Asp Asp Lys Ile

290 295 300

Met Met Pro Gln Asp Asp Leu Val Ile Ser Glu Ile Asp Phe Asn Asn

305 310 315 320

Asn Ser Ile Thr Leu Gly Lys Cys Glu Ile Trp Arg Met Glu Gly Gly

325 330 335

Ser Gly His Thr Val Thr Asp Asp Ile Asp His Phe Phe Ser Ala Pro

340 345 350

Ser Ile Thr Tyr Arg Glu Pro His Leu Ser Ile Tyr Asp Val Leu Glu

355 360 365

Val Gln Lys Glu Glu Leu Asp Leu Ser Lys Asp Leu Met Val Leu Pro

370 375 380

Asn Ala Pro Asn Arg Val Phe Ala Trp Glu Thr Gly Trp Thr Pro Gly

385 390 395 400

Leu Arg Ser Leu Glu Asn Asp Gly Thr Lys Leu Leu Asp Arg Ile Arg

405 410 415

Asp Asn Tyr Glu Gly Glu Phe Tyr Trp Arg Tyr Phe Ala Phe Ile Ala

420 425 430

Asp Ala Leu Ile Thr Thr Leu Lys Pro Arg Tyr Glu Asp Thr Asn Ile

435 440 445

Arg Ile Asn Leu Asp Ser Asn Thr Arg Ser Phe Ile Val Pro Val Ile

450 455 460

Thr Thr Glu Tyr Ile Arg Glu Lys Leu Ser Tyr Ser Phe Tyr Gly Ser

465 470 475 480

Gly Gly Thr Tyr Ala Leu Ser Leu Ser Gln Tyr Asn Met Asn Ile Asn

485 490 495

Ile Glu Leu Asn Glu Asn Asp Thr Trp Val Ile Asp Val Asp Asn Val

500 505 510

Val Arg Asp Val Thr Ile Glu Ser Asp Lys Ile Lys Lys Gly Asp Leu

515 520 525

Ile Glu Asn Ile Leu Ser Lys Leu Ser Ile Glu Asp Asn Lys Ile Ile

530 535 540

Leu Asp Asn His Glu Ile Asn Phe Ser Gly Thr Leu Asn Gly Gly Asn

545 550 555 560

Gly Phe Val Ser Leu Thr Phe Ser Ile Leu Glu Gly Ile Asn Ala Val

565 570 575

Ile Glu Val Asp Leu Leu Ser Lys Ser Tyr Lys Val Leu Ile Ser Gly

580 585 590

Glu Leu Lys Thr Leu Met Ala Asn Ser Asn Ser Val Gln Gln Lys Ile

595 600 605

Asp Tyr Ile Gly Leu Asn Ser Glu Leu Gln Lys Asn Ile Pro Tyr Ser

610 615 620

Phe Met Asp Asp Lys Gly Lys Glu Asn Gly Phe Ile Asn Cys Ser Thr

625 630 635 640

Lys Glu Gly Leu Phe Val Ser Glu Leu Ser Asp Val Val Leu Ile Ser

645 650 655

Lys Val Tyr Met Asp Asn Ser Lys Pro Leu Phe Gly Tyr Cys Ser Asn

660 665 670

Asp Leu Lys Asp Val Lys Val Ile Thr Lys Asp Asp Val Ile Ile Leu

675 680 685

Thr Gly Tyr Tyr Leu Lys Asp Asp Ile Lys Ile Ser Leu Ser Phe Thr

690 695 700

Ile Gln Asp Glu Asn Thr Ile Lys Leu Asn Gly Val Tyr Leu Asp Glu

705 710 715 720

Asn Gly Val Ala Glu Ile Leu Lys Phe Met Asn Lys Lys Gly Ser Thr

725 730 735

Asn Thr Ser Asp Ser Leu Met Ser Phe Leu Glu Ser Met Asn Ile Lys

740 745 750

Ser Ile Phe Ile Asn Ser Leu Gln Ser Asn Thr Lys Leu Ile Leu Asp

755 760 765

Thr Asn Phe Ile Ile Ser Gly Thr Thr Ser Ile Gly Gln Phe Glu Phe

770 775 780

Ile Cys Asp Lys Asp Asn Asn Ile Gln Pro Tyr Phe Ile Lys Phe Asn

785 790 795 800

Thr Leu Glu Thr Lys Tyr Thr Leu Tyr Val Gly Asn Arg Gln Asn Met

805 810 815

Ile Val Glu Pro Asn Tyr Asp Leu Asp Asp Ser Gly Asp Ile Ser Ser

820 825 830

Thr Val Ile Asn Phe Ser Gln Lys Tyr Leu Tyr Gly Ile Asp Ser Cys

835 840 845

Val Asn Lys Val Ile Ile Ser Pro Asn Ile Tyr Thr Asp Glu Ile Asn

850 855 860

Ile Thr Pro Ile Tyr Glu Ala Asn Asn Thr Tyr Pro Glu Val Ile Val

865 870 875 880

Leu Asp Thr Asn Tyr Ile Ser Glu Lys Ile Asn Ile Asn Ile Asn Asp

885 890 895

Leu Ser Ile Arg Tyr Val Trp Ser Asn Asp Gly Ser Asp Phe Ile Leu

900 905 910

Met Ser Thr Asp Glu Glu Asn Lys Val Ser Gln Val Lys Ile Arg Phe

915 920 925

Thr Asn Val Phe Lys Gly Asn Thr Ile Ser Asp Lys Ile Ser Phe Asn

930 935 940

Phe Ser Asp Lys Gln Asp Val Ser Ile Asn Lys Val Ile Ser Thr Phe

945 950 955 960

Thr Pro Ser Tyr Tyr Val Glu Gly Leu Leu Asn Tyr Asp Leu Gly Leu

965 970 975

Ile Ser Leu Tyr Asn Glu Lys Phe Tyr Ile Asn Asn Phe Gly Met Met

980 985 990

Val Ser

<210> 29

<211> 993

<212> PRT

<213> Artificial sequence

<220>

<223> Fragment of 841-1833 bits of TcdB2

<400> 29

Ile Glu Glu Ala Lys Ser Leu Thr Ser Asp Ser Ile Asn Tyr Ile Lys

1 5 10 15

Asn Glu Phe Lys Leu Ile Glu Ser Ile Ser Asp Ala Leu Tyr Asp Leu

20 25 30

Lys Gln Gln Asn Glu Leu Glu Glu Ser His Phe Ile Ser Phe Glu Asp

35 40 45

Ile Leu Glu Thr Asp Glu Gly Phe Ser Ile Arg Phe Ile Asp Lys Glu

50 55 60

Thr Gly Glu Ser Ile Phe Val Glu Thr Glu Lys Ala Ile Phe Ser Glu

65 70 75 80

Tyr Ala Asn His Ile Thr Glu Glu Ile Ser Lys Ile Lys Gly Thr Ile

85 90 95

Phe Asp Thr Val Asn Gly Lys Leu Val Lys Lys Val Asn Leu Asp Ala

100 105 110

Thr His Glu Val Asn Thr Leu Asn Ala Ala Phe Phe Ile Gln Ser Leu

115 120 125

Ile Glu Tyr Asn Ser Ser Lys Glu Ser Leu Ser Asn Leu Ser Val Ala

130 135 140

Met Lys Val Gln Val Tyr Ala Gln Leu Phe Ser Thr Gly Leu Asn Thr

145 150 155 160

Ile Thr Asp Ala Ala Lys Val Val Glu Leu Val Ser Thr Ala Leu Asp

165 170 175

Glu Thr Ile Asp Leu Leu Pro Thr Leu Ser Glu Gly Leu Pro Val Ile

180 185 190

Ala Thr Ile Ile Asp Gly Val Ser Leu Gly Ala Ala Ile Lys Glu Leu

195 200 205

Ser Glu Thr Ser Asp Pro Leu Leu Arg Gln Glu Ile Glu Ala Lys Ile

210 215 220

Gly Ile Met Ala Val Asn Leu Thr Ala Ala Thr Thr Ala Ile Ile Thr

225 230 235 240

Ser Ser Leu Gly Ile Ala Ser Gly Phe Ser Ile Leu Leu Val Pro Leu

245 250 255

Ala Gly Ile Ser Ala Gly Ile Pro Ser Leu Val Asn Asn Glu Leu Ile

260 265 270

Leu Arg Asp Lys Ala Thr Lys Val Val Asp Tyr Phe Ser His Ile Ser

275 280 285

Leu Ala Glu Ser Glu Gly Ala Phe Thr Ser Leu Asp Asp Lys Ile Met

290 295 300

Met Pro Gln Asp Asp Leu Val Ile Ser Glu Ile Asp Phe Asn Asn Asn

305 310 315 320

Ser Ile Thr Leu Gly Lys Cys Glu Ile Trp Arg Met Glu Gly Gly Ser

325 330 335

Gly His Thr Val Thr Asp Asp Ile Asp His Phe Phe Ser Ala Pro Ser

340 345 350

Ile Thr Tyr Arg Glu Pro His Leu Ser Ile Tyr Asp Val Leu Glu Val

355 360 365

Gln Lys Glu Glu Leu Asp Leu Ser Lys Asp Leu Met Val Leu Pro Asn

370 375 380

Ala Pro Asn Arg Val Phe Ala Trp Glu Thr Gly Trp Thr Pro Gly Leu

385 390 395 400

Arg Ser Leu Glu Asn Asp Gly Thr Lys Leu Leu Asp Arg Ile Arg Asp

405 410 415

Asn Tyr Glu Gly Glu Phe Tyr Trp Arg Tyr Phe Ala Phe Ile Ala Asp

420 425 430

Ala Leu Ile Thr Thr Leu Lys Pro Arg Tyr Glu Asp Thr Asn Ile Arg

435 440 445

Ile Asn Leu Asp Ser Asn Thr Arg Ser Phe Ile Val Pro Val Ile Thr

450 455 460

Thr Glu Tyr Ile Arg Glu Lys Leu Ser Tyr Ser Phe Tyr Gly Ser Gly

465 470 475 480

Gly Thr Tyr Ala Leu Ser Leu Ser Gln Tyr Asn Met Asn Ile Asn Ile

485 490 495

Glu Leu Asn Glu Asn Asp Thr Trp Val Ile Asp Val Asp Asn Val Val

500 505 510

Arg Asp Val Thr Ile Glu Ser Asp Lys Ile Lys Lys Gly Asp Leu Ile

515 520 525

Glu Asn Ile Leu Ser Lys Leu Ser Ile Glu Asp Asn Lys Ile Ile Leu

530 535 540

Asp Asn His Glu Ile Asn Phe Ser Gly Thr Leu Asn Gly Gly Asn Gly

545 550 555 560

Phe Val Ser Leu Thr Phe Ser Ile Leu Glu Gly Ile Asn Ala Val Ile

565 570 575

Glu Val Asp Leu Leu Ser Lys Ser Tyr Lys Val Leu Ile Ser Gly Glu

580 585 590

Leu Lys Thr Leu Met Ala Asn Ser Asn Ser Val Gln Gln Lys Ile Asp

595 600 605

Tyr Ile Gly Leu Asn Ser Glu Leu Gln Lys Asn Ile Pro Tyr Ser Phe

610 615 620

Met Asp Asp Lys Gly Lys Glu Asn Gly Phe Ile Asn Cys Ser Thr Lys

625 630 635 640

Glu Gly Leu Phe Val Ser Glu Leu Ser Asp Val Val Leu Ile Ser Lys

645 650 655

Val Tyr Met Asp Asn Ser Lys Pro Leu Phe Gly Tyr Cys Ser Asn Asp

660 665 670

Leu Lys Asp Val Lys Val Ile Thr Lys Asp Asp Val Ile Ile Leu Thr

675 680 685

Gly Tyr Tyr Leu Lys Asp Asp Ile Lys Ile Ser Leu Ser Phe Thr Ile

690 695 700

Gln Asp Glu Asn Thr Ile Lys Leu Asn Gly Val Tyr Leu Asp Glu Asn

705 710 715 720

Gly Val Ala Glu Ile Leu Lys Phe Met Asn Lys Lys Gly Ser Thr Asn

725 730 735

Thr Ser Asp Ser Leu Met Ser Phe Leu Glu Ser Met Asn Ile Lys Ser

740 745 750

Ile Phe Ile Asn Ser Leu Gln Ser Asn Thr Lys Leu Ile Leu Asp Thr

755 760 765

Asn Phe Ile Ile Ser Gly Thr Thr Ser Ile Gly Gln Phe Glu Phe Ile

770 775 780

Cys Asp Lys Asp Asn Asn Ile Gln Pro Tyr Phe Ile Lys Phe Asn Thr

785 790 795 800

Leu Glu Thr Lys Tyr Thr Leu Tyr Val Gly Asn Arg Gln Asn Met Ile

805 810 815

Val Glu Pro Asn Tyr Asp Leu Asp Asp Ser Gly Asp Ile Ser Ser Thr

820 825 830

Val Ile Asn Phe Ser Gln Lys Tyr Leu Tyr Gly Ile Asp Ser Cys Val

835 840 845

Asn Lys Val Ile Ile Ser Pro Asn Ile Tyr Thr Asp Glu Ile Asn Ile

850 855 860

Thr Pro Ile Tyr Glu Ala Asn Asn Thr Tyr Pro Glu Val Ile Val Leu

865 870 875 880

Asp Thr Asn Tyr Ile Ser Glu Lys Ile Asn Ile Asn Ile Asn Asp Leu

885 890 895

Ser Ile Arg Tyr Val Trp Ser Asn Asp Gly Ser Asp Phe Ile Leu Met

900 905 910

Ser Thr Asp Glu Glu Asn Lys Val Ser Gln Val Lys Ile Arg Phe Thr

915 920 925

Asn Val Phe Lys Gly Asn Thr Ile Ser Asp Lys Ile Ser Phe Asn Phe

930 935 940

Ser Asp Lys Gln Asp Val Ser Ile Asn Lys Val Ile Ser Thr Phe Thr

945 950 955 960

Pro Ser Tyr Tyr Val Glu Gly Leu Leu Asn Tyr Asp Leu Gly Leu Ile

965 970 975

Ser Leu Tyr Asn Glu Lys Phe Tyr Ile Asn Asn Phe Gly Met Met Val

980 985 990

Ser

<210> 30

<211> 550

<212> PRT

<213> Artificial sequence

<220>

<223> Fragment of 1284-1833 bits of TcdB2

<400> 30

Asp Thr Asn Ile Arg Ile Asn Leu Asp Ser Asn Thr Arg Ser Phe Ile

1 5 10 15

Val Pro Val Ile Thr Thr Glu Tyr Ile Arg Glu Lys Leu Ser Tyr Ser

20 25 30

Phe Tyr Gly Ser Gly Gly Thr Tyr Ala Leu Ser Leu Ser Gln Tyr Asn

35 40 45

Met Asn Ile Asn Ile Glu Leu Asn Glu Asn Asp Thr Trp Val Ile Asp

50 55 60

Val Asp Asn Val Val Arg Asp Val Thr Ile Glu Ser Asp Lys Ile Lys

65 70 75 80

Lys Gly Asp Leu Ile Glu Asn Ile Leu Ser Lys Leu Ser Ile Glu Asp

85 90 95

Asn Lys Ile Ile Leu Asp Asn His Glu Ile Asn Phe Ser Gly Thr Leu

100 105 110

Asn Gly Gly Asn Gly Phe Val Ser Leu Thr Phe Ser Ile Leu Glu Gly

115 120 125

Ile Asn Ala Val Ile Glu Val Asp Leu Leu Ser Lys Ser Tyr Lys Val

130 135 140

Leu Ile Ser Gly Glu Leu Lys Thr Leu Met Ala Asn Ser Asn Ser Val

145 150 155 160

Gln Gln Lys Ile Asp Tyr Ile Gly Leu Asn Ser Glu Leu Gln Lys Asn

165 170 175

Ile Pro Tyr Ser Phe Met Asp Asp Lys Gly Lys Glu Asn Gly Phe Ile

180 185 190

Asn Cys Ser Thr Lys Glu Gly Leu Phe Val Ser Glu Leu Ser Asp Val

195 200 205

Val Leu Ile Ser Lys Val Tyr Met Asp Asn Ser Lys Pro Leu Phe Gly

210 215 220

Tyr Cys Ser Asn Asp Leu Lys Asp Val Lys Val Ile Thr Lys Asp Asp

225 230 235 240

Val Ile Ile Leu Thr Gly Tyr Tyr Leu Lys Asp Asp Ile Lys Ile Ser

245 250 255

Leu Ser Phe Thr Ile Gln Asp Glu Asn Thr Ile Lys Leu Asn Gly Val

260 265 270

Tyr Leu Asp Glu Asn Gly Val Ala Glu Ile Leu Lys Phe Met Asn Lys

275 280 285

Lys Gly Ser Thr Asn Thr Ser Asp Ser Leu Met Ser Phe Leu Glu Ser

290 295 300

Met Asn Ile Lys Ser Ile Phe Ile Asn Ser Leu Gln Ser Asn Thr Lys

305 310 315 320

Leu Ile Leu Asp Thr Asn Phe Ile Ile Ser Gly Thr Thr Ser Ile Gly

325 330 335

Gln Phe Glu Phe Ile Cys Asp Lys Asp Asn Asn Ile Gln Pro Tyr Phe

340 345 350

Ile Lys Phe Asn Thr Leu Glu Thr Lys Tyr Thr Leu Tyr Val Gly Asn

355 360 365

Arg Gln Asn Met Ile Val Glu Pro Asn Tyr Asp Leu Asp Asp Ser Gly

370 375 380

Asp Ile Ser Ser Thr Val Ile Asn Phe Ser Gln Lys Tyr Leu Tyr Gly

385 390 395 400

Ile Asp Ser Cys Val Asn Lys Val Ile Ile Ser Pro Asn Ile Tyr Thr

405 410 415

Asp Glu Ile Asn Ile Thr Pro Ile Tyr Glu Ala Asn Asn Thr Tyr Pro

420 425 430

Glu Val Ile Val Leu Asp Thr Asn Tyr Ile Ser Glu Lys Ile Asn Ile

435 440 445

Asn Ile Asn Asp Leu Ser Ile Arg Tyr Val Trp Ser Asn Asp Gly Ser

450 455 460

Asp Phe Ile Leu Met Ser Thr Asp Glu Glu Asn Lys Val Ser Gln Val

465 470 475 480

Lys Ile Arg Phe Thr Asn Val Phe Lys Gly Asn Thr Ile Ser Asp Lys

485 490 495

Ile Ser Phe Asn Phe Ser Asp Lys Gln Asp Val Ser Ile Asn Lys Val

500 505 510

Ile Ser Thr Phe Thr Pro Ser Tyr Tyr Val Glu Gly Leu Leu Asn Tyr

515 520 525

Asp Leu Gly Leu Ile Ser Leu Tyr Asn Glu Lys Phe Tyr Ile Asn Asn

530 535 540

Phe Gly Met Met Val Ser

545 550

Claims (23)

1. An isolated polypeptide that specifically binds to TcdB2 and/or TcdB4, wherein the isolated polypeptide comprises: (i) the amino acid sequence of any one of SEQ ID NOs: 1-5; or (ii) an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to any one of SEQ ID NOs: 1-5; or (iii) an amino acid sequence having less than 10, less than 9, less than 8, less than 7, less than 6, less than 5, less than 4, less than 3 or less than 2 mutated amino acid residues in any of SEQ ID NOs: 1-5. 2. The isolated polypeptide according to claim 1, wherein the isolated polypeptide comprises R135, R140, I138, I133, M162 and L159 of TFPI, the numbering being according to SEQ ID NO: 22 or 23. 3. The isolated polypeptide according to claim 1, wherein the mutated amino acid residue is generated by substitution, insertion or deletion of an amino acid residue in any one of SEQ ID NOs: 1-5, preferably, the substitution is a conservative substitution. 4. The isolated polypeptide according to claim 1, wherein the isolated polypeptide consists of the amino acid sequence of any one of SEQ ID NOs: 1-5. 5. The isolated polypeptide of claim 1, wherein the isolated polypeptide is cross-linked, cyclized, conjugated, acylated, carboxylated, lipidated, acetylated, thioglycolic acid amidated, alkylated, methylated, polyglycylated, glycosylated, polysialylated, phosphorylated, adenylated, pegylated, or any combination thereof. 6. The isolated polypeptide of claim 1, wherein the isolated polypeptide comprises a modification at the C-terminus or the N-terminus. 7. The isolated polypeptide according to claim 1, wherein the isolated polypeptide further contains a fusion domain, optionally wherein the fusion domain is selected from the group consisting of polyhistidine, Glu-Glu, glutathione S transferase (GST), thioredoxin, protein A, protein G, immunoglobulin heavy chain constant region (Fc), maltose binding protein (MBP) or human serum albumin, preferably, the isolated polypeptide further contains the Fc portion of human IgG1. 8. A fusion protein comprising: (i) an isolated polypeptide as described in any one of claims 1 to 7; and (ii) a fusion domain, optionally, wherein the fusion domain is selected from the group consisting of polyhistidine, Glu-Glu, glutathione S transferase (GST), thioredoxin, protein A, protein G, immunoglobulin heavy chain constant region (Fc), maltose binding protein (MBP) or human serum albumin, preferably, the fusion domain is the Fc part of an immunoglobulin, more preferably, the fusion domain is the Fc part of human IgG1. 9. A conjugate comprising an isolated polypeptide according to any one of claims 1 to 7 conjugated to a moiety, optionally wherein the moiety is a labeling molecule, an imaging agent, a toxin, a radionuclide, or an agent capable of preventing or treating Clostridioides difficile infection (CDI) or other bacterial infection. 10. A chimeric molecule comprising a first portion and a second portion, wherein the first portion comprises the isolated polypeptide according to any one of claims 1 to 7, and wherein the second portion is a different molecule from the isolated polypeptide according to any one of claims 1 to 7. 11. The chimeric molecule of claim 10, wherein the first portion is a monomer or polymer of the isolated polypeptide according to any one of claims 1 to 7. 12. The chimeric molecule of claim 10, wherein the second portion is a portion that is capable of increasing the serum half-life of the isolated polypeptide, antibacterial agent, anti-TcdB immune molecule or ankyrin repeat sequence. 13. The isolated polypeptide according to any one of claims 1 to 7, the fusion protein according to claim 8, the conjugate according to claim 9 or the chimeric molecule according to any one of claims 10 to 12, for use in preventing and/or treating a disease caused by TcdB2 and/or TcdB4 in a subject, preferably Clostridium difficile infection (CDI) caused by branch 2 strains. 14. An isolated nucleic acid molecule comprising a polynucleotide encoding the isolated polypeptide according to any one of claims 1 to 7. 15. A vector or expression cassette comprising a polynucleotide encoding the isolated polypeptide according to any one of claims 1 to 7. 16. A recombinant host cell comprising the isolated nucleic acid molecule according to claim 14 or the vector or expression cassette according to claim 15. 17. A neutralizing molecule that targets one or more sites selected from E1433, D1467, D1468, E1469, S1598, L1599, L1434, K1435, M1438, V1492, L1494, I1496, L1489, P1506 or Y1510 of TcdB4 (SEQ ID NO:9). 18. A neutralizing molecule that targets one or more sites selected from E1432, D1466, D1467, S1597, L1598, L1433, K1434, M1437, V1491, L1493 or L1488 of TcdB2 (SEQ ID NO: 7). 19. A pharmaceutical composition comprising the isolated polypeptide according to any one of claims 1 to 7, or the fusion protein according to claim 8, or the conjugate according to claim 9, or the chimeric molecule according to claims 10 to 12, or the neutralizing molecule according to claim 17 or 18, and a pharmaceutically acceptable carrier. 20. A method for preventing and/or treating a disease caused by TcdB2 and/or TcdB4 in a subject in need thereof, wherein the method comprises administering to the subject a therapeutically effective amount of an isolated polypeptide according to any one of claims 1 to 7, or a fusion protein according to claim 8, or a conjugate according to claim 9, or a chimeric molecule according to any one of claims 10 to 12, or a neutralizing molecule according to claim 17 or 18, or a pharmaceutical composition according to claim 19. 21. The method of claim 20, wherein the disease caused by TcdB2 and/or TcdB4 is Clostridium difficile infection (CDI) caused by clade 2 strains. 22. The method of claim 20, wherein the subject is a mammal, preferably a human or a non-human primate. 23. The method of claim 20, further comprising administering to the subject an additional therapeutic agent or polypeptide.