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WO2015078840A1 - Sécrétion totale ou partielle d'une protéine et présentation à la surface d'une cellule au moyen d'un système de sécrétion de type iii - Google Patents

Sécrétion totale ou partielle d'une protéine et présentation à la surface d'une cellule au moyen d'un système de sécrétion de type iii Download PDF

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Publication number
WO2015078840A1
WO2015078840A1 PCT/EP2014/075478 EP2014075478W WO2015078840A1 WO 2015078840 A1 WO2015078840 A1 WO 2015078840A1 EP 2014075478 W EP2014075478 W EP 2014075478W WO 2015078840 A1 WO2015078840 A1 WO 2015078840A1
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Prior art keywords
protein
t3ss
cell
secretion
antibody
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Inventor
Thomas MARLOVITS
Lisa KOENIGSMAIER
Julia Radics
Daniel STREBINGER
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IMBA Institut fur Molekulare Biotechonologie GmbH
Boehringer Ingelheim International GmbH
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IMBA Institut fur Molekulare Biotechonologie GmbH
Boehringer Ingelheim International GmbH
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1037—Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
    • C07K14/255—Salmonella (G)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/035—Fusion polypeptide containing a localisation/targetting motif containing a signal for targeting to the external surface of a cell, e.g. to the outer membrane of Gram negative bacteria, GPI- anchored eukaryote proteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/10—Fusion polypeptide containing a localisation/targetting motif containing a tag for extracellular membrane crossing, e.g. TAT or VP22

Definitions

  • the present invention generally relates to the field of recombinant biotechnology and cell culture technology, and more specifically to cells constructed to export desired biomolecules from the cells to extracellular environment such as cell supernatant or another cell.
  • the present invention also relates to polypeptides exported for medical purposes such as for treating a disease. It concerns a method of generating novel host cells for biopharmaceutical manufacturing.
  • the present invention is related to the field of displaying polypeptides on the surface of a cell membrane, a method for producing a library of cells displaying proteins or polypeptides on the cell surface and a method of screening them.
  • the plasma membranes of cells present a barrier to the passage of intracellular proteins. Protein secretion is a useful tool for applications in biotechnology when proteins need to be exported for their function or for purification. Highly charged molecules in particular experience difficulty in passing across membranes, and many therapeutic proteins recombinantly expressed in host cells cannot be obtained unless the cells are disrupted and they are often engineered to be exported via a secretion path. Thus, there is a constant need for reliable means of exporting therapeutic proteins from cells.
  • T3SS type-3 secretion system
  • type III secretion system T3SS
  • T3SS is a molecular machine that exports proteins through both membranes to the extracellular environment.
  • T3SS is central to the virulence of many bacteria, including animal pathogens in the genera Salmonella, Yersinia, Shigella, and Escherichia and plant pathogens in the genera Pseudomonas, Erwinia, Xanthomonas, Ralstonia and Pantoea. These pathogens use T3SS to inject a series of proteins, so called bacterial "effector proteins," into the cytosol of host cells.
  • T3SS from Vibrio parahaemolyticus was cloned into the nonpathogenic Escherichia coli K-12 strain to explore its ability to translocate to foreign proteins into host cell (Akeda et al., "Functional cloning of Vibrio parahaemolyticus type III secretion system 1 in Escherichia coli K-12 strain as a molecular syringe," Biochem Biophys Res Commun 427(2):242-7 (2012)). Akeda et al.
  • T3SS polypeptide that can be exported through the T3SS. It has been found that proteins having fewer than 550 amino acids can be reliably secreted. However, secretion declines significantly after about 700 amino acids and proteins larger than 864 amino acids were not transported at all (Widmaier et al., "Quantification of the physiochemical constraints on the export of spider silk proteins by Salmonella type III secretion," Microb Cell Fact 9:78 (2010)). No successful studies have been published which uses T3SS system to export proteins which are larger than 864 amino acids. [006] Because of its ability to export protein to the extracellular environment, T3SS represents a powerful tool in basic and clinical biotechnology strategies.
  • A, B and/or C means A, B, C, A+B, A+C, B+C and A+B+C.
  • the term "about” or “approximately” as used herein means within 20%, preferably within 10%, and more preferably within 5% of a given value or range. It includes also the concrete number, e.g., about 20 includes 20.
  • the term “comprising” can be substituted with “containing”, “composed of”, “including”, “having” or “carrying.”
  • “consisting of” excludes any integer or step not specified in the claim/item.
  • “consisting essentially of” does not exclude integers or steps that do not materially affect the basic and novel characteristics of the claim/item. In each instance herein any of the terms “comprising”, “consisting essentially of” and “consisting of may be replaced with either of the other two terms.
  • the present invention provides for the first time a cell which is able to secrete large protein via the type III secretion system.
  • the present invention is based in part on the surprising finding that the size of the protein does not limit its ability to engage with the secretion system as observed before.
  • the inventors discovered that entry into the secretion system requires the substrate to be at least in partially unfolded state, and by fusing the substrate C-terminally to a conformationally stable moiety which is resistant to unfolding enables partial secretion as the moiety plugs the entry point of the needle complex and prevents substrate to depart fully from the needle complex.
  • the present invention provides one or more non-pathogenic cells for secreting therapeutic proteins via a type III secretion system (T3SS), wherein the cell comprises (i) a nucleotide sequence encoding a type III secretion system and (ii) a nucleotide sequence encoding a protein comprising a) an N-terminal secretion tag recognized by T3SS and b) the therapeutic protein.
  • T3SS type III secretion system
  • the protein is more than 864 amino acids long.
  • the cell secreting a therapeutic protein via a type III secretion system comprises:
  • (iii) optionally a nucleotide sequence encoding a chaperone capable of binding to said chaperone binding domain.
  • the T3SS system comprised in the cell of the present invention is encoded by genes from Salmonella Pathogenicity Island 1 (SPI-1 ) locus and more preferably that from Salmonella typhimurium.
  • the nucleotide sequence (ii) further comprises an anchoring sequence at its C-terminus.
  • Such cells can be used in a method for the production and secretion of the therapeutic protein as well as for the treatment of disease where the secretion of the therapeutic protein is desired.
  • the present invention also provides a pharmaceutical or diagnostic composition comprising the cell which secretes therapeutic proteins via a type III secretion system (T3SS).
  • T3SS type III secretion system
  • the complete polypeptide or protein may have a length of more than 864 amino acid residues via T3SS, such as more than 865, 866, 867, 868, 869, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000, 1 100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000 residues or more.
  • the present invention cell encodes in particular proteins or polypeptides that are heterologous to the cell. Particularly preferred proteins are antibodies or fragments thereof.
  • peptide As used throughout this specification, the terms “peptide”, “polypeptide”, and “protein” are used synonymously and refer to any proteinaceous compound comprising an ammo acid sequence of two or more ammo acid residues.
  • Therapeutic proteins secreted or produced may have a length of more than 629 amino acid residues via T3SS, such as more than 630, 631 , 632, 633, 634, 635, 636, 637, 638, 639, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800,
  • the present invention discloses a cell comprising a type III secretion system (T3SS) for partial secretion.
  • T3SS type III secretion system
  • partial secretion refers to the secretion of a partial stretch of a polypeptide through T3SS.
  • full secretion means that the substrate enters and completely departs from the T3SS to enter into extracellular environments.
  • Substrates for partial secretion comprise an N-terminal secretion tag, a protein of interest, one or more effector domains of a T3SS effector protein to span the needle complex, and finally, a final portion (anchoring sequence) for plugging the entry point of the needle complex.
  • Such cells are particularly useful for producing a library of cells to display polypeptides at cell surface and for screening a polypeptide of interest.
  • the present invention provides a cell comprising: a type III secretion system (T3SS), and
  • a protein comprising, from N- to C-terminus, an N-terminal secretion tag recognized by said T3SS, optionally a chaperone binding domain, a protein of interest, one or more effector domains of a T3SS effector protein having a length in amino acids that has at least the length of at least three SptP effector domains, and a protein having a globular structure or zinc finger domain.
  • the invention is also based partially on the discovery that by fusing effector domains having a given length C terminally to the protein allows the protein to pass through the needle complex. At the same time, to retain the protein on the cell surface, a protein having a globular structure or zinc finger domain is present C-terminally to the effector domains to prevent the protein of interest from fully departing the needle complex (see Example 2).
  • the spanning sequence when unfolded in the T3SS is at least 700 A, such as at least 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900 A, such as 1000, 1 100, 1200, 1300, 1400, 1500 A. It is within the skill of the artisan to measurement such length; methods provided in the example can for example be used.
  • the present invention provides a method for identifying a cell secreting a protein of interest via a T3SS, comprising (a) providing one or more cell comprising a type III secretion system (T3SS) and a protein comprising, from N to C-terminus, an N-terminal secretion tag recognized by the T3SS, a protein of interest, one or more effector domains of a T3SS effector protein, and a protein having a globular structure or zinc finger domain; and (b) identifying said protein of interest with a detection moiety which recognizes said protein of interest.
  • the protein of interest is an antibody or an antigen-binding fragment of an antibody.
  • Figure 1 (a) Designed substrates used for secretion test. One, three or five tandemly repeated effector domains from the natural substrate SptP (SptP1 , SptP3, SptP5) are fused downstream of a secretion signal (N-term signal). Each substrate was constructed without (Set-1 ) and with (Set-2) GFP followed by a 3x-FLAG-tag at the C-terminus. (b) Test results showing substrate secretion into supernatant and expression in the cell. Wild-type SptP, SptP1 , SptP3 and SptP5 were all secreted into the cell culture medium.
  • FIG. 2 Reconstructed cryo electron tomogram of Salmonella typhimurium cells (SB905) containing T3SSs. Cells with T3SS and expressing wild type SptP are shown in the upper panel. Cells expressing SptP3-GFP is shown in the lower panel.
  • Figure 3 (a) Overview of the experimental set-up to monitor substrate-loading, (b) Inhibitory effect of recombinant substrates on the secretion of the natural substrates SptP and SipA. Increasing levels of SptP3-GFP correlated with a decreased secretion of SptP and SipA. (c) Inhibitory effect of recombinant substrates on the secretion of the natural substrate SipB (translocase). Increasing levels of SptP3-GFP correlated with a decreased secretion of SipB.
  • FIG. 4 (a) SptP3-GFP containing needle complexes were immunopurified using anti FLAG which binds to the 3x FLAG-tag C-terminally fused to SptP3-GFP. (b) Electron microscopy of isolated needle complexes. An extra density is visible at the terminal end of the needle filament (white arrow) for substrate-trapped needle complexes.
  • Figure 5 (a) Model for immunogold labeling on GFP for substrate-trapped needle complexes. Anti GFP antibody was conjugated to colloidal gold for immunogold labeling of GFP on substrate trapped needle complexes (b) Electron microscopy of trapped SptP3-GFP labeled with an anti-GFP antibody conjugated to colloidal gold. The gold labels are exclusively located at the basal side of the needle complex.
  • Figure 6 (a) The longitudinal central section through the three-dimensional structure of substrate-free (w.t.) and substrate-trapped (+pSptP3-GFP) needle complexes reconstituted by single particle analysis on images obtained by cryo electron microscopy using 74964 and 82403 particles. Three-dimensional volumes (C3) have been filtered to 10 A of resolution (FSC 0.5).
  • Hollow spaces ('portal', 'channel', 'atrium' and 'tunnel') define the secretion path within the needle complex.
  • the narrowest part for substrate passage is the 10 A wide 'channel'.
  • the direction of substrate movement is indicated with arrows.
  • Figure 7 Comparison of needle filament lengths of substrate-free (w.t.) and substrate- trapped (+pSptP3-GFP) needle complexes.
  • Figure 8 Sequences of Spt N terminal secretion tag, effector domain, chaperone binding domain, SptP3-GFP and SptP5GFP used in the examples.
  • FIG. 9 Overview of the N signal sequences (SecSig) and chaperone binding domain (CBD), and effector domains of various Salmonella effector proteins.
  • Figure 10 (a) Schematic representation of the secretion assay in Example 7.
  • Figure 11 (a) pCASP plasmid used in the secretion test in Example 7.
  • T3SS preferably that encoded by Salmonella Pathogenicity Island 1 (SPI-1 ), said cell comprising:
  • (iii) optionally a nucleotide sequence encoding a chaperone capable of binding to said chaperone binding domain.
  • pseudotuberculosis Yersinia pestis, Shigella felxneri, Citrobacter rodentium, Escherichia coli EHEC, Escherichia colia EPEC, Pseudoman syringae, Ralstonia solanacearum, Xanthominas campestris, or Erwinia amylovora.
  • N-terminal secretion tag and chaperone binding domain is selected from SipA, SipB, SipC, SipD, SopA, SopB, SopD, SopE2, SptP, AvrA.
  • sequence (i) is integrated into the genome of said host cell.
  • nucleotide sequence (ii) and (iii) are contained in a plasmid
  • nucleotide sequence (ii) and (iii) are contained in an artificial bacterial chromosome
  • nucleotide sequence (ii) is contained in a plasmid and nucleotide sequence (iii) is contained in an artificial bacterial chromosome, or
  • nucleotide sequence (iii) is contained in a plasmid and nucleotide sequence (ii) is contained in an artificial bacterial chromosome.
  • the non-pathogenic cell of any one of the preceding items wherein nucleotide sequence (ii) and (iii) are co-expressed from the same plasmid.
  • the non-pathogenic cell of any one of the preceding items, wherein said host cell is E. coli, preferably E. coli Nissle.
  • the non-pathogenic cell of any one of the preceding items wherein said host cell is capable of injecting said therapeutic protein into eukaryotic cells via the T3SS of said host cell.
  • the non-pathogenic cell of any one of the preceding items, wherein the therapeutic protein can be cleaved off by a protease recognizing a protease cleavage site preceding the therapeutic protein.
  • the non-pathogenic cell of any one of the preceding items, wherein said therapeutic protein is in the form of a concatemer.
  • the non-pathogenic cell of item 16 wherein the concatemer contains 2, 3, 4, 5 or more copies of the therapeutic protein.
  • the non-pathogenic cell of item 17, wherein each copy of the therapeutic protein can be cleaved off by a protease recognizing a protease cleavage site preceding the therapeutic protein.
  • the non-pathogenic cell of any one of the preceding items, wherein the therapeutic protein has a length of more than 864 amino acids.
  • the therapeutic protein comprises an antibody, polyclonal antibody, monoclonal antibody, recombinant antibody, antibody fragment, Fab', F(ab') 2 , Fv, scFv, di-scFvs, bi-scFvs, tandem scFvs, bispecific tandem scFvs, sdAb, V H , V L , humanized antibody, chimeric antibody, IgA antibody, IgD antibody, IgE antibody, IgG antibody, IgM antibody, intrabody, minibody or monobody.
  • a pharmaceutical or diagnostic composition comprising a non-pathogenic cell of any one of the preceding items.
  • a non-pathogenic cell of any one of items 1-20 for use in a method of treating a disease Use of a non-pathogenic cell of any one of items 1-20 for the production and secretion of a therapeutic protein.
  • a method for the production of a protein of interest comprising culturing a nonpathogenic cell of any one of items 1 -20 and harvesting said therapeutic protein from the culture.
  • a cell comprising a type III secretion system (T3SS) and a protein comprising from N- to C-terminus an N-terminal secretion tag recognized by said T3SS, optionally a chaperone binding domain, a protein of interest, a spanning sequence having a length in amino acids of at least three SptP effector domains, and a protein having a globular structure or zinc finger domain.
  • T3SS type III secretion system
  • the spanning sequence comprises one or more effector domains a T3SS effector protein, and optionally wherein the domain is selected from SipA, SipB, SipC, SipD, SopA, SopB, SopD, SopE2, SptP and AvrA.
  • the cell of item 25 or 26, wherein the secretion tag and chaperone binding domain is selected from SipA, SipB, SipC, SipD, SopA, SopB, SopD, SopE2, SptP and AvrA.
  • T3SS is from Salmonella typhimurium, Salmonella enterica, Pseudomonas aeruginosa, Yersinia pseudotuberculosis, Yersinia pestis, Shigella felxneri, Citrobacter rodentium, Escherichia coli EHEC, Escherichia colia EPEC, Pseudoman syringae, Ralstonia solanacearum, Xanthominas campestris, or Erwinia amylovora.
  • the cell of item 28, wherein the T3SS is encoded by genes from the Salmonella typhimurium pathogenicity island 1 (SPI-1 ) locus.
  • the cell of any one of the preceding items wherein said cell comprises a chaperone selected from SicA, InvB, SicP, SigE, InvB, SicP, SigE, SicA.
  • a chaperone selected from SicA, InvB, SicP, SigE, InvB, SicP, SigE, SicA.
  • the cell of any one of the preceding items wherein said cell is capable of injecting said protein into eukaryotic cells via its T3SS.
  • the cell of any one of the preceding items, wherein the protein of interest can be cleaved off by a protease recognizing a protease cleavage site preceding and/or following the protein of interest.
  • the cell of any one of the preceding items comprising a plasmid comprising nucleotides encoding the N-terminal secretion tag, the optional chaperone binding domain, the therapeutic protein, and/or the chaperone. 36.
  • a method for identifying a cell secreting a protein of interest via a T3SS comprising
  • a T3SS comprising a partially secreted protein in its secretion pathway, wherein the protein comprises a globular structure or zinc finger domain at the C terminus.
  • the present invention provides in a non-pathogenic cell which secretes therapeutic proteins via a type III secretion system (T3SS), wherein the cell comprises (i) a nucleotide sequence encoding a type III secretion system and (ii) a nucleotide sequence encoding a protein comprising an N-terminal secretion tag recognized by T3SS and the therapeutic protein.
  • T3SS type III secretion system
  • the present invention also provides a non-pathogenic cell which secretes therapeutic proteins via a type III secretion system (T3SS), wherein the cell comprises (i) a type III secretion system and (ii) a protein comprising an N-terminal secretion tag recognized by T3SS and the therapeutic protein.
  • T3SS type III secretion system
  • the protein has a length of more than 864 amino acids.
  • a "cell” as used in the present invention should be understood broadly.
  • a “cell” refers to any type of lipid bilayer-enclosed structures such as a prokaryotic cell or eukaryotic cell, which are generally regarded as the smallest structural and functional unit of an organism which is considered alive.
  • a cell can also refer to other lipid bilayer-endosed structures which are not considered alive, such as a cell organelle like liposome, a subbacterial component like bacterial ghosts, or achromosomal cells like minicells.
  • cells used in the present invention are prokaryotes or eukaryotes.
  • examples include, but are not limited to, vertebrate cells, mammalian cells, human cells, animal cells, invertebrate cells, nematodal cells, insect cells, stem cells, yeast cell or fungal cells.
  • the cell is a prokaryotic cell such as bacterial cells from Gram- negative bacteria, including cells from Enterobacteriaceae, and E. coli, Pseudomonadaceae, e.g., P. putida as well as Gram-positive bacteria such as cells from Lactobacteriaceae or Bacillaceae.
  • the cell is E. coli, Bacillaceae, Salmonella, Serratia, or Pseudomonas species.
  • the cells are E. coli, such as Escherichia coli strain Nissle 1917, which is also known as Escherichia coli 083:K24:H31 . It is non-pathogenic and has been used as probiotic agents in medicine for the treatment of various gastroenterological diseases, including inflammatory bowel disease.
  • the T3SS can be present in a lipid bilayer-endosed structures which are not considered alive such as minicells.
  • T3SS engineered in minicells is known in the art and has for example been described in Carleton et al., "Engineering the type III secretion system in non-replicating bacterial minicells for antigen delivery," Nature Commun. 4, 1590 (2013).
  • non-pathogenic means that the cells do not cause significant disease in healthy animals such as human.
  • the cell used in the present invention is especially not a pathogenic strain of E. coli such as enteropathogenic or enterohemorrhagic E. coli.
  • enteropathogenic E. coli strains include strains from the serogroup 0127 such as 0127:1-16.
  • enterohemorrhagic E. coli strains include strains from the serogroup 0157 such as 0157:1-17.
  • Enteropathogenic or enterohemorrhagic E. coli is known to cause acute gastroenteritis in humans.
  • Enteropathogenic E. coli is a frequent cause of infantile diarrhea and enterohemorrhagic E. coli causes a wide spectrum of illnesses ranging from mild diarrhea to hemorrhagic colitis and hemolytic uremic syndrome.
  • the cell of the present invention is preferably non-pathogenic.
  • a non-pathogenic cell is not a pathogenic bacterium which is attenuated because attenuated pathogenic bacteria whose pathogenicity characteristics have not been fully characterized may have unknown harmful effects if administrated to mammalian culture cells or the living body.
  • T3SS type III secretion system
  • Type-3 secretion systems is known to involve 3.5 MDa syringe-like, membrane embedded injectisomes containing needle complex to connect intracellular compartments of infectious bacteria and hosts.
  • T3SS are found in a variety of gram-negative pathogens such as Yersinia pestis, Yersinia pseudotuberculosis, Yersinia enterocolitica, Pseudomonas, Shigella flexneri, Shigella dysenteriae, Xanthomonas and some Salmonella sp.
  • Effective proteins are injected by the bacteria via T3SS into the cytosol of host cells, which in turn modulate eukaryotic regulatory or signaling pathways during bacterial infection in the host cell.
  • the system resembles a large supramolecular cylindrical structure embedded in both bacterial membranes. A needle filament protruding from the bacterial surface allows the transfer of proteins directly from inside the bacteria to the cytoplasm.
  • the needle complex includes Salmonella enterica, Salmonella typhimurium, Salmonella typhi, Salmonella enteritica as well as other pathogens including Vibrio cholerae, Hafnia alvei, Bordetella sp. and Chlamydia species.
  • "Effector proteins” are injected by the bacteria via T3SS into the cytosol of host cells, which in turn modulate eukaryotic regulatory or signaling pathways during bacterial infection in the host cell.
  • the system resembles a large supramolecular cylindrical structure embedded in both bacterial membranes. A needle filament protruding from the
  • T3SS The hallmark of T3SS is the needle complex (referred to also as NC) (also called injectisome when the ATPase is excluded).
  • NC needle complex
  • the needle complex is embedded within the inner and outer bacterial membrane, spans the periplasmic space, and extends into the extracellular environment with a needle-like filament.
  • the cylindrically shaped needle complex (referred to as "injectisome”) is composed of structural proteins forming a multi-ring base associated to the bacterial envelope and a needle-like extension that protrudes several nanometers from the bacterial surface.
  • the needle is anchored to the base through another substructure, the inner rod, which together with the needle filament forms a channel that serves as conduit for the traveling of the effector proteins (Marlovits et al., Science 306, 1040-1042 (2004)).
  • the needle provides a smooth passage through the highly selective and almost impermeable membranes.
  • a single bacterium can have several hundred needle complexes on its membrane.
  • the needle complex is believed to be universal to all T3SSs.
  • the needle complex shares similarities with bacterial flagella.
  • the base of the needle complex is structurally very similar to the flagellar base; the needle itself is analogous to the flagellar hook, which is a structure connecting the base to the flagellar filament.
  • the cell of the present invention may in some aspects comprise (i) a nucleotide sequence encoding T3SS.
  • Such nucleotide sequences encode structural proteins making up the type III secretion system.
  • structural proteins as defined here includes proteins which form the base, the inner rod and the needle of T3SS.
  • This nucleotide sequence (i) can encodes any type III secretion system known in the art, such as from Yersinia, Salmonella, Bordetella, Pseudomonas, Chlamydia, Burkholderia, Escherichia, Shigella, Erwinia, Ralstonia, Xanthomonas and Rhizobium species.
  • T3SS from Salmonella typhimurium, Salmonella enterica, Pseudomonas aeruginosa, Yersinia pseudotuberculosis, Yersinia pestis, Shigella flexneri, Citrobacter rodentium, Escherichia coli EHEC, Escherichia coli EPEC, Pseudomona syringae, Ralstonia solanacearum, Xanthomonas campestris, and Erwinia amylovora.
  • Structural proteins forming the T3SS do not have to all origin from the same pathogen. Because type III secretion systems are highly conserved in a variety of gram- negative pathogenic bacteria, it is possible that the structural proteins are derived from different pathogens.
  • the nucleotide sequence (i) is preferably integrated into the genome of said cell. It may be present in in the chromosome and/or on a plasmid or vector in the cell. Preferably, the nucleotide sequence (i) is integrated into the chromosome of said cell. In other embodiments, the nucleotide sequence can be integrated in a plasmid or vector.
  • nucleotide sequence refers to either DNA or RNA.
  • Nucleic acid sequence or polynucleotide sequence refers to a single or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases read from the 5' to the 3' end. It includes both self -replicating plasmids, infectious polymers of DNA or RNA, and nonfunctional DNA or RNA.
  • nucleotide encoding the T3SS comprises genes which encode structural proteins for the needle monomer, the inner rod of the needle, the ring proteins, two translocators, the needle-tip protein, the ruler protein, the ATPase, the export apparatus proteins and the sorting platform.
  • a skilled person in the art can readily determine the nucleotides encoding such genes as they are known in the art and conserved between various T3SS systems (see for example, Kosarewicz et al., "The blueprint of the type-3 injectisome,” Philos Trans R Soc Lond B Biol Sci. 367(1592):1140-54 (2012)).
  • the following proteins encode a T3SS in salmonella (listed with SwissProt accession number):
  • Salmonella size [kDa] function Acc. Nr.
  • Table 1 structural proteins of a T3SS system in Salmonella Salmonella Pathogenicity Island 1 (SPI-1 ).
  • Nucleotide sequence (i) encoding T3SS may be obtained from T3SS genetic locus in a pathogen which expressed T3SS.
  • the nucleotide sequence (i) encoding the T3SS is derived from Salmonella Pathogenicity Island 2 (SPI-2) or more preferably from Salmonella Pathogenicity Island 1 (SPI-1 ) in Salmonella.
  • SPI-1 Salmonella Pathogenicity Island 1
  • T3SS encoded by SPI-1 is decribed herein in more detail. It is an approximately 40 kilobase (kb) gene segment, which is found in all organisms of the genus Salmonella and acquired by a lateral gene transfer event early in Salmonella evolution.
  • SPI-1 encodes all structural genes required for a T3SS in three operons: PhoP repressed genes (prg operon) (Miller et al., "The phop virulence regulon and live oral salmonella vaccines” Vaccine 1 1 (2): 122-5 (1993)), surface presentation of antigens proteins (spa operon) and Invasion proteins (inv operon) (Groiman et al., "Cognate gene clusters govern invasion of host epithelial cells by Salmonella typhimurium and Shigella flexnerf EMBO J 12(10):3779-87 (1993)).
  • T3SS effector proteins and their cognate chaperones are encoded in the Salmonella invasion proteins (sip operon) (Kaniga et al., "Identification of two targets of the type iii protein secretion system encoded by the inv and spa loci of Salmonella typhimurium that have homology to the Shigella ipad and ipaa proteins.” J Bacteriol 177(24):7078-85 (1995)).
  • the last operon in SPI-1 is the hyperinvasion locus (hil), which encodes the regulatory transcription factors for the T3SS (Bajaj et al., "hila is a novel ompr/toxr family member that activates the expression of salmonella typhimurium invasion genes," Mol Microbiol 18(4):715-27 (1995)).
  • Environmental conditions such as growth phase, pH, oxygen tension, and osmolarity regulate expression of hilA, the positive regulator for the whole SPI-1 T3SS regulon. It upregulates the inv, prg and sic operon leading to the overexpression of the structural components of the needle complex and indirectly to the increased expression of the natural effector proteins.
  • T3SS machinery recognizes signals present in the effector proteins which are typically referred to as type III signal sequences. In general, the signal sequences tend to be located near the N-terminal end of the effector protein and form the first 15-30 amino acids of the effector protein.
  • the cell according to the present invention comprises (ii) a nucleotide sequence encoding a protein comprising an N-terminal secretion tag and the therapeutic protein. The N-terminal secretion tag is required for the T3SS to recognize and secrete the therapeutic protein.
  • the N-terminal secretion tag can be derived, for example, from the N-terminal end of any type III secretion effectors (the type III signal sequence), because the effectors are readily recognized by T3SS in natural context.
  • These secretion tags have been studied and are known in the art. They can also be identified by examining sequence similarity shared between effectors (Lower et al., "Prediction of Type III Secretion Signals in Genomes of Gram-Negative Bacteria," PLoS One 4(6): e5917 (2009)).
  • Use of secretion tags is known and has been described, for example, in WO2000/059537, WO2008/110653 and WO2012/012605.
  • Table 2 Effector Proteins and cognate chaperon proteins in Salmonella.
  • Fig. 9 provides an overview of the signal sequences, chaperone binding domain and effector domains of the Salmonella effector proteins.
  • some effector proteins depend on their interaction with specific T3SS chaperones for their secretion, known as class I chaperones.
  • the nucleotide sequence (ii) of the present invention can as an optional feature additionally comprise a chaperone binding domain (CBD) after the N-terminal secretion tag for engaging the chaperons.
  • CBD chaperone binding domain
  • the chaperone binding domains of class I chaperones are generally located immediately after the N-terminal signal sequence and are usually approximately 50-100 amino acid long.
  • the nucleotide sequence (ii) includes a chaperone binding domain. However, in other embodiments, the sequence (ii) does not include the chaperone binding domain.
  • the nucleotide sequence (ii) is obtained from the first 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 1 10, 120, 130, 140 or 150 amino acids of a T3SS effector protein or homologues thereof.
  • the N-terminal secretion tag is derived from SipA, SipB, SipC, SipD, InvJ, SpaO, AvrA, SopE2 and SptP proteins of Salmonella, the YopE, YopH, YopM and YpkA proteins of Yersinia, the I pa proteins of Shigella, or the ExoS proteins of Pseudomonas aeruginosa.
  • the N-terminal secretion tag is obtained from SipA, SipB, SipC, SipD, SopA, SopB, SopD, AvrA, SopE2 and most preferably from SptP.
  • These effector proteins are described in detail in the later sections of the present specification.
  • the inventors used the first 36 amino acid residues at the N-terminal end of SptP (as shown in SEQ ID NO: 1 in Fig 8) to direct secretion via Salmonella SPI-1 T3SS, although shorter sequence can be used, such as at least the first 30, 31 , 32, 33, 34 and 35 amino acid residues of SEQ ID NO: 1 .
  • the N-terminal secretion tag comprises an amino acid sequence at least 90%, such as at least 91 , 92, 93, 94, 95, 96, 97, 98, 99 and 100% to the amino acid sequence as shown in SEQ ID NO: 1.
  • the cell of the present invention further comprises, as an optional feature, a nucleotide sequence (iii) which encodes a chaperone which is capable of binding to the chaperone binding domain encoded by the nucleotide sequence (ii). Chaperones which bind to the chaperone binding domain of effector proteins have been studied.
  • the chaperone binding domain encoded by nucleotide sequence (ii) is preferably selected from the chaperone binding domain in SipA, SipB, SipC, SipD, SopA, SopB, SopD, SopE2, SptP, AvrA.
  • the chaperone encoded by nucleotide sequence (iii) is preferably selected from one of the following the Salmonella chaperone protein SicA, InvB, SicP, SigE, InvB, SicP, SigE, SicA.
  • N-terminal secretion tag and chaperone binding domain/chaperones is encoded by the nucleotides in the cell: SipA/lnvB, SipC/SicA, SopA lnvB, SopE2/lnvB, SptP/SicP, SopB/SigE.
  • SipA lnvB the N-terminal secretion tag and chaperone binding domain is that from SipA
  • the chaperone protein is InvB.
  • the chaperone binding domain shown in Fig. 8 (SEQ ID NO: 3) was used.
  • the N-terminal secretion tag comprises an amino acid sequence at least 90%, such as at least 91 , 92, 93, 94, 95, 96, 97, 98, 99 and 100% to the amino acid sequence as shown in SEQ ID NO: 3.
  • promoter refers to a region that facilitates the transcription of a particular gene.
  • a promoter is preferably operatively linked to the adjacent nucleotide sequence which is to be expressed.
  • a promoter typically increases the amount of recombinant product expressed from a nucleotide sequence as compared to the amount of the expressed recombinant product when no promoter exists.
  • a promoter from one organism can be utilized to enhance recombinant product expression from a sequence that originates from another organism.
  • one promoter element can increase the amount of products expressed for multiple sequences attached in tandem. Hence, one promoter element can enhance the expression of one or more recombinant products. Multiple promoter elements are well known to persons of ordinary skill in the art.
  • the expression of the nucleotide sequences described herein can be driven by a “constitutive” or “inducible” promoter.
  • the term “expression” as used herein refers to the transcription and stable accumulation of mRNA from a given nucleotide sequence.
  • the promoter could be a "inducible promoter” or “constitutive promoter.”
  • “Inducible promoter” refers to a promoter which can be induced by the presence or absence of certain factors, and “constitutive promoter” refers to an unregulated promoter that allows for continuous transcription of its associated gene.
  • both the nucleotide sequences (i) and (ii) is driven by an inducible promoter. Upon induction of the promoter, the T3SS and the therapeutic protein with the secretion tag are expressed.
  • the nucleotide sequence (ii) and (iii) are contained in a plasmid or a bacterial artificial chromosome (BAC).
  • bacterial artificial chromosome BAC refers to a cloning vector derived from a bacterial chromosome, into which large DNA sequences from bacterial or nonbacterial sources can be inserted.
  • plasmid refers to an autonomous circular DNA molecule capable of replication in a cell. Most plasmids exist in only one copy per bacterial cell. Some plasmids, however, exist in higher copy numbers. For example, the plasmid ColE1 typically exists in 10 to 20 plasmid copies per chromosome in E. coli.
  • the plasmid preferably has a copy number of 20-30, 30-100 or more per host cell. With a high copy number of plasmids, it is possible to increase the amount therapeutic proteins expressed by the cell. Large numbers of suitable plasmids or vectors are known to those of skill in the art and many are commercially available. Examples of suitable vectors are provided in Sambrook et al, eds., Molecular Cloning: A Laboratory Manual (2 nd Ed.), Vols. 1-3, Cold Spring Harbor Laboratory (1989), and Ausubel et al, eds., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York (1997).
  • Embodiments in this connection include that (1 ) nucleotide sequence (ii) and (iii) are contained in a plasmid, (2) nucleotide sequence (ii) and (iii) are contained in an artificial bacterial chromosome, (3) nucleotide sequence (ii) is contained in a plasmid and nucleotide sequence (iii) is contained in an artificial bacterial chromosome, or (4) nucleotide sequence (iii) is contained in a plasmid and nucleotide sequence (ii) is contained in an artificial bacterial chromosome.
  • nucleotide sequence (ii) and (iii) are co- expressed from the same plasmid.
  • Co-expression refers to the expression of two or more nucleic acid sequences at the same time.
  • a "therapeutic protein” means any polypeptide, protein, protein variant, fusion protein and/or fragment thereof which may be administered to a mammal as a medicament. It is envisioned but not required that therapeutic protein according to the present invention is heterologous to the cell.
  • proteins that can be produced by the cell of the present invention are, without limitation, enzymes, regulatory proteins, receptors, peptides, e.g. peptide hormones, growth factors, cytokines, structural proteins, lymphokines, adhesion molecules, receptors, membrane or transport proteins, and any other polypeptides that can serve as agonists or antagonists and/or have therapeutic or diagnostic use.
  • the proteins of interest may be antigens as used for vaccination, vaccines, antigen-binding proteins, immune stimulatory proteins. It may also be an antigen-binding fragment of an antibody, which can include any suitable antigen-binding antibody fragment known in the art.
  • an antibody fragment may include but not limited to Fv (a molecule comprising the VL and VH), single-chain Fv (scFV) (a molecule comprising the VL and VH connected with by peptide linker), Fab, Fab', F(ab')2, single domain antibody (sdAb) (molecules comprising a single variable domain and 3 CD ), and multivalent presentations thereof.
  • the antibody or fragments thereof may be murine, human, humanized or chimeric antibody or fragments thereof.
  • therapeutic proteins include an antibody, polyclonal antibody, monoclonal antibody, recombinant antibody, antibody fragment, Fab', F(ab') 2 , Fv, scFv, di-scFvs, bi-scFvs, tandem scFvs, bispecific tandem scFvs, sdAb, V H , V L , humanized antibody, chimeric antibody, IgA antibody, IgD antibody, IgE antibody, IgG antibody, IgM antibody, intrabody, minibody or monobody.
  • a "therapeutic protein" in the sense of the present invention is preferably not an effector protein of T3SS or mutants thereof.
  • Such therapeutic proteins include, but are not limited to, insulin, insulin-like growth factor, hGH, tPA, cytokines, e.g. interleukines such as IL-1 , IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-1 1 , IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, interferon (IFN) alpha, IFN beta, IFN gamma, IFN omega or IFN tau, tumor necrosisfactor (TNF) TNF alpha and TNF beta, TRAIL; G-CSF, GM-CSF, M-CSF, MCP-1 and VEGF.
  • interleukines such as IL-1 , IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-1 1 , IL-12, IL
  • the therapeutic protein have a length of more than 629 amino acid residues via T3SS, such as more than 630, 631 , 632, 633, 634, 635, 636, 637, 638, 639, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000, 1 100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000 amino acid residues or more.
  • the complete polypeptide may have a length of more than 864 amino acid residues via T3SS, such as more than 865, 866, 867, 868, 869, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000 amino acid residues or more.
  • Particularly preferred therapeutic proteins are antibodies or fragments thereof which are has a length of more than 629 amino acids.
  • the therapeutic protein is an antibody, nanobody or monobody.
  • antibodies that can be used in the invention include chimeric antibodies, non-human antibodies, human antibodies, humanized antibodies, and domain antibodies (dAbs).
  • a "nanobody” is a small functional antibody fragment composed of the VHH domain of Camelidae heavy chain antibodies. They are homodimers of two heavy chains which are missing the first constant domain (CH1 ) due to a splice site mutation. The variable domain of these antibodies(denoted as VHH) binds to epitopes with a comparable affinity to conventional antibodies.
  • a “monobody” refers to an artificial or synthetic single domain antibody or antibody mimic. Also known as an ADNECTINTM (e.g., see U.S. Patent No. 7,1 15,396), monobodies are genetically engineered proteins that can bind to antigens. They are based on the structure of human fibronectin, more specifically on its tenth extracellular type III domain.
  • the expressed therapeutic proteins do not have basic amino acid stretches, oligomerization interfaces or a stable tertiary structure.
  • therapeutic protein contains basic amino acid stretches (e.g., His, Arg or Lys) of more than 6 amino acid residues in length, the basic stretch might form a positively-charged domain which interacts with negatively charged cell components like nucleic acids or the surface of cell membrane or nuclear membrane.
  • An example of basic amino acid stretch is a nuclear localisation signal.
  • the therapeutic protein does not contain oligomerization interfaces such as a dimerization interface like leucine zipper because oligomerization may abolish secretion upon oligomerization.
  • oligomerization domains include, but are not limited to, coiled-coil domains, alpha- helical coiled-coil domains, collagen domains, collagen-like domains. Further examples include helix-loop-helix domains and
  • the therapeutic protein does not have a stable tertiary structure.
  • the "term "stable tertiary structure” refers to a tertiary structure that cannot be unfolded by the ATPase of T3SS.
  • the green fluorescent protein (GFP) and ubiquitin is known to have a very compact and stable tertiary structure which cannot be unfolded by the ATPase.
  • GFP green fluorescent protein
  • ubiquitin is known to have a very compact and stable tertiary structure which cannot be unfolded by the ATPase.
  • a skilled person is able to determine whether a given therapeutic protein has a stable tertiary structure for the purpose of the present invention.
  • the therapeutic protein encoded by the nucleotide sequence (ii) can be in the form of a concatemer.
  • a "concatemer” as used herein refers to a long continuous nucleic molecule that contains multiple copies of the same nucleic acid sequence, such as 2, 3, 4, 5 or more, linked in series.
  • the chaperone binding domain and the therapeutic protein are separated by a linker.
  • linker refers to an innocuous length of nucleotide sequences or protein that joins two nucleotide sequences or proteins.
  • the therapeutic protein can be cleaved off from the secretion tag and the optional chaperone binding domain by a protease recognizing a protease cleavage site preceding the therapeutic protein. It may be desirable that the secretion tag and chaperon binding domain are cleaved after the protein is secreted via T3SS: A protease cleavage site is a specific amino acid sequence recognized by the protease for proteolytic cleavage.
  • protease cleavage sites are known in the art (see, e.g., Matayoshi et al., Science 247: 954 (1990); Dunn et al., Meth Enzymol 241 : 254 (1994); Seidah et al., Meth Enzymol 244: 175 (1994); Thomberry, Meth Enzymol 244: 615 (1994); Weber et al., Meth Enzymol 244: 595 (1994); Smith et al., Meth Enzymol 244: 412 (1994); and Bouvier et al., Meth Enzymol 248: 614 (1995)).
  • the protease cleavage site can also be contained within the linker.
  • each copy of the therapeutic protein can be cleaved off by a protease recognizing a protease cleavage site preceding each copy of the therapeutic protein.
  • the present cells can be used to generate of one or several therapeutic proteins for diagnostic purposes, research purposes or manufacturing of therapeutic proteins either on the market or in clinical development.
  • a pharmaceutical or diagnostic composition comprising the non-pathogenic cell of the present invention.
  • the nonpathogenic cell or composition may be administered in a therapeutically effective amount in any conventional dosage form in any conventional manner to treat a disease.
  • Routes of administration include, but are not limited to, intravenously, intramuscularly, subcutaneously, intrasynovially, by infusion, sublingually, transdermally, orally, topically or by inhalation, tablet, capsule, caplet, liquid, solution, suspension, emulsion, lozenges, syrup, reconstitutable powder, granule, suppository and transdermal patch.
  • Methods for preparing such dosage forms are known (see, for example, Ansel et al., Pharmaceutical Dosage Forms and Drug Delivery Systems 5th ed., Lea and Febiger (1990)).
  • a therapeutically effective amount can be determined by a skilled artisan based on factors such as weight, metabolism, and severity of the affliction etc.
  • the active compound is dosed at about 1 mg to about 500 mg per kilogram of body weight on a daily basis. More preferably the active compound is dosed at about 1 mg to about 100 mg per kilogram of body weight on a daily basis.
  • composition may be administered alone or in combination with adjuvants to enhance the stability of the therapeutic proteins, to facilitate administration, to provide increased dissolution or dispersion, to increase the activity, to provide adjunct therapy and the like.
  • adjuvants may utilize lower dosages of the active ingredient, thus reducing possible toxicity and adverse side effects.
  • This invention allows the cell to be used as production factories of recombinant proteins that can be secreted into the extracellular medium without the need for cell lysis.
  • the present invention thus provides nonpathogenic cell used for the production and secretion of a therapeutic protein.
  • the method for the production of the protein comprises culturing the non-pathogenic cell of the present invention and harvesting said therapeutic protein from the culture.
  • Another possible application of the cells is for the delivery of therapeutic proteins into the target eukaryotic cells.
  • the cells should be cultured under conditions suitable for the expression of the aforementioned nucleotide sequences. Technique of cell culture is well-established in the art.
  • the therapeutic protein is then secreted via T3SS into the culture medium which can then be harvested.
  • the therapeutic proteins in the medium can be optionally treated by protease to cleave secretion tag off. It may be desirable to purify the protein to obtain substantially homogenous preparations of the protein. In general, methods which are routinely applied in the expression of recombinant proteins in a host cell can be employed.
  • the current invention also provides in another aspect use of the present cells to deliver therapeutic protein into eukaryotic cells.
  • the cells can be brought into contact with eukaryotic cells in vivo or in vitro under suitable conditions for the therapeutic protein to be injected into host cell cytoplasm via T3SS.
  • the present invention relates to the display of proteins on the cell surface.
  • Bacterial surface display had been achieved using chimeric genes derived from bacterial outer membrane proteins, lipoproteins, fimbria proteins, and flagellar proteins.
  • the present invention provides yet a novel means of surface display achieved by a partial secretion of a given protein via T3SS. It is generally considered that the expression of heterologous proteins on the surface of cells provides a powerful tool for diverse activities as obtaining specific antibodies, determining enzyme specificity, exploring protein-protein interactions, and introducing new functions into proteins.
  • One advantage of the present invention is the ability to display large proteins.
  • the present invention provides one or more cell comprising a type III secretion system and a protein comprising from N to C-terminus, (1 ) an N-terminal secretion tag recognized by said T3SS, (2) optionally a chaperone binding domain, (3) a protein of interest, (4) optionally a sequence for spanning the secretion path of T3SS (a "spanning sequence") having a length in amino acids that has at least the length of at least three SptP effector domains, and (5) an "anchoring sequence" which enables partial secretion of the protein.
  • a spanning sequence can be used so to enable the protein of interest to pass through T3SS and enter the extracellular environment.
  • the present invention provides a T3SS comprising a partially secreted protein comprising an anchoring sequence at the C-terminus of the protein.
  • the cell comprises a type III secretion system (T3SS) and a protein comprising, from N to C-terminus, (1 ) an N-terminal secretion tag recognized by said T3SS, (2) optionally a chaperone binding domain, (3) a protein of interest, (4) as the "spanning sequence" one or more effector domains of a T3SS effector protein having a length in amino acids that has at least the length of at least three SptP effector domains, and (5) an anchoring sequence encoding for example a protein having a globular structure or zinc finger domain.
  • T3SS type III secretion system
  • a protein comprising, from N to C-terminus, (1 ) an N-terminal secretion tag recognized by said T3SS, (2) optionally a chaperone binding domain, (3) a protein of interest, (4) as the "spanning sequence" one or more effector domains of a T3SS effector protein having a length in amino acids that has at least the length of at least three SptP effector
  • cell display libraries include phage displayed peptide libraries, bacterial surface displayed polypeptides and monoclonal antibody libraries.
  • the protein of interest could be for example an antibody, polyclonal antibody, monoclonal antibody, recombinant antibody, antibody fragment, Fab', F(ab') 2 , Fv, scFv, di-scFvs, bi-scFvs, tandem scFvs, bispecific tandem scFvs, sdAb, V H , V L , humanized antibody, chimeric antibody, IgA antibody, IgD antibody, IgE antibody, IgG antibody, IgM antibody, intrabody, minibody or monobody.
  • proteins that can be produced by the cell of the present invention are any polypeptides, with or without biological functions, including enzymes, regulatory proteins, receptors, peptides, e.g. peptide hormones, growth factors, cytokines, structural proteins, lymphokines, adhesion molecules, receptors, membrane or transport proteins, and any other polypeptides that can serve as agonists or antagonists and/or have therapeutic or diagnostic use.
  • the proteins of interest may be antigens as used for vaccination, vaccines, antigen-binding proteins, immune stimulatory proteins. It may also be an antigen-binding fragment of an antibody, which can include any suitable antigen-binding antibody fragment known in the art.
  • an antibody fragment may include but not limited to Fv (a molecule comprising the VL and VH), single- chain Fv (scFV) (a molecule comprising the VL and VH connected with by peptide linker), Fab, Fab', F(ab')2, single domain antibody (sdAb) (molecules comprising a single variable domain and 3 CDR), and multivalent presentations thereof.
  • Fv a molecule comprising the VL and VH
  • scFV single- chain Fv
  • Fab Fab'
  • F(ab')2 single domain antibody
  • sdAb single domain antibody
  • therapeutic proteins include an antibody, polyclonal antibody, monoclonal antibody, recombinant antibody, antibody fragment, Fab', F(ab') 2 , Fv, scFv, di-scFvs, bi-scFvs, tandem scFvs, bispecific tandem scFvs, sdAb, V H , V L , humanized antibody, chimeric antibody, IgA antibody, IgD antibody, IgE antibody, IgG antibody, IgM antibody, intrabody, minibody or monobody.
  • Further proteins of interest include, but are not limited to, insulin, insulin-like growth factor, hGH, tPA, cytokines, e.g.
  • interleukines such as IL-1 , IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11 , IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, interferon (IFN) alpha, IFN beta, IFN gamma, IFN omega or IFN tau, tumor necrosisfactor (TNF) TNF alpha and TNF beta, TRAIL; G-CSF, GM-CSF, M-CSF, MCP-1 and VEGF as well as homologues thereof.
  • the display system mainly involves (1 ) a N-terminal secretion tag recognized by T3SS, (4) one or more effector domains of a T3SS effector protein having a certain length for secretion, and (5) an anchoring sequence which anchors the recombinant proteins onto the surface of transfected cells.
  • N-terminal secretion tag recognized by said T3SS and the optional (2) chaperone binding domain as well as other characterization of the cells have been described earlier in the present specification and are applicable for constructing cells for surface display.
  • the (3) protein of interest to be displayed is not limited to therapeutic proteins. Since cell display libraries are particularly useful for generating protein-specific affinity reagents for therapeutics and drug discovery, any type of proteins or polypeptides can be displayed by the cells according to the present invention.
  • the surprising discovery of the inventors has furthermore made it possible for the first time to partially secrete large proteins via T3SS.
  • the present invention enables a method for displaying large polypeptides in a cell surface display system.
  • the T3SS comprised in the cell is preferably from Salmonella typhimurium, Salmonella enterica, Pseudomonas aeruginosa, Yersinia pseudotuberculosis, Yersinia pestis, Shigella flexneri, Citrobacter rodentium, Escherichia coli EHEC, Escherichia coli EPEC, Pseudomona syringae, Ralstonia solanacearum, Xanthomonas campestris, and Erwinia amylovora.
  • the N-terminal secretion tag is preferably obtained from SipA, SipB, SipC, SipD, SopA, SopB, SopD, AvrA, SopE2 and most preferably from SptP.
  • the chaperone binding domain is preferably from one of SipA, SipB, SipC, SipD, SopA, SopB, SopD, SopE2, SptP, AvrA.
  • the cell may optionally comprise a chaperone selected from SicA, InvB, SicP, SigE, InvB, SicP, SigE, SicA.
  • one of the following N-terminal secretion tag and chaperone binding domain/chaperones is encoded by the nucleotides in the cell: SipA/lnvB, SipC/SicA, SopA lnvB, SopE2/lnvB, SptP/SicP, SopB/SigE.
  • the chaperone binding domain and the protein of interest are preferably separated by a linker.
  • the protein of interest can be cleaved off from the secretion tag and the optional chaperone binding domain by a protease recognizing a protease cleavage site preceding the protein of interest. Overview of the signal sequences, chaperone binding domain and effector domains of various Salmonella effector proteins is provided in Fig. 9.
  • the cell comprises a type III secretion system (T3SS) and a protein comprising from N- to C-terminus an N-terminal secretion tag recognized by said T3SS, a chaperone binding domain, a protein of interest, three Spt effector domains from Salmonella, and a protein having a globular structure or zinc finger domain.
  • T3SS type III secretion system
  • the secretion tag and chaperone binding domain can optionally be selected from SipA, SipB, SipC, SipD, SopA, SopB, SopD, SopE2, SptP and AvrA from Salmonella.
  • the cell comprises a type III secretion system (T3SS) and a protein comprising from N- to C-terminus an N-terminal secretion tag recognized by said T3SS, optionally a chaperone binding domain, a protein of interest, three Spt effector domains from Salmonella and a GFP or ubiquitin.
  • T3SS type III secretion system
  • the cell can optionally comprise a chaperone selected from SicA, InvB, SicP, SigE, InvB, SicP, SigE, SicA.
  • the cell comprises a type III secretion system (T3SS) and a protein comprising from N- to C-terminus an N-terminal secretion tag recognized by said T3SS, optionally a chaperone binding domain, a protein of interest, one or more effector domains of a T3SS effector protein having a length in amino acids that has at least the length of at least three SptP effector domains and a protein having a globular structure or zinc finger domain, wherein the following N-terminal secretion tag and chaperone binding domain/chaperones are applied SipA lnvB, SipC/SicA, SopA lnvB, SopE2/lnvB, SptP/SicP, SopB/SigE.
  • T3SS type III secretion system
  • a protein comprising from N- to C-terminus an N-terminal secretion tag recognized by said T3SS, optionally a chaperone binding domain, a protein of interest, one or more
  • T3SS is preferably a salmonella T3SS derived from the Salmonella Pathogenicity Island 1 (SPI-1 ) locus.
  • the protein of interest can be cleaved off from the secretion tag, the optional chaperone binding domain, and/or the effector domains by a protease recognizing a protease cleavage site preceding the protein of interest and/or following. Successful cleavage will allow the protein of interest to be delivered into a host cell such as eukaryotic cells via its T3SS.
  • the present cell surface display system is characterized in a spanning sequence comprising one or more effector domains have at least a given length to span the T3SS secretion path and expose the preceding protein of interest to the extracellular environment.
  • the term "spanning sequence" is a stretch of amino acid sequence of the partially secreted protein that occupies the secretion path when partial secretion is attained.
  • the spanning sequence can be composed of any sequence which does not have a stable structure which cannot be unfolded by ATPase of the T3SS system. A skilled person can readily determine when needed by way of assay whether a given spanning sequence is unfoldable.
  • the spanning sequence comprises one or more "effector domains," which refer to one or more of “complete, partial or homologous sequences" of the portion in a given effector protein because such sequences are transportable by T3SS.
  • effector domains refer to one or more of “complete, partial or homologous sequences" of the portion in a given effector protein because such sequences are transportable by T3SS.
  • homologous it is meant that the sequences are at least 5%, such as at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 97, 98 and 99% homologous in comparison.
  • the spanning sequence comprises at least 1 , 2, 3, 4 or 5 effector domains from any one of SipA, SipB, SipC, SipD, SopA, SopB, SopD, AvrA, SopE2 and SptP from Salmonella.
  • the "effector domain" for the purpose of the present invention does not include the N terminal secretion sequence.
  • the spanning sequence comprises 1 , 2, 3, 4, 5, 6, or 7 effector domains from SipA, SipB, SipC, SipD, SopA, SopB, SopD, AvrA, SopE2 and/or SptP. Sequences of these proteins are provided in Table 2 with reference to SwissProt accession numbers.
  • the spanning sequence comprises effector domains from one effector protein as repeats. However, it is also possible to combine effector domains from different effector proteins. Furthermore, it is not necessary that the "effector domain" retains its biological activity as an effector protein.
  • the inventors have compared and analyzed the structural differences between empty T3SS and T3SS with partially secreted substrate (also referred to as substrate- trapped needle complexes) and it was surprisingly found that this secretion path of the needle complex is longer than empty T3SS.
  • substrate- trapped needle complexes partially secreted substrate
  • this secretion path of the needle complex is longer than empty T3SS.
  • the path is about 800 A and at least three SptP effector domains is sufficient to span the secretion path of the T3SS needle complex (see Example 5- 7).
  • the spanning sequence comprises one or more effector domains where each effector domain comprises an amino acid sequence which is at least 10, 20, 30, 40, 50, 60, 70, 80, 90 91 , 92, 93, 94, 95, 96, 97, 98, 99 and 100% homologous to SEQ ID NO: 2.
  • sequence based alignment methodologies which are well known to those skilled in the art, are useful in determining homology among sequences. These include, but not limited to, the local identity/homology algorithm of Smith, F. and Waterman, M. S. (1981 ) Adv. Appl. Math. 2: 482-89, homology alignment algorithm of Peason, W. R. and Lipman, D. J. (1988) Proc. Natl. Acad. Sci. USA 85: 2444-48, Basic Local Alignment Search Tool (BLAST) described by Altschul, S. F. et al. (1990) J. Mol. Biol. 215: 403-10, or the Best Fit program described by Devereau, J. et al.
  • BLAST Basic Local Alignment Search Tool
  • homology is calculated by Fast alignment algorithms based upon the following parameters: mismatch penalty of 1.0; gap size penalty of 0.33, joining penalty of 30 (see “Current Methods in Comparison and Analysis” in Macromolecule Sequencing and Synthesis: Selected Methods and Applications, p. 127-149, Alan R. Liss, Inc., 1998).
  • Another example of a useful algorithm is PILEUP. PILEUP creates multiple sequence alignment from a group of related sequences using progressive, pairwise alignments. It can also plot a tree showing the clustering relationships used to create the alignment.
  • PILEUP uses a simplification of the progressive alignment method of Feng, D. F. and Doolittle, R. F. (1987) J. Mol. Evol. 25, 351-60, which is similar to the method described by Higgins, D. G. and Sharp, P. M. (1989) CABIOS 5: 151-3.
  • Useful parameters include a default gap weight of 3.00, a default gap length weight of 0.10, and weighted end gaps.
  • Another example of a useful algorithm is the family of BLAST alignment tools initial described by Altschul et al. (see also Karlin, S. et al. (1993) Proc. Natl. Acad. Sci. USA 90: 5873-87).
  • WU-BLAST-2 program described in Altschul, S. F. et al. (1996) Methods Enzymol. 266: 460-80.
  • WU-BLAST uses several search parameters, most of which are set to default values.
  • the HSP S and HSP S2 parameters are dynamic values and are established by the program itself depending upon the composition of the particular sequence and composition of the particular database against which the sequence of interest is being searched; however, the values may be adjusted to increase sensitivity.
  • An additional useful algorithm is gapped BLAST as reported by Altschul, S. F. et al. (1997) Nucleic Acids Res. 25: 3389-402.
  • Gapped BLAST uses BLOSSOM-62 substitution scores; threshold parameter set to 9; the two-hit method to trigger ungapped extensions; charges gap lengths of k at cost of 10+k; Xu set to 16, and Xg set to 40 for database search stage and to 67 for the output stage of the algorithms. Gapped alignments are triggered by a score corresponding to -22 bits. Speific programs have been developed to may and assemble NGS data, e.g. the program BOWTIE.
  • Salmonella invasion protein A plays a vital role in the invasion process, via actin binding and in inducing inflammation.
  • the C-terminal entity of SipA is important for F-actin bundling, inhibits actin depolymerization and potentiates the activity of SipC, thereby supporting the invasion process.
  • the protein is processed by Caspase3 between residues 431 to 434, resulting in two fragments.
  • the C-terminal fragment contains the actin interacting domains, whereas the N-terminal fragment is important for the inflammatory response.
  • SipA induces CXC chemokine and IL- 8 expression by phosphorylation of JUN and p38MAPK and activates NF K B via NOD1/NOD2 signaling.
  • SipB is one of the two hydrophobic translocases.
  • the secretion sequence was found to be at residues 3 to 8 of the SipB protein and the chaperone binding domain is located around somewhere between residues 80 and 100 (see for example Kim et al., "Analysis of functional domains present in the N-terminus of the SipB protein", Microbiology 153, 2998-3008 (2007)).
  • the translocase has two transmembrane helices in the C-terminal region, which are inserted into the host membrane. Upon insertion of the transmembrane helices a hydrophilic loop of SipB is located in the mammalian cytoplasm. SipB has been shown to induce apoptosis in macrophages in a caspasel -dependent and a caspasel - independent manner.
  • SipC is the second hydrophobic translocase and exhibits actin nucleation, actin bundling and translocation functions.
  • the protein forms homooligomers in the translocation pore and interacts with SipB. Its secretion depends on the first 120 amino acids of SipC, where the secretion signal and the chaperone binding domain are located.
  • the translocation activity of SipC depends on its interaction with SipB, however the mechanism for membrane insertion remains elusive. SipB and SipC have been shown to form extracellular complexes during secretion.
  • Salmonella Invasion Protein D (SipD) is part of the tip complex and is denoted the hydrophilic translocase protein. It has two functional domains, comprising an N-terminal secretion signal and a C-terminal functional domain.
  • SopA has a 45 N-terminal domain containing the secretion signal and the chaperone binding domain (CBD).
  • the C-terminal domain contains the HECT-like motif, composed of a substrate binding-helix, an extended central domain, the N-lobe and a globular C-terminal C-lobe.
  • the mammalian ubiquitin system uses a triade of enzymes to transfer the small post-translational modification to proteins.
  • the first enzyme is an E1 ubiquitin-activating enzyme, which transfers the protein to an E2 ubiquitin conjugating enzyme. From E2 the ubiquitin is transferred to an E3 ubiquitin-ligase, which subsequently transfers the modification to a lysine of the target protein.
  • SopA is an E3- ubiquitin-protein ligase, which can transfer ubiquitin to target proteins. SopA uses UbcH5a, UbcH5c and UbcH7 as E2 ubiquitin-conjugating enzymes. These E2s have been shown to be involved in inflammation. SopA induces inflammation and polymorphonuclear transmigation, thereby promoting enteritidis.
  • SopB The N-terminus of Salmonella outer protein B (SopB) is characterized by the presence of a secretion signal and the chaperone binding domain (CBD), which is followed by a guanine dissociation inhibitor (GDI) domain comprising residues 1 17 to 168.
  • CBD chaperone binding domain
  • GDI guanine dissociation inhibitor
  • the C- terminal moiety carries two inositol-4-phosphatase domains and a very C-terminal synaptojanin-homologous region (residues 357 - 561 ).
  • SopB has sequence homology to mammalian inositol polyphosphate 4-phosphatases and that recombinant SopB has inositol phosphate phosphatase activity in vitro. SopB mediates virulence by interdicting inositol phosphate signaling pathways.
  • Salmonella outer protein D is a 317 amino acid polymer and has a weight of 36.141 kDa. SopD is involved in membrane fission and macropinosome formation. It is recruited to membranes in a SopB-dependent manner and thus these two effectors act cooperatively to encourage host-cell membrane internalization and sealing. Loss of SopD leads to delayed membrane fission kinetics, but has little impact on the uptake efficiency of Salmonella.
  • N-terminal approximately 100 amino acids of the guanine nucleotide exchange factor SopE serve as secretion signal and chaperone binding domain (CBD) for the chaperone InvB, whereas the enzymatic activity is lying in the C-terminal part (from residues 78 to 240).
  • the Avirulence Protein A (AvrA) is a ubiquitin-like protein cystein protease and exhibits acetyltransferase activity. It enhances proliferation and inhibits inflammation by stabilizing ⁇ -catenin and inhibiting JNK. The latter one is a consequence of the acetylation of upstream activators (MKKs) on serines or threonines, which inhibits their activation. Additionally AvrA deubiquitinizes Wnts and therefore dampens inflammatory responses. Recently AvrA has been shown to be important for the establishment of chronic infections.
  • the Salmonella protein tyrosine phosphatase has an N-terminal secretion signal followed by a chaperone binding domain of 139 residues, which is bound by Salmonella invasion chaperone P (SicP).
  • the central domain of the protein has GTPase activating protein (GAP) function from residues 174 to 290, which induces small GTPases to hydrolyse their bound GTP and renders them inactive.
  • GTPase activating protein (GAP) function from residues 174 to 290, which induces small GTPases to hydrolyse their bound GTP and renders them inactive.
  • the very C-terminal residues 340 to 543 have tyrosine-protein phosphatase (PTPase) activity.
  • SptP has GAP activity for Rac1 and Cdc42 and thereby antagonizes the effect of SopE, reconstituting the cytoskeleton. Additionally, the inactivation of the Rho GTPases leads to the downregulation of JNK phosphorylation, which is induced by activated ho GTPases and leads to the expression of inflammatory genes. As SptP can be detected in cells up to eight hours post infection, it is also involved in the formation of the intracellular niche Salmonella uses to survive (SCV). SptP interacts with the AAA+ ATPase Valosin- containing protein (VCP) and dephosphorylates it.
  • VCP Valosin- containing protein
  • SptP targets VCP on membranes, distinct from the SCV, dephosphorylates it, which in turn allows the interaction of VCP with adaptor proteins and cofactors leading to the formation of Sa/mone//a-induced filaments.
  • the function of SptP is therefore important for the maintenance of the SCV and allows the acquisition of nutrients.
  • the complete polypeptide length preceding the anchoring sequence may have a length of more than 864 amino acid residues, such as more than 865, 866, 867, 868, 869, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000, 1 100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000 residues or more.
  • a protein having a globular structure is expressed C-terminally to the one or more effector domains for anchoring.
  • Globular structure can be determined by X-ray crystallography or NMR spectroscopy.
  • GFP is a globular protein of 27 kDa with a diameter of about 35x50 A.
  • Ubiquitin is another example of globular protein which is about 8 kDa and has a diameter of about 20x30 A.
  • the globular structure should have a minimum diameter of at least 10 A, such as at least 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30 A, so it will anchor the sequence to the needle complex at the basal side of the complex.
  • the term globular protein refers to proteins which has a generally round structure.
  • the present invention provides a method for identifying a cell secreting a protein of interest via T3SS comprising (a) providing one or more cells on which proteins are partially secreted via T3SS, ; and (b) identifying said protein of interest with a detection moiety which recognizes said protein of interest. Identification can be performed by using any known means in the field of cell surface display.
  • a zinc finger is a small protein structural motif that is characterized by the coordination of one or more zinc ions to form a stable compact fold.
  • Zinc fingers coordinate zinc ions with a combination of cysteine and histidine residues.
  • Classes of zinc fingers include Cys2His2, Gag knuckle, Treble clef, Zinc ribbon, Zinc ribbon, Zn2/Cys6, and TAZ2 domain like zinc fingers.
  • the term "zinc finger domain" as used within the present invention refers to a protein domain that comprises a zinc ion and is capable of binding to a specific three basepair DNA sequence. Proteins comprising zinc fingers are known in the art.
  • T3SS Any cells can be used for cell surface displayed employing T3SS as described herein.
  • Pathogens which inherently express T3SS can advantageously employed, although non-pathogenic cells may be preferred for reduced harmful effects due to the pathogenicity.
  • Cells suitable for cell surface display of proteins of interest include Salmonella typhimurium, Bacillus subtilis, Pseudomonas aeruginosa, Vibrio cholerae, Klebsiella pneumonia, Neisseria gonorrhoeae, Neisseria meningitidis, Bacteroides nodosus, Moraxella bovis, and Escherichia coli. Examples
  • [001 15] We designed a series of substrates to demonstrate properties allowing or precluding entry into and secretion from the needle complex. Overview of the design can be seen in Set-1 of Fig. 1 a.
  • the non-flagellated Salmonella typhimurium strain SB905 was derived from SJW2941 as described in Sukhan et al. J Bacterid 183, 1 159-1 167 (2001 ). This strain carried the plasmid pSB3291 (AmpR) expressing the transcriptional regulator hilA under the araBAD promoter for overexpression of needle complex proteins.
  • Substrates were based on SptP, containing an N-terminal signal (35 amino acids), a chaperone binding domain and one or multiples of the effector domain organized as tandem repeats (SptP1 , SptP3, SptP5). Sequences for 3x-FLAG-tags were introduced at the C-terminal end of the proteins. To maintain a balanced ratio of designed substrates and the chaperone SicP, co-expression was carried out by cloning both constructs into a single plasmid (pACYCDuet-1 (CmR) (Merck Chemicals Ltd.).
  • Natural, wild-type SptP (60.1 kD) served as a positive control for secretion. Position of migration of various SptP constructs in 4-20% SDS-PAgels is indicated on the right-hand side of the individual gels.
  • Fig. 1 d summarizes the result as well as the size of the substrates in terms of residue and the molecular weight. This example demonstrates that the protein size does not dictate T3SS secretion and substrate of large size and concatemeric design can be transported through T3SS.
  • globular proteins fused at the N terminus of the substrate such as GFP (26kD; about 35x50 A) or ubiquitin (8kD; about 20x30 A) are able to hinder substrate secretion.
  • Fig. 1c Overview of the substrate design in this example is shown in Fig. 1c.
  • NC structural needle complex
  • Fig. 1 c and 1d show that only the substrate fused to ubiquitin mutant (I3G/1 13G) but not wild type ubiquitin was specifically secreted by the T3SS.
  • natural SptP see anti SptP in Fig. 1 c
  • the stability of wild-type ubiquitin prevented the substrate secretion from the T3SS.
  • the conformationally-destabilized mutant I3G/I13G was secreted by the T3SS.
  • Cryo electron tomography was performed on Salmonella typhimurium SB905 wild type and SptP3-GFP expressing cells.
  • Five ⁇ _ of osmotically shocked bacteria were applied on holey carbon grids (Quantifoil, R2/1 , Mo, 400 mesh) and then mixed with 10 nm gold particles used as fiducial markers and subsequently vitrified automatically in liquid ethane using the Leica grid plunger.
  • Vitrified samples were transferred under liquid nitrogen temperatures into a 300 kV field emission gun (FEG) Polara transmission electron microscope (FEI). Tilt series were acquired using the software SerialEM covering an angular range of -60 to +60 degrees and with tilt increments of 2 degrees.
  • FEG field emission gun
  • FEI Polara transmission electron microscope
  • the upper panel shows the reconstructed cryo electron tomogram of the cell containing T3SSs in wild type cells.
  • the lower panel shows cells expressing SptP3-GFP (lower panel)).
  • the T3SSs in the presence of the GFP-fused substrates were strikingly similar.
  • SB905 cells expressing SptP3-GFP show additional densities at the needle tip of injectisomes (see arrow).
  • Fig. 3a Overview of the design can be seen in Fig. 3a.
  • the substrates used are wildtype SptP, Spt3, and Spt3-GFP. Methods and materials used are as described under Example 1 and 2, except that IPTG was added after 4 hours arabinose induction (time point Omin) for a another growth period of 60, 120, or 180 minutes before harvesting to allow measurement for incremental secretion (one hour before harvesting fresh and pre-warmed (37 ° C) LB medium (+ 0.3mM NaCI) was exchanged).
  • Fig. 3b Fig. 3b
  • SipB Fig. 3c
  • Expression of needle complex proteins InvG, PrgH, and PrgK monitored by immunoblotting using rabbit anti needle complex (NC) antibodies.
  • Fig. 3c shows that SptP3-GFP also has an inhibitory effect on the secretion of the natural substrate SipB. Increasing expression of SptP3-GFP over time (180min) in cells correlated with a decrease of SipB secreted in the cell culture supernatant.
  • Substrate trapped complexes were then immuno-purified from the isolated needle complexes by using anti-FLAG M2 magnetic beads (Sigma-Aldrich).
  • buffer A (10 mM Tris, 500 mM NaCI, 5 mM EDTA, 0.1 % (w/v) LDAO (n-Dodecyl-N,N-Dimethylamine-N- Oxide), pH 8.0) equilibrated beads (250 ⁇ _, 50% (v/v) slurry) were transferred into a 0.7 ml_ tube and equilibrated 3x10 min with 500 ⁇ _ buffer A.
  • CsCI fractions 2-5 of previously purified needle complexes were pooled (about 120 ⁇ _), added to the equilibrated beads and the volume adjusted to 700 ⁇ _ using buffer A. The solution was kept at room temperature under gentle agitation for 2-3 hours. Thereafter, beads were washed 10x using 400 ⁇ _ buffer A each under gentle agitation for 10 min. Bound complexes were eluted with 150 ⁇ _ 3x FLAG peptide (400 ng/ ⁇ ) in buffer A for 45 min at room temperature. The elution fractions were subsequently analyzed by Western blotting.
  • SptP3-GFP containing complexes were immunopurified using a 3x FLAG-tag fused to the C terminus of SptP3-GFP ('S' input sample, 'FT' flow-through, ⁇ ' elution).
  • As a control substrate-free wild type (w.t.) needle complex preparations were not withheld on anti FLAG-beads as analyzed by Western blotting (anti NC) monitoring for the presence of the NC structural proteins InvG, PrgH, and PrgK.
  • the simultaneous demarcation of the substrate entry and exit positions for the substrate shows that N-terminal SptP3-portion of SptP3-GFP enters needle complex and continues until the C-terminally fused GFP domain reaches the entry position because the transport is blocked by GFP due to the folded state.
  • the substrate spanning the needle and occupying the entire secretion path confirms that fully unfolded proteins are passed through T3SS. Furthermore, this also shows that the length of at least three SptP effector domains prior to the globular protein allows for the preceding polypeptides to exit and extend outside the needle complex.
  • the difference volume obtained reflects the space that is occupied by the substrate (Fig. 6c, Asub), and these dimensions imply the presence of an unfolded substrate within injectisomes. Difference mapping also revealed small changes within the larger inner ring-1 (IR1 ) of needle complexes (A IR1 ), which is distant from the secretion path.
  • the secretion path within the injectisome is characterized by areas of different diameters (Fig. 6d): the funnel-shaped 'portal' at the cytoplasmic site tapers from about a 15 to 10 A wide opening to continue into an approximately 10x10 A constriction ('channel') connected to a about 40 A wide space ('atrium'). This means the protein which is used to plug the opening should have a minimal diameter of more than 10-15 A so it would not pass the opening and/or the channel. From the atrium, the secretion path continues with an about 20 A wide 'tunnel', which is defined by the inner rod and the needle filament.
  • the 'portal', 'channel', and 'atrium' are determined by the centrally located cup and socket substructures (Fig. 6d), Surprisingly, we observed that the dimensions of the 'channel', the narrowest part of the secretion path, stay largely invariant during substrate transport. Therefore, the constricting dimensions of the 'channel' can be plugged by an unfolded domain which is located C-terminally from the substrate.
  • Fig. 7 shows that the most frequent needle length for substrate-trapped NCs is 41.4 nm and for substrate-free NCs 29.9 nm. It is surprisingly found that substrate-containing complexes have longer needles.
  • vgfp 323 36.194 binds eGFP SEQ ID NO . 6
  • Nanobodies vamy 330 37,069 binds Amylase SEQ I • 7
  • HEL 321 35.520 binds Lysozyme SEQ ID NO • 9
  • Adnl 315 35.324 binds EGFR SEQ ID NO 10
  • HA4-7cl 2 420 46.025 binds Bcr-Abl SEQ ID NO 12
  • Ubiquitin 283 31.91.2 mammalian, protein SEQ ID NO 16
  • the transgenic Salmonella strains were subsequently used for a secretion assay.
  • Salmonella Typhimurium is grown in T3SS inducing medium (LB supplemented with 0.3M NaCI) for six hours, allowing the bacteria to secrete T3S proteins into the cell culture supernatant.
  • T3SS inducing medium LB supplemented with 0.3M NaCI
  • the bacteria to secrete T3S proteins into the cell culture supernatant.
  • the cells are separated from the supernatant by centrifugation and subsequently the supernatant and cells are subjected to SDS-PAGE and immunoblotting.
  • supernatant and cells were probed with a polyclonal SptP antibody (aSptP) and with a monoclonal Flag antibody (aFlag).
  • aSptP polyclonal SptP antibody
  • aFlag monoclonal Flag antibody
  • the SptP antibody allows the direct detection of the SptP moiety.
  • Flag blot additionally allows the detection of the carboxyterminal end of the secreted effectors and therefore the combination ensures that the correct constructs are expressed and secreted.
  • Scheme of the assay is provided in Fig. 10a and the result of the secretion is shown in Fig. 10b.
  • Fig. 10B The blots in Fig. 10B indicate that all tested nanobody effectors (vgfp, vamy, perennial and HEL; bands around 36kDa) and all tested monobodies (Adn1 and HA4; 34:5/ Da) were secreted. Furthermore, T3SS also successfully secreted the tandem monobody (HA4- 7c12, tandem), which gives a band at 46/ Da. In a separate experiment it was found that the secreted nanobodies and monobodies are functional after secretion into cell supernatant.
  • thermodynamically less stable mutant ubiquitin (I3G/I13G, Ubi3,13) which could be unfolded was secreted.
  • the stably folded wild-type ubiquitin (Ubi) was not secreted.
  • the proteins Granzyme B (GrmB) and Oct4 also showed no bands in the supernatant and are therefore considered to be not secreted.
  • Granzyme B is a trypsin-like serine proteinase, which is the major activator of apoptotic pathways. It is secreted by natural killer (NK) cells and cytotoxic T leukocytes (CTL) to kill transformed or infected cells and activate the apoptotic program.
  • NK natural killer
  • CTL cytotoxic T leukocytes
  • the protein has a stable complex structure forming two ⁇ barrels connected by loops as in GFP: As shown above GFP cannot be secreted via Salmonella Typhimurium T3SS.
  • Granzyme contains basic amino acid stretches which diminish its secretion efficiency.
  • Oct4 is a transcription factor of the POU (Pit, Oct, Unc) homeobox family.
  • the expression of Oct4 is tightly regulated and high expression is usually found in pluripotent stem cells, where it is responsible to keep the cells in an undifferentiated state.
  • Oct4 contains two helix-loop-helix DNA binding motifs which forms dimers by packing the second helix against the same helix of another molecule. It is also believed that the non-secretion by Salmonella T3SS is due to the in the basic helix establishing a DNA contact.
  • Fig. 10B bottom shows the cells probed with a polyclonal needle complex antibody (aNC) used as a loading control to estimate the amount of loaded needle complexes (NCs) and to allow the evaluation of needle complex induction and check if cell lysis appeared by probing the supernatant.
  • aNC polyclonal needle complex antibody
  • Multiple prominent bands, corresponding to the major building blocks of the needle complex, are detected by the antibody such as InvG (62/fDa), PrgH (45/ Da) and PrgK (28/cDa).
  • the aNC in the supernatant shows no bands, meaning that no cells were lysed.
  • the transcription activator-like endonucleases (AbrBs3 and variants) cannot be amplified by PCR during cloning and was therefore sub-cloned to pCASP(MCS2) (Fig. 1 1 a and b) by gateway cloning and the restriction site used for the excision of the full-length construct lies behind a stop codon.
  • the soluble fraction of the AbrBs3 and variants was precipitated by salting out with ammonium sulfate and subjected to SDS-page and immunoblotting.
  • the blots for the AvrBs3 variants are shown in Figure 10c for SptP and NC, respectively.
  • the needle complex blots served as loading and induction control.
  • AvrBs3 and AvrBs3-VP64 TAL-TF, 142kDa, see Fig. 10c) can be secreted, whereas AvrBs3-Fokl (TALEN, 157kDa) was not secreted.
  • the endonuclease domain of Fokl from Flavobacterium okeanokoites is known to form dimers.
  • the lack of secretion observed for AvrBs3-Fokl demonstrates that an oligomerization interface interferes with T3SS secretion.

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Abstract

La présente invention concerne une cellule qui sécrète totalement ou partiellement une protéine d'intérêt via un système de sécrétion de type III (T3SS). Les cellules comprenent un système de sécrétion de type III et une protéine d'intérêt présentant une étiquette de sécrétion N-terminale. On obtient une sécrétion partielle au moyen d'une séquence d'ancrage qui bouche le point d'entrée du T3SS afin d'empêcher la sécrétion complète de la protéine d'intérêt.
PCT/EP2014/075478 2013-11-26 2014-11-25 Sécrétion totale ou partielle d'une protéine et présentation à la surface d'une cellule au moyen d'un système de sécrétion de type iii Ceased WO2015078840A1 (fr)

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WO2021256244A1 (fr) * 2020-06-16 2021-12-23 出光興産株式会社 Agent comprenant de la guadinomine en tant que principe actif pour prévenir une infection bactérienne à gram négatif et les dégâts provoqués par une maladie due à une infection bactérienne à gram négatif
WO2021257803A1 (fr) * 2020-06-17 2021-12-23 Flagship Pioneering Innovations Vi, Llc Adas comprenant des systèmes de sécrétion bactérienne
CN113838520A (zh) * 2021-09-27 2021-12-24 电子科技大学长三角研究院(衢州) 一种iii型分泌系统效应蛋白识别方法及装置
EP3932937A1 (fr) * 2020-07-03 2022-01-05 Universitätsklinikum Hamburg-Eppendorf Nouveau domaine de translocation de protéines
US11471494B2 (en) 2017-01-06 2022-10-18 Synlogic Operating Company, Inc. Microorganisms programmed to produce immune modulators and anti-cancer therapeutics in tumor cells
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US11723932B2 (en) 2016-01-11 2023-08-15 Synlogic Operating Company, Inc. Microorganisms programmed to produce immune modulators and anti-cancer therapeutics in tumor cells
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WO2021256244A1 (fr) * 2020-06-16 2021-12-23 出光興産株式会社 Agent comprenant de la guadinomine en tant que principe actif pour prévenir une infection bactérienne à gram négatif et les dégâts provoqués par une maladie due à une infection bactérienne à gram négatif
WO2021257803A1 (fr) * 2020-06-17 2021-12-23 Flagship Pioneering Innovations Vi, Llc Adas comprenant des systèmes de sécrétion bactérienne
EP3932937A1 (fr) * 2020-07-03 2022-01-05 Universitätsklinikum Hamburg-Eppendorf Nouveau domaine de translocation de protéines
CN113838520A (zh) * 2021-09-27 2021-12-24 电子科技大学长三角研究院(衢州) 一种iii型分泌系统效应蛋白识别方法及装置
CN113838520B (zh) * 2021-09-27 2024-03-29 电子科技大学长三角研究院(衢州) 一种iii型分泌系统效应蛋白识别方法及装置

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