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WO2010054445A1 - Detection of tannerella forsythia infection - Google Patents

Detection of tannerella forsythia infection Download PDF

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Publication number
WO2010054445A1
WO2010054445A1 PCT/AU2009/001495 AU2009001495W WO2010054445A1 WO 2010054445 A1 WO2010054445 A1 WO 2010054445A1 AU 2009001495 W AU2009001495 W AU 2009001495W WO 2010054445 A1 WO2010054445 A1 WO 2010054445A1
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WO
WIPO (PCT)
Prior art keywords
protein
peptide
fragment
individual
tannerella
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/AU2009/001495
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French (fr)
Inventor
Eric Charles Reynolds
Paul Veith
Neil Martin O'brien-Simpson
Stuart Geoffrey Dashper
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Oral Health Australia Pty Ltd
Original Assignee
Oral Health Australia Pty Ltd
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Filing date
Publication date
Priority claimed from AU2008905956A external-priority patent/AU2008905956A0/en
Application filed by Oral Health Australia Pty Ltd filed Critical Oral Health Australia Pty Ltd
Publication of WO2010054445A1 publication Critical patent/WO2010054445A1/en
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911—Bacteria
    • G01N33/56955—Bacteria involved in periodontal diseases
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/118—Prognosis of disease development
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/142—Toxicological screening, e.g. expression profiles which identify toxicity

Definitions

  • the invention relates to detecting Tannerella forsythia infections and related pathologies.
  • Chronic periodontitis is an inflammatory disease of the supporting tissues of the teeth leading to resorption of alveolar bone and eventual tooth loss.
  • the disease is a major public health problem in all societies and is estimated to affect up to 15% of the adult population with severe forms affecting 5-6%.
  • T. forsythia (previously named Tannerella forsythensis and Bacteroides forsythus) is a Gram-negative, anaerobic, fusiform bacterium that is found associated with Porphyromonas gingivalis and Treponema denticola in sub-gingival plaque. Together they are implicated as periodontal pathogens.
  • T. forsythia A distinctive feature of the T. forsythia proteome is the presence of a major double band above 200 kDa in SDS- PAGE profiles of whole cell extracts. These bands correspond to two proteins designated TfsA and TfsB which are the major constituents of the surface layer (S- layer), a distinct layer beyond the OM that can be clearly seen in electron micrographs. Recently, these proteins were reported to play an important role in the adherence of the bacteria to gingival epithelial cells using deletion mutants.
  • BspA a cell surface antigen containing a leucine-rich repeat (LRR) domain and which binds to fibrinogen and fibronectin.
  • LRR leucine-rich repeat
  • T. forsythia also possesses trypsin-like activity and a sialidase (neuraminidase) but the importance of these potential virulence factors or their co- relation to specific protein sequences is not yet clear.
  • T. forsythia which can be used to diagnose or monitor T. forsythia related diseases and conditions such as periodontal disease.
  • a "fragment" of a protein or peptide as used herein is an amino acid sequence that is characteristic of the protein or peptide from which it is derived. In some embodiments, the fragment is least 5 amino acids in length, preferably greater than 10 amino acids, preferably greater than 20 amino acids in length.
  • the invention provides a method for determining whether an individual is susceptible to periodontal disease, or likely to develop periodontal disease or for screening for early stage periodontal disease including:
  • the invention provides a method for determining the risk of an individual developing periodontal disease including: - selecting an individual;
  • the methods of the invention are used for determining whether a periodontal site has a Tannerella infection, is susceptible to periodontal disease, or is likely to develop periodontal disease, or for screening for early stage periodontal disease, for monitoring Tannerella infection in a periodontal site receiving treatment for periodontal disease, for determining risk of a periodontal site developing periodontal disease and/or for determining the risk of periodontal disease progression at a periodontal site.
  • a use of a polynucleotide or fragment thereof for detecting a nucleic acid that encodes or controls the expression of a protein described in Table 1 or 2 herein for determining whether an individual has a Tannerella infection in another embodiment there is provided a use of a polynucleotide or fragment thereof for detecting a nucleic acid that encodes or controls the expression of a protein described in Table 1 or 2 herein for determining whether an individual has a Tannerella infection.
  • a kit for determining whether an individual has a Tannerella infection including:
  • polynucleotide or fragment thereof capable of detecting a nucleic acid encoding or controlling the expression of a protein, peptide or fragment thereof described in Table 1 or 2 herein.
  • the kit may further include one or more Tannerella proteins, peptides or fragment thereof for use as a control and written instructions for use of the kit in a method as described herein.
  • the protein, peptide or fragment thereof is arranged on a solid phase to permit capture of an antibody in a sample taken from a person for whom Tannerella infection is to be detected.
  • the antibody is arranged on a solid phase to permit capture of a protein, peptide or fragment thereof in a sample taken from a person for whom Tannerella infection is to be detected.
  • the invention provides a substantially non-glycosylated protein, peptide or fragment thereof described in Table 1 or 2 herein.
  • the protein, peptide or fragment thereof described in Table 1 or 2 herein does not contain any glycosylated amino acids.
  • the substantially non-glycosylated protein is a non-glycosylated form of a protein selected from the group consisting of TF1342, TF0090, TF0091 , TF1331 TF2123, TF1056 and TF0945.
  • the protein, peptide or fragment thereof described in Table 1 or 2 herein that does not contain any glyclosylated amino acids is selected from the group consisting of TF1342, TF0090, TF0091 , TF1331 TF2123, TF1056 and TF0945.
  • the protein, peptide or fragment thereof of the invention is in a composition.
  • the composition further comprises a carrier.
  • Substantially non-glycosylated protein, peptide or fragments thereof are described further herein.
  • the antiserum has specificities that react with non-glycosylated protein, peptide or fragments thereof described in Table 1 or 2 herein.
  • the antiserum is polyclonal. In another embodiment the antiserum is monoclonal.
  • a substantially non-glycosylated protein, peptide or fragment thereof described in Table 1 or 2 herein in the preparation of a medicament for the treatment or prevention of a Tannerella infection (and/or the other conditions identified herein as suitable for treatment).
  • the medicament is for the treatment or prevention of periodontal disease.
  • a substantially non-glycosylated protein, peptide or fragment thereof described in Table 1 or 2 herein for use in the treatment or prevention of a Tannerella infection (and/or the other conditions identified herein as suitable for treatment).
  • the invention provides a composition for the treatment or prevention of periodontal disease (and/or the other conditions identified herein as suitable for treatment) comprising as an active ingredient a substantially non- glycosylated protein, peptide or fragment thereof described in Table 1 or 2 herein. Also provided is a use of an antigen in the form of:
  • Tannerella For determining whether an individual is infected with a Tannerella.
  • kits for determining whether an individual is infected with a Tannerella including:
  • an antigen being a protein, peptide or fragment thereof that is expressed in a Tannerella
  • an antigen being a protein, peptide or fragment thereof described in Table 1 or 2 herein;
  • polynucleotide capable of detecting a nucleic acid encoding or controlling the expression of at least one of the above described antigens.
  • a kit of the invention may further include written instructions for use of the kit in a method as described herein.
  • an immune complex including an antigen being a protein, peptide or fragment thereof described in Table 1 or 2 herein bound to an antibody specific for said protein, peptide or fragment thereof.
  • Fig 1 Representative 2D gel of T. forsythia outer membrane. Spots are numbered 1-54 with each number corresponding to a unique protein. Adjacent spots corresponding to the same protein are joined by lines, or marked with an * indicating that only a fragment of the protein was identified. Spots labelled 'N' and 'C represent N- and C-terminal fragments of surface layer protein B respectively.
  • Fig 2 Carbohydrate staining of 2D gel of T. forsythia outer membrane. 2D gel stained with Pro-Q Emerald fluorescent carbohydrate stain (Right) prior to staining with Coomassie Blue (Left).
  • Fig 3 Multiple alignment of CTD family proteins from T. forsythia and P. gingivalis.
  • CTD family proteins from T. forsythia were identified by multiple BLAST searches against the T. forsythia database from the oralgen website (www.oralgen.lanl.gov). To reduce redundancy, six CTD sequences TF2592, TF2339, TF1591 , TF2998, TF3080 and TF0541 were excluded due to their high sequence identity to the exhibited CTD sequences TF1741 , TF1259, TF1589, TF1843, TF1458, TF0537 respectively. Multiple alignment was conducted separately for T. forsythia and P.
  • PG gingivalis sequences using the ClustalW program. Invariant residues within the T. forsythia sequences are marked with an asterisk (*), strongly conserved positions are marked with a colon (:), and moderately conserved positions are marked with a period (.) Deletions of two residues and four residues from the P. gingivalis alignment are marked with triangles ( ⁇ ). Residues underlined represent the most C-terminal peptides identified by MS/MS (Table 3). Residues in bold represent C-terminal peptides identified from unmodified proteins isolated from P. gingivalis strain HG66 (unpublished data).
  • Fig 4 TonB-dependent receptor containing loci identified.
  • the solid arrows (left) represent the genes of TonB-dependent receptors (R), while the dashed arrows represent adjacent genes that encode lipoproteins (L).
  • Each gene is labeled with its TIGR accession number, which is highlighted if the encoded protein was identified (see Tables 1 & 2).
  • the lipoproteins were always found to be downstream of their respective receptors, and the lipoproteins directly downstream all share sequence similarity to each other with the exception of TF2606.
  • the receptor labeled 1506/1507 is annotated as two separate receptors, however BLAST searching indicates that they represent two halves of a single receptor suggesting a mutation or sequencing error in the TF1506 stop codon.
  • Fig 5 Heat-modifiability and the effect of reduction on the MW of TF1331.
  • TF1331 enriched sample was subjected to SDS-PAGE.
  • A 100° C, + DTT;
  • B 50° C, + DTT;
  • C 100° C;
  • D 50° C.
  • Arrows represent bands identified as TF1331 by MS.
  • BenchmarkTM protein standards were used containing proteins of MW 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 160, and 220 kDa. The 10, 15 and 25 kDa markers are not shown.
  • Fig 6 LIFT-TOF/TOF data for m/z 3516.6 di-sulfide bonded peptide from TF1331.
  • A Full spectrum. Boxed areas are presented enlarged in panels B and C.
  • C Zoomed in spectrum showing backbone fragmentation pattern and its assignment to the RPEFCPECPKCPEVK peptide from TF1331.
  • Fig 7 Western Blot Analysis. 2D-Westerns of T. forsythia outer membrane were performed and probed with (A) antisera raised against the outer membrane preparation and (B) antisera raised against formalin killed T. forsythia cells. C. Image from (A) was coloured red and overlayed with Coomassie Blue stained 2D gel (blue). The scales of the images were manipulated by linear stretches only. The identity of numbered spots are presented in Table 4.
  • Fig. 8 SDS-PAGE gel of T. forsythia outer membrane. Identical samples of T. forsythia OM were separated on a 10% PA Bis-Tris gel with MOPs running buffer. The gel was stained with Coomassie Blue G250. Fifty-five contiguous gel segments were excised from four identical lanes. Two lanes are shown in order to clearly mark the centre of each excised segment.
  • a Tannerella infection is an infection that is characterised by invasion or contamination of tissues or cells by a Tannerella.
  • a Tannerella infection may have clinical or subclinical manifestations including acute or chronic inflammation of the gingiva. The hallmarks of acute inflammation may be present including an increased movement of plasma and leukocytes from the blood into the injured tissues. Clinical signs of acute infection of the gingiva may also be present including rubor (redness), calor (increased heat), tumor (swelling), dolor (pain), and function laesa (loss of function).
  • Chronic inflammation may be characterised by leukocyte cell (monocytes, macrophages, lymphocytes, plasma cells) infiltration. Tissue and bone loss may be observed.
  • a Tannerella infection may also be characterised by an increased level of Tannerella bacteria, particular T. forsythia, above a normal range observed in individuals without a Tannerella infection.
  • a Tannerella infection may be manifested in an individual at a periodontal site that has greater than approximately 10 3 to 10 5 Tannerella cells/mg of subgingival plaque.
  • a Tannerella infection may be manifested in an individual at a periodontal site that has greater than approximately 10 4 Tannerella cells/mg of subgingival plaque.
  • the "measuring for the expression" of a target protein in a sample is the detection of the degree or level or amount or concentration of a protein, peptide or fragment thereof described in Table 1 or 2 herein.
  • An individual that is selected and from whom a sample is obtained may be an individual that has a previous history of Tannerella infection or periodontal disease or a person already diagnosed with periodontal disease. Alternatively, the individual may not have had a prior Tannerella infection or experienced periodontal disease previously.
  • the target protein is a protein, peptide or fragment thereof described in Table 1 or 2.
  • Detection of a protein, peptide or fragment thereof indicates the presence of a Tannerella bacteria, in particular T. forsythia.
  • the detection of a level or degree of expression of a protein, peptide or fragment thereof may indicate the level of Tannerella bacteria, in particular T. forysthia, in a sample which in turn may indicate the occurrence of, or degree of, or progression of a Tannerella infection or periodontal disease.
  • a strong level of expression, increased amount or concentration of a protein, peptide or fragment thereof in a sample taken from an individual compared to an uninfected control may indicate a high level of Tannerella in the sample.
  • the detection of the presence or level or degree of expression of a protein, peptide or fragment thereof may correlate with the risk of developing a Tannerella infection or periodontal disease.
  • Tannerella infection in an individual or an individual “infected with a Tannerella” generally refers to:
  • Tannerella may be Tannerella forsythia.
  • the sample obtained from the individual is a body fluid or fraction thereof.
  • the body fluid or fraction thereof from the individual may be an oral fluid taken from the oral cavity.
  • an oral fluid may be saliva, gingival crevicular fluid or blood.
  • oral fluids for example saliva, are a combination of secretions from a number of sources such as parotid, submandibular, sublingual, accessory glands, gingival mucosa and buccal mucosa and the term oral fluid includes the secretion of each of these sources individually or in combination.
  • the saliva may be stimulated or in a preferred embodiment unstimulated. Stimulation of the saliva in the individual may occur by allowing the individual to chew on sugar-free gum, a piece of paraffin film or tart candy. Unstimulated saliva means that the subject will expectorate into a collection vessel without stimulation of salivary flow.
  • Saliva specimens for testing can be collected following various methods known in the art, for example, stimulated or unstimulated saliva can be sampled by the individual expectorating into a collection vessel or using a swab or syringe to extract the saliva. Other ways for obtaining unstimulated saliva are known in the art. (Nazaresh and Christiansen, J. Dent. Res. 61 : 1158- 1162 (1982)). Methods and devices for collecting saliva have also been described. (See also, U. S. Patent No. 5,910, 122).
  • the methods of the present invention can also be practiced by analyzing stimulated saliva.
  • the methods of the present invention are not limited to performing salivary analysis immediately after collection of the sample.
  • salivary analysis following the methods of the present invention can be performed on a stored saliva sample.
  • the saliva sample for testing can be preserved using methods and apparatuses known in the art. (See e.g., U. S. Patent No. 5,968, 746).
  • the methods of the present invention be used to perform salivary analysis on saliva samples that have been treated to reduce its viscosity.
  • the viscous nature of saliva due to the nature of mucopolysaccharides, makes testing of these fluids difficult.
  • the saliva may be rendered sufficiently fluid (i.e. viscosity must be reduced) and free from debris. Techniques used to remove debris include centrifugation and filtration.
  • the viscosity of saliva can also be reduced by mixing a saliva sample with a cationic quaternary ammonium reagent. (See, U. S. Patent No. 5,112, 758).
  • the sample from an individual may be taken from the crypts of the dorsum of the tongue.
  • the sample from the individual may be taken from a specific periodontal site.
  • a periodontal site is a region within the oral cavity.
  • a periodontal site is region around a tooth including disto-buccal, mid-buccal, mesio-buccal, mesio-palatal, mid- palatal and disto-palatal and disto-lingual, mid-lingual and mesio-lingual.
  • the sample may be taken from a periodontal site that exhibits clinical signs of periodontal disease or Tannerella infection. A method, use, protein or composition of the invention could then be used to determine whether the periodontal site is infected with Tannerella, or at risk of periodontal disease progression.
  • the sample from an individual may be a sample of a tissue.
  • the tissue or part thereof may be from the oral cavity.
  • the tissue is gingival.
  • the gingival tissue may be from various sites around a tooth including disto- buccal, mid-buccal, mesio-buccal, mesio-palatal, mid-palatal and disto-palatal and disto- lingual, mid-lingual and mesio-lingual.
  • the tissue may be obtained by normal biopsy or may be obtained from an extracted tooth.
  • the sample from the individual may be dental plaque.
  • the plaque may be subgingival or supragingival.
  • Subgingival plaque may be sampled using a sterile curette or paper point.
  • Supragingival plaque may be removed using standard techniques known in the art.
  • the subgingival plaque may be collected from various sites around a tooth including disto-buccal, mid-buccal, mesio-buccal, mesio-palatal, mid- palatal and disto-palatal and disto-lingual, mid-lingual and mesio-lingual periodontal sites.
  • the subgingival plaque samples may be obtained during the normal dental examination provided by a qualified dentist or periodontist.
  • the plaque sample may be analysed as is or treated to extract the protein, peptide or fragment thereof of interest using an extraction buffer.
  • An extraction buffer could contain a pH buffer (e.g. phosphate, HEPES, etc), salts (e.g. NaCI) to maintain ionic strength and protein solubilising agents (e.g. detergents (SDS, Triton X100, etc)), reducing agents (e.g. dithiothreitol, cysteineHCI) and/or chaotropic agents (e.g. urea, guanidinium chloride, lithium perchlorate).
  • a pH buffer e.g. phosphate, HEPES, etc
  • salts e.g. NaCI
  • protein solubilising agents e.g. detergents (SDS, Triton X100, etc)
  • reducing agents e.g. dithiothreitol, cysteineHCI
  • chaotropic agents e.g. urea, gu
  • an "uninfected control” is a sample from an individual or represents a set of parameters previously defined from individuals that do not have all the attributes of an individual with a Tannerella infection.
  • the individual from whom the "uninfected control" sample is derived generally does not have inflammation of the gums, antibodies directed against Tannerella in their blood, have an amount of Tannerella above approximately 10 3 to 10 5 cells/mg of subgingival plaque and/or have an amount of Tannerella above approximately 10 3 cells/ml of saliva.
  • the "uninfected control" sample may be taken from the oral cavity of a subject that, but for an absence of a Tannerella infection, is generally the same or very similar to the individual selected for determination of whether they have a Tannerella infection (the latter otherwise known as the "test sample").
  • the measurement of the level of expression of the target in the sample from the oral cavity of the subject for deriving the uninfected control is generally done using the same assay format that is used for measurement of the target protein in the test sample.
  • the control sample may also be taken from the same individual from which the test sample is taken, but at a different time-point, or periodontal site, in order to determine the risk of, or progression of the Tannerella infection or periodontal disease.
  • individuals with a healthy oral cavity may contain a low level of Tannerella present, in particular T. forsythia. This low level or normal level of bacterial colonisation may be sampled and is also within the scope of "uninfected control".
  • determination of whether an individual has a Tannerella infection or periodontial disease includes (1 ) an elevated level of Tannerella in a sample taken from the individual compared to the uninfected control sample, or (2) an increased proportion of Tannerella bacteria in a sample taken from the individual compared to the total level of bacteria in the uninfected control sample, or (3) an increased proportion of Tannerella bacteria relative to one or more other bacteria species in a sample taken from the individual when compared with the uninfected control sample.
  • the method includes measuring the expression of the target in the uninfected control to compare the measured level in the test sample with the level in the uninfected control.
  • an internal standard is applied. This may be used to ensure that the method operates within accepted decision limit quality control criteria.
  • the assay then provides a value/number/result or other output for each test sample. That output is then deemed to represent infection or non infection based on independent data derived from frequency distributions of results from an uninfected control.
  • This control may describe distributions of results obtained from more than one uninfected subject, for example, from an uninfected population which may be of the same species, geographic origin, age, sex as the test sample.
  • a positive -negative cut -point for the method is determined from these distributions to provide defined levels of diagnostic sensitivity and specificity for the method.
  • the internal standard or reference may obviate the need to physically provide an uninfected control in the form of uninfected cells or otherwise to physically measure the level of the target in an uninfected control.
  • the level of expression in an uninfected control has been predetermined and may be provided for example in the form of written information that is supplied with a diagnostic kit.
  • the measurement of the level of expression of the target in the subject for deriving the uninfected control is generally done using the same assay format as that that is used for measurement of the target in the test sample. However, it is not necessary to use the same assay when an internal standard that can be used to compare data obtained from different assay formats is or has been applied.
  • the subject from which the uninfected control is derived and the individual selected for determination of whether they have an infection are of the same family.
  • the individual for determination of whether they have a Tannerella infection is a human
  • the negative control is generally derived from the measurement of the level of expression of the target in a human.
  • the individual selected for determination of whether they have an infection and the subject from which the negative control is to be derived are from the same species. It may not be necessary that they be of similar age or sex or have been exposed to similar environmental influence.
  • the detection of expression of the protein in the individual is assessed by the steps of:
  • An "infected control” is a sample from an individual or represents a set of parameters previously defined from individuals that do have all the attributes of an individual with a Tannerella infection.
  • the individual from whom the "infected control" sample is derived generally does have subgingival plaque, inflammation of the gums, antibodies directed against Tannerella in their blood, have an amount of Tannerella at a level above approximately 10 3 to 10 5 cells/mg of subgingival plaque and/or have an amount of Tannerella at a level above approximately 10 3 cells/ml of saliva.
  • the "infected control" sample may be taken from the oral cavity of a subject that, but for the presence of a Tannerella infection, is generally the same or very similar to the individual selected for determination of whether they have a Tannerella infection (the latter otherwise known as the "test sample").
  • the measurement of the level of expression of the target in the sample from the oral cavity of the subject for deriving the infected control is generally done using the same assay format that is used for measurement of the target protein in the test sample.
  • the control sample may also be taken from the same individual from which the test sample is taken, but at a different time-point, or periodontal site, in order to determine progression of the Tannerella infection or periodontal disease.
  • the method includes measuring the level of expression of the target in the infected control to compare the measured level in the test sample with the level in the infected control.
  • an internal standard may be applied that obviates the need to provide an infected control or otherwise to measure the level of the target in an infected control.
  • the measurement of the level of expression of the target in the tissue of the subject for deriving the infected control is generally done using the same assay format as that that is used for measurement of the target in the test sample. However, again it is not necessary to use the same assay when an internal standard that can be used to compare data obtained from different assay formats is or has been applied.
  • the subject from which the infected control is derived and the individual selected for determination of infection are of the same family.
  • the infected control is generally derived from the measurement of the level of expression of the target in a human.
  • the individual selected for determination of whether they have an infection and the subject from whom the positive control is to be derived are from the same species. It may not be necessary that they be of similar age or sex or have been exposed to similar environmental influence.
  • the measurement of the level of one or more Tannerella protein, peptide or fragments thereof in the "test sample” may be compared with standard measurements of the same protein, peptide or fragments thereof associated with a series of known Tannerella levels, for example, 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 or 10 9 cells grown in the laboratory and cell numbers verified using FACS or real time PCR.
  • the antibodies used to capture and detect the protein, peptide or fragment thereof will be used at dilutions to ensure that any chairside test for the bacteria in a sample (e.g.
  • saliva and/or subgingival plaque sample will give a positive reaction (e.g. a noticeable colour) only for concentrations above certain amounts of cells, e.g. 10 4 Tannerella cells per mg of subginigval plaque sample.
  • the intensity of the colour reaction will be a guide to the clinician on the level of the Tannerella at the periodontal site. This will be provided as a colour guide with a kit of the invention described further herein.
  • the positive reading will be taken by the clinician to indicate a Tannerella infection at that periodontal site. Hence that site will be treated (e.g. debridement of the tooth root to remove all subgingival plaque) and the patient, depending on the severity of the infection, may be prescribed a course of antibiotic therapy.
  • the target protein is directly detected to determine whether the individual has a Tannerella infection. This is otherwise known as a "direct detection" of the target to measure the level of expression of the target.
  • the target protein, peptide or fragment thereof described in Table 1 or 2 herein may be detected.
  • the level of expression of a molecule, the expression of which is modulated in accordance with the expression of the target is measured. This is otherwise known as an "indirect detection" of the target to measure the level of expression of the target.
  • a nucleic acid contained in the individual selected for determination of whether they have a Tannerella infection that encodes a target protein, or that is complementary to a nucleic acid that encodes a target protein is measured.
  • the nucleic acid may be one which can be used to determine the presence of a given protein, or level of expression of a given protein in an individual as per a molecular genetic approach.
  • a polynucleotide that is complementary to a nucleic acid (DNA, RNA, cDNA) that encodes a target protein is hybridised to the nucleic acid and hybridisation is detected.
  • quantitative PCR Others include quantitative Northern and Southern blotting, and microarray.
  • the method includes the step of detecting a target protein, or peptide or fragment thereof in a sample from the individual to assess the level of expression of the target in the individual.
  • the presence of a given protein, or level of expression of a given protein in an individual can be detected by any number of assays. Examples include immunoassays, chromatography and mass spectrometry. One example of an immunoassay that is particular preferred is FACS.
  • Immunoassays i.e. assays involving an element of the immune system are particularly preferred. These assays may generally be classified into one of:
  • purified antigen for example, an antigen that is expressed in a Tannerella
  • purified antigen is bound to solid phase by adsorption or indirectly through another molecule and serum from an individual is applied followed by another antibody for detecting presence or absence of host antibody;
  • purified antigen for example, an antigen that is expressed in a Tannerella
  • immune cells such as T and B lymphocytes.
  • peripheral white cells are purified from an individual and cultured with purified antigen. The presence or absence of one or factors indicating immunity are then detected.
  • assays that measure cell proliferation include assays that measure cell proliferation (lymphocyte proliferation or transformation assays) following exposure to purified antigen, and assays that measure cell death (including apoptosis) following exposure to purified antigen;
  • the level of expression of the target protein may be measured by obtaining a sample from an individual selected for assessment and determining the level of expression of the target in the sample.
  • the level of expression could be determined in vivo, for example by providing labelled antibodies to the individual which can be visualised in vivo.
  • Various assays that can be used to detect the presence of a target protein in a sample include:
  • Enzyme linked immunosorbent assay This method involves fixation of a sample, for example saliva, containing a target protein, peptide or fragment thereof to a surface such as a well of a microtiter plate.
  • a target protein specific antibody coupled to an enzyme is applied and allowed to bind to the target protein, peptide or fragment thereof. Presence of the antibody is then detected and quantitated by a colorimetric reaction employing the enzyme coupled to the antibody.
  • Enzymes commonly employed in this method include horseradish peroxidase and alkaline phosphatase. If well calibrated and within the linear range of response, the amount of target protein, peptide or fragment thereof present in the sample is proportional to the amount of color produced.
  • a target protein, peptide or fragment thereof standard is generally employed to improve quantitative accuracy.
  • Western blot This method involves separation of a target protein, peptide or fragment thereof from other protein by means of an acrylamide gel followed by transfer of the protein, peptide or fragment thereof to a membrane (e.g., nylon or PVDF). Presence of the target protein, peptide or fragment thereof is then detected by antibodies specific to the target protein, peptide or fragment thereof, which are in turn detected by antibody binding reagents.
  • Antibody binding reagents may be, for example, protein A, or other antibodies.
  • Antibody binding reagents may be radiolabeled or enzyme linked as described hereinabove. Detection may be by autoradiography, colorimetric reaction or chemiluminescence. This method allows both quantitation of an amount of target protein, peptide or fragment thereof and determination of its identity by a relative position on the membrane which is indicative of a migration distance in the acrylamide gel during electrophoresis.
  • Radio-immunoassay In one version, this method involves precipitation of the desired target protein, peptide or fragment thereof with a specific antibody and radiolabeled antibody binding protein (e.g., protein A labelled with I 125 ) immobilized on a precipitable carrier such as agarose beads. The number of counts in the precipitated pellet is proportional to the amount of target protein, peptide or fragment thereof.
  • a specific antibody and radiolabeled antibody binding protein e.g., protein A labelled with I 125
  • a labelled target protein, peptide or fragment thereof and an unlabelled antibody binding protein are employed.
  • a sample containing an unknown amount of a target protein, peptide or fragment thereof is added in varying amounts.
  • the decrease in precipitated counts from the labelled target protein, peptide or fragment thereof is proportional to the amount of target protein, peptide or fragment thereof in the added sample.
  • Fluorescence activated cell sorting This method involves detection of a target protein, peptide or fragment thereof in situ in cells by target protein, peptide or fragment thereof specific antibodies.
  • the target protein, peptide or fragment thereof specific antibodies are linked to fluorophores. Detection is by means of a cell sorting machine which reads the wavelength of light emitted from each cell as it passes through a light beam. This method may employ two or more antibodies simultaneously.
  • lmmunohistochemical analysis This method involves detection of a target protein, peptide or fragment thereof in situ in fixed cells by target protein, peptide or fragment thereof specific antibodies.
  • the target protein, peptide or fragment thereof specific antibodies may be enzyme linked or linked to fluorophores. Detection is by microscopy and subjective or automatic evaluation. If enzyme linked antibodies are employed, a colorimetric reaction may be required. It will be appreciated that immunohistochemistry is often followed by counterstaining of the cell nuclei using for example Hematoxyline or Giemsa stain.
  • In situ activity assay According to this method, a chromogenic substrate is applied on the cells containing an active enzyme and the enzyme catalyzes a reaction in which the substrate is decomposed to produce a chromogenic product visible by a light or a fluorescent microscope.
  • In vitro activity assays In these methods the activity of a particular enzyme is measured in a protein mixture extracted from the cells. The activity can be measured in a spectrophotometer well using colorimetric methods or can be measured in a non- denaturing acrylamide gel (i.e., activity gel). Following electrophoresis the gel is soaked in a solution containing a substrate and colorimetric reagents. The resulting stained band corresponds to the enzymatic activity of the protein of interest. If well calibrated and within the linear range of response, the amount of enzyme present in the sample is proportional to the amount of colour produced. An enzyme standard is generally employed to improve quantitative accuracy.
  • the assay is a point-of-care or point-of-use diagnostic test.
  • Point-of-care testing is defined as diagnostic testing at or near the site of patient care or tissue or body fluid or plaque sampling. The motivation behind POCT is to bring the test conveniently and immediately to the patient, which in turn increases the likelihood that the patient will receive the results in a timely manner. Therefore, treatment can immediately follow diagnosis.
  • a non-limiting example of a point-of-care test is a lateral flow test.
  • Lateral flow tests also known as lateral flow immunochromatographic assays are a simple device intended to detect the presence (or absence) of a target protein, peptide or fragment thereof in sample.
  • Lateral flow tests are a form of immunoassay in which the test sample flows along a solid substrate, for example paper strip, via capillary action. After the sample is applied to the test it encounters a coloured reagent which mixes with the sample and transits the substrate encountering lines or zones which have been pretreated with an antibody or antigen. Depending upon the analytes present in the sample the coloured reagent can become bound at the test line or zone.
  • the antibody-antigen binding can release some material that has been pre- bound to the antibody, such as gold or colloid nanoparticles.
  • the colloid in turn may produce a visible line on the substrate which can be detected either by the naked eye or imaging device such as silicon photodiode or CCD device. Lateral flow tests can operate as either competitive or sandwich assays.
  • any coloured particle can be used, however in a preferred embodiment either latex (blue colour) or nanometer sized particles of gold (red colour) are used.
  • the gold particles are red in colour due to localised surface plasmon resonance. Fluorescent or magnetic labelled particles can also be used in combination with an electronic reader to assess the test result.
  • the sample first encounters coloured particles which are labelled with antibodies raised to the target protein, peptide or fragment thereof.
  • the test line will also contain antibodies to the same target, although it may bind to a different epitope on the target protein, peptide or fragment thereof.
  • the test line will show as a coloured band in positive samples.
  • the sample first encounters coloured particles which are labelled with the target protein, peptide or fragment thereof or an analogue.
  • the test line contains antibodies to the target/its analogue. Unlabelled target protein, peptide or fragment thereof in the sample will block the binding sites on the antibodies preventing uptake of the coloured particles and the test line will show as a coloured band in negative samples.
  • one version could be used to assess the risk of future periodontal disease development in an individual (e.g., high, medium or low risk for future periodontal disease development).
  • test version could be used to quantitate periodontal disease risk leading to the prediction of periodontal disease experience at subsequent ages. This test might be administered in a dentist's office where appropriate countermeasures could be initiated. Yet another test version would be diagnostic and used with medically compromised patients, such as those suffering from diabetes or AIDS. Still another test version would feature multiple sample, high throughput characteristics. The use of this test version would be targeted to screening populations of samples, for example saliva samples, such as those used for epidemiological surveys. It will be appreciated that the tools necessary for detecting the presence of a target protein, peptide or fragment thereof in a sample from an individual may be provided as a kit, which may contain one or more unit dosage form containing an active ingredient for detection of the target protein, peptide or fragment thereof.
  • the kit may comprise means for collecting the sample and specific detection means packaged separately.
  • the kit may be accompanied by instructions for use.
  • the kit may include devices such as a dipstick or a cartridge, (optionally comprised in a housing) which the individual or clinician places into the oral cavity or sample obtained from the individual.
  • the device may comprise any agent capable of specifically detecting the target proteins, peptides or fragments thereof.
  • the device may comprise one or a combination of monoclonal and polyclonal antibody reagents or fragments thereof and an indicator for detecting binding.
  • Antibody supports are known in the art. In an embodiment of this invention, antibody supports are absorbent pads to which the antibodies are removably or fixedly attached.
  • the device of the invention is a lateral flow device comprising an inlet means for flowing a fluid, for example body fluid, into contact with one or more agents, for example a polyclonal or monoclonal antibody or fragment thereof, capable of detecting the proteins, peptides or fragments thereof of the present invention.
  • the test device can also include a flow control means for assuring that the test is properly operating.
  • Such flow control means can include control proteins, peptides or fragments thereof bound to a support which capture detection antibodies as a means of confirming proper flow of sample fluid through the test device.
  • the flow control means can include capture antibodies in the control region which capture the detection antibodies, again indicating that proper flow is taking place within the device.
  • the kit comprises a monoclonal target protein, peptide or fragment thereof coloured conjugate and polyclonal anti-target protein coated on a membrane test area.
  • the sample migrates over the test area and reacts with the impregnated reagents to form visible coloured bands in the test window.
  • the presence of the target protein, peptide or fragment thereof in concentrations above normal will result in the formation of a distinct coloured band in the test area thus indicating a positive result for a Tannerella infection or risk of progression of periodontal disease or any other outcome described herein. Conversely, if no line appears in the test area, the test is negative.
  • the bacteria may be a species or subspecies of Tannerella including Tannerella forsythia.
  • Tannerella is of the species forsythia (Tannerella forsythia).
  • the test for a Tannerella may be used in combination with a method detection of other bacteria known to effect periodontal disease initiation or progression.
  • the bacteria may be Porphyromonas gingivalis or Treponema denticola.
  • the methods of the present invention may be performed at the same time as analysis of clinical parameters.
  • clinical parameters include modified gingival index (Lobene et al. Clin Rev Dent 1986: 8:3-6), plaque index (Silness et al. Acta Odontol Scand 1964: 22 121-135), pocket depth, recession, clinical attachment level, bleeding on probing and suppuration.
  • the method may be useful for assessing a response of an individual to administration of a protein or substance representing part or all of a Tannerella.
  • the protein or substance is administered to an individual and the expression of a target protein described in Table 1 or 2 herein is assessed to determine a response to the Tannerella protein or substance.
  • the individual is a human.
  • the human may be either male or female.
  • the age of the human can be between 18 and 35 years old, between 2 and 45 years old; between 2 and 80 years old or above; or between 15 and 60 years old or above. It is not intended that the methods of the invention are limited to determining Tannerella infection in individuals within a particular age group.
  • the individual may be an animal including a domestic animal.
  • the animal is a cat, dog, sheep, cow or horse.
  • the invention provides a substantially non-glycosylated protein, peptide or fragment thereof described in Table 1 or 2 herein.
  • the protein, peptide or fragment thereof described in Table 1 or 2 herein does not contain any glycosylated amino acids.
  • the substantially non-glycosylated protein is a non-glycosylated form of TF1342, TF0090, TF0091 , TF1331 , TF2123.
  • the amino acid sequences of these proteins, and others, of the invention are listed in Table 6 and referenced by the SEQ ID NOs: 1 to 5 shown therein.
  • the invention provides an antigenic region of a substantially non- glycosylated protein of the invention.
  • An antigenic region of a protein may be determined using various algorithms including EMBOSS Antigenic (Kolaskar.AS and Tongaonkar.PC (1990). A semi-empirical method for prediction of antigenic determinants on protein antigens. FEBS Letters 276: 172-174) or Antigenicity Plot (Hopp.T.P. and Woods.K.R. (1981 ) Prediction of protein antigenic determinants from amino acid sequences. Proc Natl Acad Sci USA 86:152-156).
  • the fragment of a substantially non-glycosylated protein of the invention is a region of the protein that is exposed on the surface of the bacteria when the protein is in its native conformation in the bacteria.
  • Tannerella have an outer membrane and outer membrane proteins are those which are embedded in or protrude from the outer membrane.
  • a protein, peptide or fragment thereof of the invention has a sequence that has 60, 70, 80, 90, 91 , 92, 93, 94, 95, 96, 97, 98 or 99% amino acid identity to a protein, peptide or fragment thereof described in Table 1 or 2 herein.
  • the protein, peptide or fragment thereof is substantially non- glycosylated.
  • a protein, peptide or fragment thereof of the invention has a sequence that has 60, 70, 80, 90, 91 , 92, 93, 94, 95, 96, 97, 98 or 99% amino acid identity to TF1342, TF0090, TF0091 , TF1331 TF2123.
  • the protein, peptide or fragment thereof is substantially non-glycosylated.
  • the protein, peptide or fragment thereof is in a composition.
  • substantially all of the protein in a composition of the invention is substantially non-glycosylated.
  • all of the protein in a composition of the invention is substantially non-glycosylated.
  • the composition consists solely of protein.
  • the composition of the invention may include, further to protein, other cellular components such as fragments or parts of a membrane or cell wall.
  • the composition may include lipids, carbohydrates and nucleic acids.
  • a composition of the invention may be used to generate antibodies in an animal, for example a mouse, rat, rabbit, sheep or human.
  • the composition may be used to generate polyclonal or monoclonal antibodies.
  • the antibodies have a number of utilities including detection of an immune response in an individual generated against a Tannerella or other in vitro or in vivo applications.
  • a protein or composition of the invention may also be used to detect an immune response in an individual generated against a Tannerella.
  • the composition further comprises a carrier, diluent preservative or other component that could be used to modify the immune response in an animal having received the composition.
  • the carrier, diluent, preservative or other component is particularly useful for increasing the likelihood of generating an antibody response in an animal to a protein, peptide or fragment thereof or composition of the invention.
  • composition of the invention comprises a substantially non- glycosylated protein, peptide or fragment thereof differing from a protein, peptide or fragment thereof described in Table 1 or 2 by conservative amino acid substitutions.
  • Substantially non-glycosylated with respect to a protein, peptide or fragment thereof described in Table 1 or 2 herein, is defined as less than half the maximum level of glycosylation of the protein, peptide or fragment thereof predicted by any available computer software anaylsis, for example on http://www.expasy.ch/tools/ or http://www.cbs.dtu.dk/services/.
  • “Substantially non-glycosylated” in certain embodiments is also defined as less than half the maximum level of glycosylation of the protein, peptide or fragment thereof as determined experimentally.
  • Substantially non- glycosylated may refer to a reduced number of amino acids per protein, peptide or fragment thereof that are glycosylated or to reduced number of saccharides on each glycosylated amino acid.
  • Substantially non-glycosylated may also refer to a protein, peptide or fragment thereof that has been treated with one or more glycosidases or may refer to a protein, peptide or fragment that has a glycosylation profile that is the same or similar to the glycosylation profile of a protein, peptide or fragment thereof that has been treated with a glycosidase.
  • Percent (%) amino acid identity or " percent (%) identical” with respect to a protein, peptide or polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the specific protein, peptide or polypeptide sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity.
  • PSI-Blast can be used to perform an iterated search that detects distant relationships between molecules. See Altschul et al. (1997) supra.
  • the default parameters of the respective programs e.g., BLASTX and BLASTN
  • Alignment may also be performed manually by inspection.
  • Another non- limiting example of a mathematical algorithm utilized for the comparison of sequences is the ClustalW algorithm (Higgins et al. (1994) Nucleic Acids Res. 22:4673- 4680).
  • ClustalW compares sequences and aligns the entirety of the amino acid or DNA sequence, and thus can provide data about the sequence conservation of the entire amino acid sequence.
  • the ClustalW algorithm is used in several commercially available DNA/amino acid analysis software packages, such as the ALIGNX module of the Vector NTI Program Suite (Invitrogen Corporation, Carlsbad, CA). After alignment of amino acid sequences with ClustalW, the percent amino acid identity can be assessed.
  • a non- limiting examples of a software program useful for analysis of ClustalW alignments is GENEDOCTM or JalView (http://www.jalview.org/). GENEDOCTM allows assessment of amino acid (or DNA) similarity and identity between multiple proteins.
  • the outer membrane (OM) of T. forsythia was separated by 2D-PAGE and the spots were identified by MALDI-TOF/TOF MS.
  • the sixty-nine putative outer membrane proteins (Omps) that were identified represent a sub-proteome that includes many unique features that are compared and contrasted to Omps identified from the related Porphyromonas and Bacteroides genuses.
  • T. forsythia strain ATCC43037 was grown anaerobically in brain heart infusion media supplemented with 5 ⁇ g/ml hemin, 0.5 ⁇ g/ml menadione, 0.001% w/w N-acetyl muramic acid and 5% fetal bovine serum.
  • the conditions for the cultivation of T. forsythia mimic the environment of a site affected by periodontal disease in an oral cavity.
  • T. forsythia outer membrane was prepared by the sarkosyl method (Filip, C et al. (1973). Solubilization of the cytoplasmic membrane of Escherichia coli by the ionic detergent sodium-lauryl sarcosinate. J Bacteriol 115, 717- 722). Briefly, harvested cells were resuspended in 35 ml buffer (50 mM Tris pH 8.0, 150 mM NaCI, 5 mM MgCI 2 ), and sonicated on ice for 15 min at 50% power and a duty cycle of 5 in a Branson Sonifier. The membranes were collected by centrifugation at 40,000 g, and washed twice with buffer.
  • the membranes were then resuspended in an equal volume of buffer and 2% sodium-lauryl sarcosinate (aq) and incubated at 37 0 C for 1 h to facilitate inner membrane solubilisation.
  • the OM was pelleted at 40,000 g, washed with buffer and stored at -7O 0 C.
  • TF1331 enrichment OM sample from above was incubated in solubilizer 2 (Invitrogen) at 50° C for 10 min and centrifuged at 50,000 g for 15 min. The pellet was resuspended in solubilizer 2, centrifuged again and the supernatant discarded. The pellet containing mainly TF1331 was analysed by SDS-PAGE using a precast NuPAGE Bis-Tris mini gel with MOPS running buffer (Invitrogen).
  • Two-dimensional polyacrylamide gel electrophoresis 2D-PAGE was performed using pH4-7 IPG strips in the first dimension and 4-12% Bis-Tris Zoom gels in the second dimension according to the manufacturer's instructions (Invitrogen).
  • Solubiliser 2 (Invitrogen) was used to solubilise the outer membrane samples, and IPG strips were reduced with DTT and alkylated with iodoacetamide before the second dimension.
  • Gels were stained for protein with colloidal Coomassie Blue (Molloy, M. P., et al. (2000). Proteomic analysis of the Escherichia coli outer membrane. Eur J Biochem 267, 2871-2881.) or for carbohydrate with Pro-Q Emerald 300 fluorescent stain according to the manufacturer's protocol (Invitrogen) and imaged with a LAS 3000 (Fuji).
  • the Coomassie stained gel was placed in an SP gel frame of cut-out size 76x70 mm and placed onto the scanner of the Proteineer SP spot picker (Bruker Daltonics) where it was submerged under water.
  • the gel was scanned and analyzed using Proteomweaver software (Biorad) to enable spot detection and gel calibration with respect to pi and MW.
  • the spotlist was transferred to the spot picker and spot picking was performed using a 1mm diameter cutting tool and the preset "pick pick" method for non-backed gels.
  • the gel plugs were transferred to 96 well digest adaptors (Bruker Daltonics).
  • Trypsin digestion and MALDI target preparation This procedure was performed in a Class III cabinet (Laminar flow is sufficient) to minimize keratin contamination. After spot picking, the 96 well plates containing the gel plugs were removed from the digest adaptors and blotted onto lint-free tissue and alternately washed with 30 ⁇ l per well of solution A (8% CH 3 CN in 20 mM NH 4 HCO 3 ) and 30 ⁇ l per well of solution B (50% CH 3 CN in 20 mM NH 4 HCO 3 ) and finally dehydrated in solution C (100% CH 3 CN) for a total of eight washes (ABABABCC). Each wash was for 5 min duration after which the wash solution was removed by blotting.
  • a 600 ⁇ m anchorchip MALDI target was manually prepared with HCCA matrix using the thinlayer technique according to the anchorchip manual (Bruker Daltonics) just prior to sample deposition. Digested peptides (4 ⁇ l) were deposited onto the target and after 10 min adsorption the samples were collectively washed by the rapid pouring of at least 100 ml of 0.1 % TFA over the plate. Automated TOF/TOF MS
  • a single set of 50 spectra were accumulated for the parent ion with a S/N threshold of 3 at a fixed laser power followed by 14 sets of 50 fragmentation spectra at a laser power elevated by 30%, each set being acquired at a different raster position.
  • the MS/MS spectra were smoothed, baseline subtracted and peaks were detected as above except that the quality factor threshold was reduced to 30. Peaklists were automatically sent to ProteinScape as above.
  • ProteinScape v1.2 was used for internal calibration of MS spectra, removal of calibrants and contaminants from the MS peaklists and submission of peaklists to Mascot 2.1 (Matrix Science) for both PMF and MS/MS ions searches (Chamrad, D. C, Koerting, G., Gobom, J., Thiele, H., Klose, J., Meyer, H. E. & Blueggel, M. (2003). Interpretation of mass spectrometry data for high-throughput proteomics. Analytical and bioanalytical chemistry 376, 1014-1022. Interpretation of mass spectrometry data for high-throughput proteomics. Analytical and bioanalytical chemistry 376, 1014-1022). All searches were against the T.
  • Bacterial cell cultures were grown to a cell density of ⁇ 7 x 10 8 cells/mL and harvested by centrifugation (7,000 g, 20 min, 4 0 C). The cells were then washed once in TC150 (50 mM Tris, 150 mM NaCI, 5 mM CaCI 2 , pH 7.4) and suspended in a 2-5% vol. of formal saline (0.5% v/v formaldehyde in saline, 150 mM NaCI). Cells were incubated overnight at room temperature under agitation, then washed and suspended in TC150. After preparation, formalin-killed cells were stored at 4 0 C.
  • mice 25 BALB/c female mice (6-8 weeks old) were immunized twice, one week apart with 2 x
  • the gels were equilibrated for 15 min in transfer buffer (25 mM Tris, 192 mM glycine pH 8.3, 2% v/v methanol) before being electroblotted onto a PVDF membrane prewetted with methanol. Electroblotting was conducted for 2 h at 70 V using a Transblott Cell (Bio- Rad) filled with transfer buffer at 4 0 C.
  • transfer buffer 25 mM Tris, 192 mM glycine pH 8.3, 2% v/v methanol
  • Western blots were performed using a transblot cell (Biorad).
  • the primary antibody used was pooled sera collected from mice as described above at a 1/100 dilution, and goat anti-mouse horseradish peroxidase conjugate was used as a secondary antibody at a dilution of 1/10,000.
  • the membranes were developed using SuperSignal® West Pico Chemiluminescent Substrate (Pierce).
  • the antigenic proteins determined by Western blot analysis were compared with the corresponding 2D gel by overlapping the images using Photoshop software. The images were aligned by linear stretches of one image with respect to the other image until the best fit was obtained.
  • Gel spots were excised from the gels with a robot, manually digested and analysed automatically using a MALDI-TOF/TOF mass spectrometer. In all, 56 non-redundant proteins were reproducibly identified and mapped to one or more spots, as shown on the master 2D gel and table (Fig 1 , Table 2). For simplicity, multiple spots matching the same protein were labelled with the same number. To be included in this list, each spot had to be identified from at least two 2D gels. A further eleven proteins were identified from a single 2D gel only (Table 2) and an additional ten proteins were identified only from 1D gels (Table 3).
  • CTD C-terminal domain
  • T. forsythia appears to prefer neutral hydrophilic residues such as Asn, Thr or Ser.
  • the overall length of the T. forsythia CTD is similar to the average length in P. gingivalis, however in P. gingivalis the spacing of the motifs is more heterogeneous.
  • a total of 42 putative lipoproteins were identified based on the presence of a signal sequence suitable for cleavage by lipoprotein signal peptidase.
  • the majority of the lipoproteins are currently annotated as hypothetical, or conserved hypothetical indicating that their function is as yet unknown.
  • the signal sequences of these proteins contained preferably serine, or alternatively glycine or alanine in the -1 position relative to the predicted cleavage site .
  • Lipoproteins identified that have a functional name were a peptidyl-prolyl cis-trans isomerase (TF0304), proteinases (TF2531 , TF0749, TF1033), a TPR domain protein (TF1940), a thiol:disulfide interchange protein (TF3165) and an exo-alpha-sialidase (or neuraminidase, TF2207).
  • TF0447 with a predicted mass of 10.7 kDa (Table 3) was identified in 1 D gel bands corresponding to a mass of about 120 kDa suggesting this predicted lipoprotein is part of an SDS-resistant complex, perhaps with itself.
  • TDR-associated lipoproteins represent some of the most intense spots on the gel, particularly within the 39-64 kDa range.
  • the MW and isoelectric points was sort of the TDRs whose genes are adjacent to the major LP-Ts that were identified (see above). It was found that the TDRs typically exhibited a MW of 80-120 kDa and a basic pi, demonstrating why they were not observed on a pH 4-7 range. Attempts to resolve the OM samples using a pH 7-11 range were unsuccessful, and therefore instead sample separated by 1 D SDS-PAGE in the appropriate MW range was analysed. In this way, after in-gel digestion and MALDI-TOF/TOF analysis, a total of ten TDRs were identified with predicted isoelectric points ranging from 7.0 to 9.5 (Table 2 & 3).
  • SusE and SusF also appeared to interact with each other and the SusC-D complex, however they were not essential for binding to starch.
  • the susB operon in T. forsythia appears to be homologous to the S. thetaiotaomicron system except that the susG gene is absent.
  • SusD (TF0092) was only identified from 1 D gels, which may be consistent with it forming a strong complex with SusC (TF0093) and therefore due to the high pi of SusC not be resolved on the pi 4-7 2D gels.
  • SusD exhibits sequence similarity to all of the other LP- Ts identified whose genes are directly downstream of their respective TDR, except TF2606 (Fig 4), however the additional LP-Ts whose genes are not directly downstream of a TDR are not well conserved. These results suggest that the primary LP-Ts identified all have a similar solute binding function associated with their respective TDRs. An overrepresentation of TDR genes was also reported for the B. thetaiotaomicron genome which encodes 106 predicted TDRs, and 57 LP-Ts (SusD paralogs).
  • T. forsythia does not appear to share the same plethora of polysaccharide utilization enzymes and most TDR loci are devoid of them.
  • Overrepresentation of TDR genes is also observed in various Proteobacteria, specifically those that share the ability to degrade a wide range of complex carbohydrates. The difference between TDR systems found in Proteobacteria compared to Bacteroidetes, is that the former do not contain SusD homologs.
  • TF1331 was partially purified and examined by SDS-PAGE in both reducing and non-reducing conditions and at both 5O 0 C and 100 0 C (Fig 5).
  • TF1331 migrated at 42 kDa when fully denatured, or 33.5 kDa when partially denatured by heating at 5O 0 C, consistent with the heat modifiability demonstrated for Omp40/41 of P. gingivalis and of OmpA.
  • TF1331 migrated to a MW of 99 kDa when fully denatured, or 72 kDa at 5O 0 C which is very similar to the MW estimated for heterodimeric Omp40/41 of P. gingivalis suggesting that the non-reduced form of TF1331 is a homodimer held together by disulfide bridges.
  • the central peak appeared to consist of three distinct peaks that correspond to the monomer peptides with each cysteine reduced (m/z 1761.8), and the presence of dehydroalanine and dithiocysteine (m/z 1759.8) and the presence of dehydroalanine, dithiocysteine and thioaldehyde (m/z 1757.8) representing LIFT-TOF/TOF fragmentation patterns that have been previously reported for di-sulfide bonded peptides.
  • the groups of peaks either side of the central peak correspond to a different mix of reduced cysteines, dithiocysteines, dehydroalanines and thioaldehydes producing a difference of 32 Da (a sulfur atom) between clusters (Fig 6).
  • Small peaks at approximately -17 Da relative to the large peaks is probably due to the loss of ammonia from the N-terminus.
  • the intensity of other fragments was generally low, however by comparing the fragmentation pattern with that of the reduced peptide it was possible to assign a significant series of a- and b-ions (Fig 6).
  • OmpA-like proteins such as TF1331
  • TF1331 it is not certain whether the main role of OmpA-like proteins such as TF1331 is simply to form a structural link between the OM and peptidoglycan wall, or whether the ability of some to form a diffusion pore is biologically important.
  • TF1331 is homologous to the Omp40 and Omp41 antigens (PG33, PG32) from P. gingivalis, and TF0090 and TF0091 are homologous to starch binding proteins in B. thetaiotaomicron .
  • OM proteome is dominated by CTD family proteins, proteins involved with TonB dependent transport and the OmpA-like protein, TF1331.
  • TF1331 is a novel di-sulfide bonded homodimer that shares the OmpA-like properties of heat modifiability and high copy number.
  • the CTD family members include the surface layer proteins which are very abundant and also the more weakly expressed BspA protein that exhibits multiple virulence properties.
  • Several other CTD-family proteins of very high abundance e.g TF2339 & TF1741 ) are implicated as being present on the surface of the cell.
  • TDR loci Seventeen TDR loci were identified including ten TDRs and 18 lipoproteins, most of which were very abundant. As there are more than 60 TDRs encoded in the T. forsythia genome, proteomic studies are essential to determine which systems are abundant, or present under given growth conditions.
  • Example 2 shows the characterisation of T. forsythia outer membrane using 1 D SDS-PAGE and LC-MALDI analysis. The results are reported here in the context of those in Example 2.
  • Polyacrylamide gel electrophoresis For SDS-PAGE 30 ⁇ g of T. forsythia OM was reduced with 50 mM DTT in LDS sample buffer at 100 0 C for 5 minutes, centrifuged to remove insoluble particles and loaded onto a 10% NuPAGE Bis-Tris gel and SDS- PAGE performed at 150 V for 50-60 min with NuPAGE MOPS SDS Running Buffer and using the XCeII SureLockTM Mini-cell system (Invitrogen, NSW 1 Australia). For 2D- PAGE the ZOOM ® IPGRunnerTM system was used and the isoelectric focusing and SDS-PAGE performed as per the manufacturer's instructions (Invitrogen, NSW, Australia). Briefly, 50 ⁇ g of T.
  • IPG strips (pH 4-7) were rehydrated with the solubilised T. forsythia OM (25°C, 18 hrs). After rehydration the IPG strips were assembled in the IPGRunner Min-Cell system and isoelectric focusing (IEF) performed (175 V, 15 min, 175-2000 V ramp for 45 min, 2000 V for 120 min).
  • IPF isoelectric focusing
  • the IPG strips were either used directly for SDS-PAGE for Western blotting and carbohydrate staining or were alkylated using 125 mM iodoacetamide in NuPAGE LDS sample buffer (15 min, 25°C) for protein identification by MALDI-TOF.
  • the IPG strips were set by 0.5% w/v agarose in running buffer on top of the ZOOM gel and SDS-PAGE performed as described above. Gels were stained for protein with colloidal Coomassie Blue 19 or for carbohydrate with Pro- Q Emerald 300 fluorescent stain (Invitrogen, NSW, Australia) according to the manufacturer's protocol and gels imaged with a LAS 3000 imaging system (Fuji, Tokyo, Japan).
  • HPLC of peptide extracts together with their deposition onto PAC target plates was performed according to Ang et. al. 23 with the following modifications. Separation was achieved using a RP column (C18 Acclaim PepmaplOO , 75 ⁇ m id * 15 cm, 5 ⁇ m, 100A, Dionex) and eluted with 0.1% TFA and a gradient of 0-64% CH3CN over 40 min followed by 64-80% CH3CN over 5 min. The flow rate was 6 ⁇ L/min through the column. Fractions were collected every 12 s from approximately 20% to 74% CH3CN. The target plate was analyzed with Bruker Ultraflex III MALDI TOF/TOF.
  • MS analysis was carried in reflectron mode measuring from 700 to 4000 Da using an accelerating voltage of 25 kV. All MS spectra were produced from five sets of 100 laser shots using random movement. Calibration of the instrument was performed externally with ions of pre-spotted internal standards. MSMS analysis was carried in LIFT mode in which the ions were accelerated to 8 kV and subsequently "lifted" to 19 kV in the LIFT cell. MSMS spectra were produced from 750 laser shots using random movement.
  • MS peak lists were generated by FlexAnalysis version 3.0.90 using SNAP algorithm, with S/N threshold 4. The peak list was filtered to remove common contaminants such as Coomassie Blue and keratin peaks. Selection of parent precursors was determined using WARP-LC software. The compounds separated by less than six fractions were considered the same and were selected as parent precursors if the SN was > 25. The MSMS peak list was also generated using SNAP algorithm after the spectra were smoothed using Savitsky-Golay algorithm (width 0.2 m/z) and baseline subtraction using TopHat algorithm. WARP-LC generated a combined MS/MS peak list which was searched using MASCOT version 2.2.04 (Matrix Science) via BioTools 3.1.0 software.
  • MS/MS ions searches against the T. forsythia database were conducted with a mass tolerance of 100 ppm on the parent and 0.5 Da on fragments.
  • One missed cleavage was considered with carbamidomethyl (C) as fixed modification, and oxidation (MHW) as variable modifications.
  • Decoy search was done automatically by MASCOT on randomized database of equal composition and size.
  • Proteins identified from a single peptide were accepted only if the peptide score was greater than 25 (p ⁇ 0.02, false positive rate measured to be 0.047). Peptides with a score less than identity threshold were still included for unique peptide count.
  • 1 ,163 peptides were non-redundant corresponding to 210 different proteins of which 134 were identified with at least two peptides above threshold (p ⁇ 0.05), and 76 were identified on the basis of having a single high scoring peptide (p ⁇ 0.02) (Table 1 ).
  • the identified proteins were grouped into categories, namely: CTD family proteins, putative lipoproteins (LP), outer membrane located proteins (OM) and TDRs, putative inner membrane proteins (IMP), putative periplasmic proteins (PP) and proteins with neither N-terminal signal nor trans-membrane helices and therefore likely to be located in the cytoplasm (Cyt) (Table 1 ).
  • the gel spots are colour coded according to these categories ( Figure 1 ).
  • CTD family proteins All of the high molecular weight proteins identified from 2D gels (MW>80 kDa) were found to share sequence similarity over approximately 60 amino acid residues at their extreme C-terminal end, which we designate the C-terminal domain (CTD) due to its similarity to the CTD of a family of proteins in P. gingivalis and certain other members of the Bacteroidetes. In P. gingivalis, the presence of the CTD has been demonstrated to be required for proper maturation of the CTD-containing proteinase, RgpB together with its correct secretion and attachment to the cell surface. The CTD may therefore be a secretion signal for a novel Bacteroidetes secretion pathway. A search within the T.
  • TfsA surface-layer protein A
  • TfsB surface-layer protein B
  • BspA BspA
  • TF1032 possible internalin-related protein
  • BspA The C-terminal -300 residues of BspA are almost identical to TF1843, which is also annotated as BspA.
  • Five other proteins in the database but not identified in this study are also annotated as BspA due to extensive sequence similarity to the original BspA.
  • mutants lacking a functional BspA gene presumably TF2998
  • TF2998 may be the only BspA protein produced at a sufficiently high enough level to allow binding and invasion to be reliably detected.
  • the surface layer of T The surface layer of T.
  • CTD family proteins not to have a MW greater than that predicted were TF3080 and the BspA proteins TF1843 and TF2998 (Table 4). Each of these had an observed MW considerably less than expected suggesting proteolysis rather than non-glycosylation.
  • the individual BspA proteins could not be distinguished, as their identification was based on a single peptide that is identical in both proteins.
  • T. forsythia This was not observed in T. forsythia however, even for C- terminal fragments of surface layer protein B which still maintained sharp spots despite a higher than expected MW ( Figure 1 ). This may suggest that the modification in T. forsythia has different properties or is less heterogeneous to that in P. gingivalis. As with P. gingivalis, the absence of CTD domain peptides suggests that the CTD is heavily glycosylated or that part or all of the CTD is removed by proteolytic processing.
  • T. forsythia appears to prefer neutral hydrophilic residues such as Asn, Thr or Ser.
  • the overall length of the T. forsythia CTD is similar to the average length in P. gingivalis, however in P. gingivalis the spacing of the motifs is more heterogeneous.
  • TF1331 is similar to the Omp40 and Omp41 antigens (PG33, PG32) from P. gingivalis, and TF0090 and TF0091 are similar to TDR-associated starch binding proteins in B. thetaiotaomicron. Apart from TF1331 , all of the identified antigens were reactive to sera raised against formalin killed whole T. forsythia cells indicating their likely cell-surface exposure.
  • the signal sequences of the putative lipoproteins contained preferably serine, or alternatively glycine or alanine in the -1 position relative to the predicted cleavage site.
  • Lipoproteins identified that have functional names include peptidyl-prolyl cis-trans isomerases (TF2214, TF0304, TF0305), proteinases (TF0749, TF1033, TF1755, TF2531 , TF3024), a TPR domain protein (TF1940), a thiol:disulfide interchange protein (TF3165), a previously described beta-N-acetylglucosaminidase (TF2925) and an exo-alpha-sialidase (or neuraminidase, TF2207).
  • TDR-associated lipoproteins represent some of the most intense spots on the 2D gel, particularly within the 39-64 kDa range.
  • TonB-dependent transport As there is a very large number (>60) of predicted TDRs in the T. forsythia database, and we could only identify fragments of three from the 2D gel, we obtained the theoretical MW and isoelectric points of the TDRs whose genes are adjacent to the major LP-Ts that were identified (see above). These TDR sequences typically predict a MW of 80-120 kDa and a basic pi, demonstrating why they were not observed in the pH 4-7 range used. LC-MALDI of 1 D gel bands however resulted in the identification of 46 putative TDRs.
  • the larger group consists of large interrelated TDRs (108-140 kDa) with adjacent and interrelated LP-Ts.
  • the remaining TDRs exhibit poorer sequence conservation, are smaller (69- 121 kDa) and are mostly 'lone' TDRs.
  • the exceptions are TF1535, TF0682, TF2597, TF0045 and the unidentified TDR, TF2606.
  • Each of these has a downstream LP that is not significantly related to the other LP-Ts identified.
  • TDR loci such as TF0094 - TF0090 which is similar to the susB operon of Bacteroides thetaiotaomicron contain multiple downstream genes that encode lipoproteins.
  • the susB operon which comprises susB through to susG is involved in starch utilization.
  • SusB and SusG are glycosidic enzymes
  • SusC is a TDR
  • SusD are OM lipoproteins.
  • SusC and SusD are reported to form a complex that is essential for binding of cells to starch.
  • SusE and SusF also appeared to interact with each other and the SusC-D complex, however they were not essential for binding to starch.
  • the susB operon in T is reported to form a complex that is essential for binding of cells to starch.
  • SusE and SusF also appeared to interact with each other and the SusC-D complex, however they were not essential for binding to starch.
  • the susB operon in T is reported to form a complex
  • SusD exhibits sequence similarity to many of the other LP-Ts identified whose genes are directly downstream of their respective TDR, however the additional lipoproteins such as TF0090 and TF0091 whose genes are not directly downstream of a TDR are not well conserved.
  • TDR genes Most sequenced Gram-negative bacteria have up to nine TDRs encoded in their genomes and more than 80% have less than 25 TDRs and only 4% have more than 60. The 60 or more present in T. forsythia therefore is unusual.
  • An overrepresentation of TDR genes was also reported for the B. thetaiotaomicron genome which encodes 106 predicted TDRs, and 57 LP-Ts (SusD paralogs). The majority of these are part of loci containing polysaccharide degrading enzymes and therefore the TDRs and LP-Ts of such loci are likely to be involved in binding to and transporting polysaccharides into the periplasm. T.
  • TDR polysaccharide utilization enzymes
  • TDR loci are devoid of them.
  • Overrepresentation of TDR genes is also observed in various Proteobacteria, specifically those that share the ability to degrade a wide range of complex carbohydrates.
  • the difference between TDR systems found in Proteobacteria compared to Bacteroidetes, is that the former do not contain SusD homologs. This is the first time to our knowledge that TDR overrepresentation has been demonstrated at the protein level.
  • proteomic techniques are currently unable to detect all proteins expressed within a proteome, the results imply that T. forsythia prefers to express the majority of its TDRs at moderate to high levels rather than expressing a small range of receptors to suit the availability of specific nutrients.
  • Omps Other proteins identified that are predicted to be integral to the OM include the P. gingivalis homologs, Omp41 (TF1331), P40 (TF2852) and P58 (TF1444), and the OM efflux proteins TF0773, TF0810, TF1409, TF1476, TF1822 which are related to ToIC. Due to the finding in P. gingivalis that Omp41 and its homolog Omp40 were heterodimeric under non-reducing conditions, held together by two disulfide bridges, and also exhibited heat-mod if iability similar to OmpA, TF1331 was partially purified and examined by SDS-PAGE in both reducing and non-reducing conditions and at both 50° C and 100° C ( Figure 5).
  • TF1331 migrated at 42 kDa when fully denatured, or 33.5 kDa when partially denatured by heating at 50° C, consistent with the heat modifiability demonstrated for Omp40/41 of P. gingivalis and of OmpA.
  • TF1331 migrated to a MW of 99 kDa when fully denatured, or 72 kDa at 50° C which is very similar to the MW estimated for heterodimeric Omp40/41 of P. gingivalis suggesting that the non-reduced form of TF1331 is a homodimer held together by disulfide bridges.
  • the central peak appeared to consist of three distinct peaks that correspond to the monomer peptides with each cysteine reduced (m/z 1761.8), and the presence of dehydroalanine and dithiocysteine (m/z 1759.8) and the presence of dehydroalanine, dithiocysteine and thioaldehyde (m/z 1757.8) representing LIFT- TOF/TOF fragmentation patterns that have been previously reported for disulfide bonded peptides.
  • the groups of peaks either side of the central peak correspond to a different mix of reduced cysteines, dithiocysteines, dehydroalanines and thioaldehydes producing a difference of 32 Da (a sulfur atom) between clusters ( Figure 6).
  • OmpA-like proteins such as TF1331
  • TF1331 it is not certain whether the main role of OmpA-like proteins such as TF1331 is simply to form a structural link between the OM and peptidoglycan wall, or whether the ability of some to form a diffusion pore is biologically important.
  • OM proteome is dominated by CTD family proteins, proteins involved with TonB dependent transport and the OmpA-like protein, TF1331.
  • TF1331 is a novel disulfide bonded homodimer that shares the OmpA-like properties of heat modifiability and high copy number.
  • the CTD family members include the surface layer proteins which are very abundant and also the more weakly expressed BspA protein that exhibits multiple virulence properties.
  • Several other CTD-family proteins of very high abundance e.g TF2339 & TF1741 ) are implicated as being present on the surface of the cell.
  • TDR loci Forty-eight TDR loci were identified including 46 TDRs and 28 lipoproteins, most of which were very abundant. Fifteen proteins were found to be antigenic, and these could be useful for developing diagnostics and therapeutics against T. forsythia infection.
  • the term “comprise” and variations of the term, such as “comprising”, “comprises” and “comprised”, are not intended to exclude further additives, components, integers or steps.
  • Table 1 Identification data for all proteins identified.
  • accession numbers and protein descriptions are from the Oralgen website (www.oralgen.lanl.gov) Hyphenated accession numbers are where two adjacent genes in the database correspond to a single protein as indicated by both proteomics and homology data. Abbreviations used are: HP, hypothetical protein; HP-C, conserved HP; and others as already defined
  • MS/MS Mascot score is the best single peptide score obtained for the protein/2D gel spot combination indicated
  • the protein score is the best total MS/MS score obtained for the indicated protein by LC- MALDI of SDS-PAGE bands g
  • the number of unique peptides identified by MS/MS is from the LC-MALDI data except for proteins identified from 2D gels only. Overlap of identical peptides is provided for TF0063 & TF0064, TF1053 & TF1057, TF2403 & TF2412, TF1259 & TF2339, TF1741 & TF2592, and TF2661-2 & TF2663.
  • the protein with accession TF extra was identified from DNA sequence obtained from TIGR (www.tigr.org) and has not yet been annotated in the Oralgen database
  • Proteins identified from 2D gel spot numbers labeled (-) were only identified from a single 2D gel, and are not included in Fig 1
  • the BspA proteins TF 1843 and TF2998 could not be distinguished as their identification was based on a single overlapping peptide.
  • Table 2 Identification data for proteins identified from 2D gels in Example 2.
  • Table 3 Identification data for selected SDS-PAGE separated proteins.

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Abstract

The present invention relates to methods for detecting Tannerella, in particular Tannerella forsythia, infections and related pathologies such as periodontal disease.

Description

Detection of Tannerella forsythia infection
Field of the invention
The invention relates to detecting Tannerella forsythia infections and related pathologies.
Background of the invention
Chronic periodontitis is an inflammatory disease of the supporting tissues of the teeth leading to resorption of alveolar bone and eventual tooth loss. The disease is a major public health problem in all societies and is estimated to affect up to 15% of the adult population with severe forms affecting 5-6%.
The development and progression of chronic periodontitis has been associated with specific Gram-negative bacteria in subgingival plaque. The presence of three Gram- negative bacterial species Tannerella forsythia (T. forsythia) , Treponema denticola and Porphyromonas gingivalis in subgingival plaque has been associated with disease.
T. forsythia (previously named Tannerella forsythensis and Bacteroides forsythus) is a Gram-negative, anaerobic, fusiform bacterium that is found associated with Porphyromonas gingivalis and Treponema denticola in sub-gingival plaque. Together they are implicated as periodontal pathogens.
One problem is that virulence factors of T forsythia are as yet poorly understood, due in part to the difficulty in culturing this fastidious organism, and the lack of genetic tools for creating mutants. For the study of virulence mechanisms, and the associated development of therapeutic strategies, an understanding of the surface structures of pathogens at the molecular level is necessary.
A proteomic approach has not yet been applied to T. forsythia and few outer membrane proteins or virulence factors have been described. A distinctive feature of the T. forsythia proteome is the presence of a major double band above 200 kDa in SDS- PAGE profiles of whole cell extracts. These bands correspond to two proteins designated TfsA and TfsB which are the major constituents of the surface layer (S- layer), a distinct layer beyond the OM that can be clearly seen in electron micrographs. Recently, these proteins were reported to play an important role in the adherence of the bacteria to gingival epithelial cells using deletion mutants. Another well-studied protein is BspA, a cell surface antigen containing a leucine-rich repeat (LRR) domain and which binds to fibrinogen and fibronectin. BspA has also been demonstrated to mediate coaggregation with other bacteria, invasion into epithelial cells and induction of alveolar bone loss in mice. T. forsythia also possesses trypsin-like activity and a sialidase (neuraminidase) but the importance of these potential virulence factors or their co- relation to specific protein sequences is not yet clear.
There is a need for markers of T. forsythia which can be used to diagnose or monitor T. forsythia related diseases and conditions such as periodontal disease.
There also exists a need to monitor the efficacy of prophylactic or therapeutic treatments of T. forsythia related diseases and conditions.
Reference to any prior art in the specification is not, and should not be taken as, an acknowledgment or any form of suggestion that this prior art forms part of the common general knowledge in Australia or any other jurisdiction or that this prior art could reasonably be expected to be ascertained, understood and regarded as relevant by a person skilled in the art.
Summary of the invention
In one embodiment the invention provides a method for determining whether an individual has a Tannerella infection including:
- selecting an individual;
-detecting whether the selected individual contains a protein, peptide or fragment thereof described in Table 1 or 2 herein, wherein detection of said protein, peptide or fragment thereof determines that the individual has a Tannerella infection,
thereby determining whether the individual has a Tannerella infection. A "fragment" of a protein or peptide as used herein is an amino acid sequence that is characteristic of the protein or peptide from which it is derived. In some embodiments, the fragment is least 5 amino acids in length, preferably greater than 10 amino acids, preferably greater than 20 amino acids in length.
In one embodiment the invention provides a method for determining whether an individual is susceptible to periodontal disease, or likely to develop periodontal disease or for screening for early stage periodontal disease including:
- selecting an individual;
-detecting whether the selected individual contains a protein, peptide or fragment thereof described in Table 1 or 2 herein, wherein detection of said protein, peptide or fragment thereof determines that the individual is susceptible to periodontal disease, likely to develop periodontal disease or has early stage periodontal disease,
thereby determining whether the individual is susceptible to periodontal disease, likely to develop periodontal disease or has early stage periodontal disease.
In one embodiment the invention provides a method for monitoring Tannerella infection in an individual receiving treatment for periodontal disease including:
- selecting an individual who is undergoing or who has undergone treatment for periodontal disease;
-detecting whether the selected individual contains a protein, peptide or fragment thereof described in Table 1 or 2 herein, wherein detection of said protein, peptide or fragment thereof determines the individuals response to treatment for periodontal disease,
thereby determining whether the individual is responding to treatment for periodontal disease.
In one embodiment the invention provides a method for determining the risk of an individual developing periodontal disease including: - selecting an individual;
-detecting whether the selected individual contains a protein, peptide or fragment thereof described in Table 1 or 2 herein, wherein detection of said protein, peptide or fragment thereof determines the risk of an individual developing periodontal disease,
thereby determining the risk of an individual developing periodontal disease.
In one embodiment the invention provides a method for determining the risk of periodontal disease progression in an individual including:
- selecting an individual;
-detecting whether the selected individual contains a protein, peptide or fragment thereof described in Table 1 or 2 herein, wherein detection of said protein, peptide or fragment thereof determines the risk of periodontal disease progression in an individual;
thereby determining the risk of periodontal disease progression in an individual.
In certain embodiments, the methods of the invention are used for determining whether a periodontal site has a Tannerella infection, is susceptible to periodontal disease, or is likely to develop periodontal disease, or for screening for early stage periodontal disease, for monitoring Tannerella infection in a periodontal site receiving treatment for periodontal disease, for determining risk of a periodontal site developing periodontal disease and/or for determining the risk of periodontal disease progression at a periodontal site.
In other embodiments there is provided a use of an antibody specific for a protein, peptide or fragment thereof described in Table 1 or 2 herein for determining whether an individual has a Tannerella infection.
In another embodiment there is provided a use of a polynucleotide or fragment thereof for detecting a nucleic acid that encodes or controls the expression of a protein described in Table 1 or 2 herein for determining whether an individual has a Tannerella infection. In further embodiments there is provided a kit for determining whether an individual has a Tannerella infection including:
- an antibody specific for a protein, peptide or fragment thereof described in Table 1 or 2 herein; or
- a polynucleotide or fragment thereof capable of detecting a nucleic acid encoding or controlling the expression of a protein, peptide or fragment thereof described in Table 1 or 2 herein.
As described further below, the kit may further include one or more Tannerella proteins, peptides or fragment thereof for use as a control and written instructions for use of the kit in a method as described herein.
In one embodiment there is provided a device or apparatus for the detection of Tannerella infection including:
- a protein, peptide or fragment thereof described in Table 1 or 2 herein;
wherein the protein, peptide or fragment thereof is arranged on a solid phase to permit capture of an antibody in a sample taken from a person for whom Tannerella infection is to be detected.
In another embodiment there is provided a device or apparatus for the detection of Tannerella infection including:
- an antibody directed to a protein, peptide or fragment thereof described in Table 1 or 2 herein;
wherein the antibody is arranged on a solid phase to permit capture of a protein, peptide or fragment thereof in a sample taken from a person for whom Tannerella infection is to be detected.
In one embodiment the invention provides a substantially non-glycosylated protein, peptide or fragment thereof described in Table 1 or 2 herein. Preferably, the protein, peptide or fragment thereof described in Table 1 or 2 herein does not contain any glycosylated amino acids. Preferably, the substantially non-glycosylated protein is a non-glycosylated form of a protein selected from the group consisting of TF1342, TF0090, TF0091 , TF1331 TF2123, TF1056 and TF0945. Preferably, the protein, peptide or fragment thereof described in Table 1 or 2 herein that does not contain any glyclosylated amino acids is selected from the group consisting of TF1342, TF0090, TF0091 , TF1331 TF2123, TF1056 and TF0945.
Typically, the protein, peptide or fragment thereof of the invention is in a composition. The composition further comprises a carrier.
"Substantially non-glycosylated" protein, peptide or fragments thereof are described further herein.
In one embodiment there is provided an antiserum raised against a protein, peptide or fragment thereof described in Table 1 or 2 herein. In one embodiment, the antiserum has specificities that react with non-glycosylated protein, peptide or fragments thereof described in Table 1 or 2 herein. In one embodiment the antiserum is polyclonal. In another embodiment the antiserum is monoclonal.
In one embodiment there is provided a use of a substantially non-glycosylated protein, peptide or fragment thereof described in Table 1 or 2 herein in the preparation of a medicament for the treatment or prevention of a Tannerella infection (and/or the other conditions identified herein as suitable for treatment). In one embodiment the medicament is for the treatment or prevention of periodontal disease.
In one embodiment there is provided a substantially non-glycosylated protein, peptide or fragment thereof described in Table 1 or 2 herein for use in the treatment or prevention of a Tannerella infection (and/or the other conditions identified herein as suitable for treatment).
In another embodiment the invention provides a composition for the treatment or prevention of periodontal disease (and/or the other conditions identified herein as suitable for treatment) comprising as an active ingredient a substantially non- glycosylated protein, peptide or fragment thereof described in Table 1 or 2 herein. Also provided is a use of an antigen in the form of:
- a protein, peptide or fragment thereof that is expressed in a Tannerella or;
- a protein, peptide or fragment thereof described in Table 1 or 2 herein
in the manufacture of means for determining whether an individual is infected with a Tannerella.
In another embodiment there is provided a use of a non-glycosylated antigen in the form of:
- a protein, peptide or fragment thereof that is expressed in a Tannerella or;
- a protein, peptide or fragment thereof described in Table 1 or 2 herein
in the manufacture of means for determining whether an individual is infected with a Tannerella.
In other embodiments there is provided a use of an antibody specific for:
- a protein, peptide or fragment thereof that is expressed in a Tannerella or;
- a protein, peptide or fragment thereof described in Table 1 or 2 herein
for determining whether an individual is infected with a Tannerella.
In another embodiment there is provided a use of a polynucleotide for detecting a nucleic acid that encodes or controls the expression of:
- a protein, peptide or fragment thereof that is expressed in a Tannerella or;
- a protein, peptide or fragment thereof described in Table 2 or 3 herein
in the manufacture of means for determining whether an individual is infected with a Tannerella. In further embodiments there is provided a kit for determining whether an individual is infected with a Tannerella including:
- an antigen being a protein, peptide or fragment thereof that is expressed in a Tannerella; or
- an antigen being a protein, peptide or fragment thereof described in Table 1 or 2 herein; or
- an ar\i\-Tannerella antibody specific for at least one of the above described antigens; or
- an antibody specific for an idiotype of an above described ant\-Tannerella antibody; or
- a polynucleotide capable of detecting a nucleic acid encoding or controlling the expression of at least one of the above described antigens.
In one embodiment there is provided a method of treating a Tannerella infection in an individual including
- detecting a Tannerella infection in an individual according to a method of the invention described herein; and
- applying a therapeutic agent to treat the Tannerella infection.
A kit of the invention may further include written instructions for use of the kit in a method as described herein.
In further embodiments there is provided an immune complex including an antigen being a protein, peptide or fragment thereof described in Table 1 or 2 herein bound to an antibody specific for said protein, peptide or fragment thereof.
Brief description of the drawings
Fig 1: Representative 2D gel of T. forsythia outer membrane. Spots are numbered 1-54 with each number corresponding to a unique protein. Adjacent spots corresponding to the same protein are joined by lines, or marked with an * indicating that only a fragment of the protein was identified. Spots labelled 'N' and 'C represent N- and C-terminal fragments of surface layer protein B respectively.
Fig 2: Carbohydrate staining of 2D gel of T. forsythia outer membrane. 2D gel stained with Pro-Q Emerald fluorescent carbohydrate stain (Right) prior to staining with Coomassie Blue (Left).
Fig 3: Multiple alignment of CTD family proteins from T. forsythia and P. gingivalis. CTD family proteins from T. forsythia (TF) were identified by multiple BLAST searches against the T. forsythia database from the oralgen website (www.oralgen.lanl.gov). To reduce redundancy, six CTD sequences TF2592, TF2339, TF1591 , TF2998, TF3080 and TF0541 were excluded due to their high sequence identity to the exhibited CTD sequences TF1741 , TF1259, TF1589, TF1843, TF1458, TF0537 respectively. Multiple alignment was conducted separately for T. forsythia and P. gingivalis (PG) sequences using the ClustalW program. Invariant residues within the T. forsythia sequences are marked with an asterisk (*), strongly conserved positions are marked with a colon (:), and moderately conserved positions are marked with a period (.) Deletions of two residues and four residues from the P. gingivalis alignment are marked with triangles (▼ ). Residues underlined represent the most C-terminal peptides identified by MS/MS (Table 3). Residues in bold represent C-terminal peptides identified from unmodified proteins isolated from P. gingivalis strain HG66 (unpublished data).
Fig 4: TonB-dependent receptor containing loci identified. The solid arrows (left) represent the genes of TonB-dependent receptors (R), while the dashed arrows represent adjacent genes that encode lipoproteins (L). Each gene is labeled with its TIGR accession number, which is highlighted if the encoded protein was identified (see Tables 1 & 2). The lipoproteins were always found to be downstream of their respective receptors, and the lipoproteins directly downstream all share sequence similarity to each other with the exception of TF2606. The receptor labeled 1506/1507 is annotated as two separate receptors, however BLAST searching indicates that they represent two halves of a single receptor suggesting a mutation or sequencing error in the TF1506 stop codon. Fig 5: Heat-modifiability and the effect of reduction on the MW of TF1331. TF1331 enriched sample was subjected to SDS-PAGE. (A) 100° C, + DTT; (B) 50° C, + DTT; (C) 100° C; (D) 50° C. Arrows represent bands identified as TF1331 by MS. Benchmark™ protein standards were used containing proteins of MW 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 160, and 220 kDa. The 10, 15 and 25 kDa markers are not shown.
Fig 6: LIFT-TOF/TOF data for m/z 3516.6 di-sulfide bonded peptide from TF1331.(A) Full spectrum. Boxed areas are presented enlarged in panels B and C. (B) Zoomed in spectrum showing products resulting from cleavage of disulfide bonds. Symbols used to represent the chemical state of the three fragmented disulfides: C = reduced cysteine; A = dehydroalanine; C = thioaldehyde; X = dithiocysteine. Other combinations besides those that are shown are possible. (C) Zoomed in spectrum showing backbone fragmentation pattern and its assignment to the RPEFCPECPKCPEVK peptide from TF1331.
Fig 7: Western Blot Analysis. 2D-Westerns of T. forsythia outer membrane were performed and probed with (A) antisera raised against the outer membrane preparation and (B) antisera raised against formalin killed T. forsythia cells. C. Image from (A) was coloured red and overlayed with Coomassie Blue stained 2D gel (blue). The scales of the images were manipulated by linear stretches only. The identity of numbered spots are presented in Table 4.
Fig. 8: SDS-PAGE gel of T. forsythia outer membrane. Identical samples of T. forsythia OM were separated on a 10% PA Bis-Tris gel with MOPs running buffer. The gel was stained with Coomassie Blue G250. Fifty-five contiguous gel segments were excised from four identical lanes. Two lanes are shown in order to clearly mark the centre of each excised segment.
Detailed description of the embodiments
It will be understood that the invention disclosed and defined in this specification extends to all alternative combinations of two or more of the individual features mentioned or evident from the text or drawings. All of these different combinations constitute various alternative aspects of the invention. The inventors have identified the major proteins and antigenic proteins of T. forsythia. Importantly, these proteins can be used to detect Tannerella infection in individuals and provide a means for diagnosing periodontitis, periodontal disease or related pathology caused, initiated or potentiated by Tannerella bacteria in an individual.
Thus in one embodiment there is provided a method for determining whether an individual has a Tannerella infection including:
-selecting an individual;
- detecting whether the selected individual contains a protein, peptide or fragment thereof described in Table 1 or 2 in the individual, wherein detection of said protein determines that the individual has a Tannerella infection.
thereby determining whether the individual has a Tannerella infection.
Typically detection of the protein, peptide or fragment thereof in the individual is assessed by the steps of:
-obtaining a sample from the individual;
- measuring for the expression or presence of the target protein in the sample; and
- comparing the measured level with an uninfected control that describes the level of expression of the target as observed in a sample from a subject that has been determined as not having a Tannerella infection.
In one embodiment the invention provides a method for:
- determining whether an individual has a Tannerella infection,
- determining whether an individual is susceptible to periodontal disease,
- determining whether an individual is likely to develop periodontal disease,
- screening for early stage periodontal disease, - monitoring Tannerella infection in an individual receiving treatment for periodontal disease,
- determining the risk of an individual developing periodontal disease, or
- determining the risk of periodontal disease progression in an individual,
comprising detecting whether an individual contains a protein, peptide or fragment thereof described in Table 1 or 2 herein, wherein detection of said protein, peptide or fragment thereof determines:
- that the individual has a Tannerella infection,
- the individual is susceptible to periodontal disease,
- the individual is likely to develop periodontal disease,
- the individual has early stage periodontal disease,
- the Tannerella infection in an individual receiving treatment for periodontal disease,
- the risk of an individual developing periodontal disease, or
- the risk of periodontal disease progression in an individual.
A Tannerella infection is an infection that is characterised by invasion or contamination of tissues or cells by a Tannerella. A Tannerella infection may have clinical or subclinical manifestations including acute or chronic inflammation of the gingiva. The hallmarks of acute inflammation may be present including an increased movement of plasma and leukocytes from the blood into the injured tissues. Clinical signs of acute infection of the gingiva may also be present including rubor (redness), calor (increased heat), tumor (swelling), dolor (pain), and functio laesa (loss of function). Chronic inflammation may be characterised by leukocyte cell (monocytes, macrophages, lymphocytes, plasma cells) infiltration. Tissue and bone loss may be observed. A Tannerella infection may also be characterised by an increased level of Tannerella bacteria, particular T. forsythia, above a normal range observed in individuals without a Tannerella infection. For example, a Tannerella infection may be manifested in an individual at a periodontal site that has greater than approximately 103 to 105 Tannerella cells/mg of subgingival plaque. A Tannerella infection may be manifested in an individual at a periodontal site that has greater than approximately 104 Tannerella cells/mg of subgingival plaque.
An "uninfected control" sample is further defined herein.
The "measuring for the expression" of a target protein in a sample is the detection of the degree or level or amount or concentration of a protein, peptide or fragment thereof described in Table 1 or 2 herein.
An individual that is selected and from whom a sample is obtained may be an individual that has a previous history of Tannerella infection or periodontal disease or a person already diagnosed with periodontal disease. Alternatively, the individual may not have had a prior Tannerella infection or experienced periodontal disease previously.
In certain embodiments, the target protein is a protein, peptide or fragment thereof described in Table 1 or 2.
Detection of a protein, peptide or fragment thereof indicates the presence of a Tannerella bacteria, in particular T. forsythia. The detection of a level or degree of expression of a protein, peptide or fragment thereof may indicate the level of Tannerella bacteria, in particular T. forysthia, in a sample which in turn may indicate the occurrence of, or degree of, or progression of a Tannerella infection or periodontal disease. For example, a strong level of expression, increased amount or concentration of a protein, peptide or fragment thereof in a sample taken from an individual compared to an uninfected control may indicate a high level of Tannerella in the sample. The detection of the presence or level or degree of expression of a protein, peptide or fragment thereof may correlate with the risk of developing a Tannerella infection or periodontal disease.
A "Tannerella infection" in an individual or an individual "infected with a Tannerella" generally refers to:
(1) an elevated level of Tannerella in a sample taken from the individual compared to an uninfected control sample; (2) an increased proportion of Tannerella bacteria in a sample taken from the individual compared to the total level of bacteria in an uninfected control sample;
(3) an increased proportion of Tannerella bacteria relative to one or more other bacteria species in a sample taken from the individual when compared to an uninfected control sample; or
(4) the presence of Tannerella bacteria in a sample compared to an uninfected control sample when Tannerella is undetectable in the uninfected control.
In any embodiment of the invention the Tannerella may be Tannerella forsythia.
In one embodiment the sample obtained from the individual is a body fluid or fraction thereof. The body fluid or fraction thereof from the individual may be an oral fluid taken from the oral cavity. In particular, an oral fluid may be saliva, gingival crevicular fluid or blood. It is recognized that oral fluids, for example saliva, are a combination of secretions from a number of sources such as parotid, submandibular, sublingual, accessory glands, gingival mucosa and buccal mucosa and the term oral fluid includes the secretion of each of these sources individually or in combination. The saliva may be stimulated or in a preferred embodiment unstimulated. Stimulation of the saliva in the individual may occur by allowing the individual to chew on sugar-free gum, a piece of paraffin film or tart candy. Unstimulated saliva means that the subject will expectorate into a collection vessel without stimulation of salivary flow.
Saliva specimens for testing can be collected following various methods known in the art, for example, stimulated or unstimulated saliva can be sampled by the individual expectorating into a collection vessel or using a swab or syringe to extract the saliva. Other ways for obtaining unstimulated saliva are known in the art. (Nazaresh and Christiansen, J. Dent. Res. 61 : 1158- 1162 (1982)). Methods and devices for collecting saliva have also been described. (See also, U. S. Patent No. 5,910, 122).
It is contemplated that the methods of the present invention can also be practiced by analyzing stimulated saliva.
Furthermore, the methods of the present invention are not limited to performing salivary analysis immediately after collection of the sample. In certain embodiments, salivary analysis following the methods of the present invention can be performed on a stored saliva sample. The saliva sample for testing can be preserved using methods and apparatuses known in the art. (See e.g., U. S. Patent No. 5,968, 746).
It is also contemplated that the methods of the present invention be used to perform salivary analysis on saliva samples that have been treated to reduce its viscosity.
The viscous nature of saliva, due to the nature of mucopolysaccharides, makes testing of these fluids difficult. In order to prepare saliva for any laboratory testing procedure, the saliva may be rendered sufficiently fluid (i.e. viscosity must be reduced) and free from debris. Techniques used to remove debris include centrifugation and filtration. The viscosity of saliva can also be reduced by mixing a saliva sample with a cationic quaternary ammonium reagent. (See, U. S. Patent No. 5,112, 758).
In another embodiment, the sample from an individual may be taken from the crypts of the dorsum of the tongue.
The sample from the individual may be taken from a specific periodontal site. A periodontal site is a region within the oral cavity. Preferably, a periodontal site is region around a tooth including disto-buccal, mid-buccal, mesio-buccal, mesio-palatal, mid- palatal and disto-palatal and disto-lingual, mid-lingual and mesio-lingual. The sample may be taken from a periodontal site that exhibits clinical signs of periodontal disease or Tannerella infection. A method, use, protein or composition of the invention could then be used to determine whether the periodontal site is infected with Tannerella, or at risk of periodontal disease progression.
In another embodiment, the sample from an individual may be a sample of a tissue. The tissue or part thereof may be from the oral cavity. In certain embodiments the tissue is gingival. The gingival tissue may be from various sites around a tooth including disto- buccal, mid-buccal, mesio-buccal, mesio-palatal, mid-palatal and disto-palatal and disto- lingual, mid-lingual and mesio-lingual. The tissue may be obtained by normal biopsy or may be obtained from an extracted tooth.
In another embodiment the sample from the individual may be dental plaque. The plaque may be subgingival or supragingival. Subgingival plaque may be sampled using a sterile curette or paper point. Supragingival plaque may be removed using standard techniques known in the art. The subgingival plaque may be collected from various sites around a tooth including disto-buccal, mid-buccal, mesio-buccal, mesio-palatal, mid- palatal and disto-palatal and disto-lingual, mid-lingual and mesio-lingual periodontal sites. The subgingival plaque samples may be obtained during the normal dental examination provided by a qualified dentist or periodontist. The plaque sample may be analysed as is or treated to extract the protein, peptide or fragment thereof of interest using an extraction buffer. An extraction buffer could contain a pH buffer (e.g. phosphate, HEPES, etc), salts (e.g. NaCI) to maintain ionic strength and protein solubilising agents (e.g. detergents (SDS, Triton X100, etc)), reducing agents (e.g. dithiothreitol, cysteineHCI) and/or chaotropic agents (e.g. urea, guanidinium chloride, lithium perchlorate).
An "uninfected control" is a sample from an individual or represents a set of parameters previously defined from individuals that do not have all the attributes of an individual with a Tannerella infection. For example, the individual from whom the "uninfected control" sample is derived generally does not have inflammation of the gums, antibodies directed against Tannerella in their blood, have an amount of Tannerella above approximately 103 to 105 cells/mg of subgingival plaque and/or have an amount of Tannerella above approximately 103 cells/ml of saliva. The "uninfected control" sample may be taken from the oral cavity of a subject that, but for an absence of a Tannerella infection, is generally the same or very similar to the individual selected for determination of whether they have a Tannerella infection (the latter otherwise known as the "test sample"). The measurement of the level of expression of the target in the sample from the oral cavity of the subject for deriving the uninfected control is generally done using the same assay format that is used for measurement of the target protein in the test sample. It will be appreciated that the control sample may also be taken from the same individual from which the test sample is taken, but at a different time-point, or periodontal site, in order to determine the risk of, or progression of the Tannerella infection or periodontal disease.
It is contemplated that individuals with a healthy oral cavity may contain a low level of Tannerella present, in particular T. forsythia. This low level or normal level of bacterial colonisation may be sampled and is also within the scope of "uninfected control". When using such an uninfected control and comparing it to a test sample, determination of whether an individual has a Tannerella infection or periodontial disease includes (1 ) an elevated level of Tannerella in a sample taken from the individual compared to the uninfected control sample, or (2) an increased proportion of Tannerella bacteria in a sample taken from the individual compared to the total level of bacteria in the uninfected control sample, or (3) an increased proportion of Tannerella bacteria relative to one or more other bacteria species in a sample taken from the individual when compared with the uninfected control sample.
In certain embodiments, the method includes measuring the expression of the target in the uninfected control to compare the measured level in the test sample with the level in the uninfected control.
In other embodiments, an internal standard is applied. This may be used to ensure that the method operates within accepted decision limit quality control criteria. The assay then provides a value/number/result or other output for each test sample. That output is then deemed to represent infection or non infection based on independent data derived from frequency distributions of results from an uninfected control. This control may describe distributions of results obtained from more than one uninfected subject, for example, from an uninfected population which may be of the same species, geographic origin, age, sex as the test sample. A positive -negative cut -point for the method is determined from these distributions to provide defined levels of diagnostic sensitivity and specificity for the method.
The internal standard or reference may obviate the need to physically provide an uninfected control in the form of uninfected cells or otherwise to physically measure the level of the target in an uninfected control. Where the internal standard or reference is used, the level of expression in an uninfected control has been predetermined and may be provided for example in the form of written information that is supplied with a diagnostic kit.
The measurement of the level of expression of the target in the subject for deriving the uninfected control is generally done using the same assay format as that that is used for measurement of the target in the test sample. However, it is not necessary to use the same assay when an internal standard that can be used to compare data obtained from different assay formats is or has been applied. Generally the subject from which the uninfected control is derived and the individual selected for determination of whether they have an infection are of the same family. For example, where the individual for determination of whether they have a Tannerella infection is a human, the negative control is generally derived from the measurement of the level of expression of the target in a human. In one embodiment, the individual selected for determination of whether they have an infection and the subject from which the negative control is to be derived are from the same species. It may not be necessary that they be of similar age or sex or have been exposed to similar environmental influence.
In another embodiment, the detection of expression of the protein in the individual is assessed by the steps of:
-obtaining a sample from the oral cavity of the individual;
- measuring for the level of expression or presence of the target protein in the sample; and
- comparing the measured level with an infected control that describes the level of expression of the target as observed in a sample from the oral cavity of a subject that has been determined as having a Tannerella infection;
thereby determining whether the individual has a Tannerella infection.
An "infected control" is a sample from an individual or represents a set of parameters previously defined from individuals that do have all the attributes of an individual with a Tannerella infection. For example, the individual from whom the "infected control" sample is derived generally does have subgingival plaque, inflammation of the gums, antibodies directed against Tannerella in their blood, have an amount of Tannerella at a level above approximately 103 to 105 cells/mg of subgingival plaque and/or have an amount of Tannerella at a level above approximately 103 cells/ml of saliva. The "infected control" sample may be taken from the oral cavity of a subject that, but for the presence of a Tannerella infection, is generally the same or very similar to the individual selected for determination of whether they have a Tannerella infection (the latter otherwise known as the "test sample"). The measurement of the level of expression of the target in the sample from the oral cavity of the subject for deriving the infected control is generally done using the same assay format that is used for measurement of the target protein in the test sample. It will be appreciated that the control sample may also be taken from the same individual from which the test sample is taken, but at a different time-point, or periodontal site, in order to determine progression of the Tannerella infection or periodontal disease.
In certain embodiments, the method includes measuring the level of expression of the target in the infected control to compare the measured level in the test sample with the level in the infected control. However, again an internal standard may be applied that obviates the need to provide an infected control or otherwise to measure the level of the target in an infected control.
The measurement of the level of expression of the target in the tissue of the subject for deriving the infected control is generally done using the same assay format as that that is used for measurement of the target in the test sample. However, again it is not necessary to use the same assay when an internal standard that can be used to compare data obtained from different assay formats is or has been applied.
Generally the subject from which the infected control is derived and the individual selected for determination of infection are of the same family. For example, where the individual for determination of whether they have a Tannerella infection is a human, the infected control is generally derived from the measurement of the level of expression of the target in a human. In one embodiment, the individual selected for determination of whether they have an infection and the subject from whom the positive control is to be derived are from the same species. It may not be necessary that they be of similar age or sex or have been exposed to similar environmental influence.
In one embodiment the method includes
- measuring the level of expression of the target protein in a sample from the oral cavity of the individual; and
- comparing the measured level with an uninfected control and an infected control. In one embodiment, the measurement of the level of one or more Tannerella protein, peptide or fragments thereof in the "test sample" may be compared with standard measurements of the same protein, peptide or fragments thereof associated with a series of known Tannerella levels, for example, 103, 104, 105, 106, 107, 108 or 109 cells grown in the laboratory and cell numbers verified using FACS or real time PCR. The antibodies used to capture and detect the protein, peptide or fragment thereof will be used at dilutions to ensure that any chairside test for the bacteria in a sample (e.g. saliva and/or subgingival plaque sample) will give a positive reaction (e.g. a noticeable colour) only for concentrations above certain amounts of cells, e.g. 104 Tannerella cells per mg of subginigval plaque sample. The intensity of the colour reaction will be a guide to the clinician on the level of the Tannerella at the periodontal site. This will be provided as a colour guide with a kit of the invention described further herein. The positive reading will be taken by the clinician to indicate a Tannerella infection at that periodontal site. Hence that site will be treated (e.g. debridement of the tooth root to remove all subgingival plaque) and the patient, depending on the severity of the infection, may be prescribed a course of antibiotic therapy.
Typically the target protein is directly detected to determine whether the individual has a Tannerella infection. This is otherwise known as a "direct detection" of the target to measure the level of expression of the target. In these embodiments, the target protein, peptide or fragment thereof described in Table 1 or 2 herein may be detected.
In certain embodiments, the level of expression of a molecule, the expression of which is modulated in accordance with the expression of the target is measured. This is otherwise known as an "indirect detection" of the target to measure the level of expression of the target.
In one embodiment, a nucleic acid contained in the individual selected for determination of whether they have a Tannerella infection that encodes a target protein, or that is complementary to a nucleic acid that encodes a target protein, is measured. In this embodiment, the nucleic acid may be one which can be used to determine the presence of a given protein, or level of expression of a given protein in an individual as per a molecular genetic approach. One example is where a polynucleotide that is complementary to a nucleic acid (DNA, RNA, cDNA) that encodes a target protein is hybridised to the nucleic acid and hybridisation is detected. One example is quantitative PCR. Others include quantitative Northern and Southern blotting, and microarray.
In another embodiment, the method includes the step of detecting a target protein, or peptide or fragment thereof in a sample from the individual to assess the level of expression of the target in the individual. The presence of a given protein, or level of expression of a given protein in an individual can be detected by any number of assays. Examples include immunoassays, chromatography and mass spectrometry. One example of an immunoassay that is particular preferred is FACS.
Immunoassays, i.e. assays involving an element of the immune system are particularly preferred. These assays may generally be classified into one of:
(i) assays in which purified antigen (for example, an antigen that is expressed in a Tannerella) is used to detect an antibody in host serum. For example, purified antigen is bound to solid phase by adsorption or indirectly through another molecule and serum from an individual is applied followed by another antibody for detecting presence or absence of host antibody;
(ii) assays in which purified antigen (for example, an antigen that is expressed in a Tannerella) is used to detect immune cells, such as T and B lymphocytes. For example, peripheral white cells are purified from an individual and cultured with purified antigen. The presence or absence of one or factors indicating immunity are then detected. Other examples include assays that measure cell proliferation (lymphocyte proliferation or transformation assays) following exposure to purified antigen, and assays that measure cell death (including apoptosis) following exposure to purified antigen;
(iii) assays in which purified antibody specific for an antigen (for example, an antigen that is expressed in a Tannerella) is used to detect an antigen in the individual. For example, purified antibody is bound to solid phase, sample from an individual is then applied followed by another antibody specific for the antigen to be detected. There are many examples of this approach including ELISA, RIA;
(iv) assays in which a purified anti-id iotypic antibody is used to detect an antibody from an individual. For example, anti-idiotypic antibody is adsorbed to solid phase, serum from an individual is added and anti-Fc antibody is added to bind to any antibodies from the individual having been bound by the anti-id iotypic antibody.
It will be understood that the level of expression of the target protein may be measured by obtaining a sample from an individual selected for assessment and determining the level of expression of the target in the sample. Alternatively, the level of expression could be determined in vivo, for example by providing labelled antibodies to the individual which can be visualised in vivo.
Various assays that can be used to detect the presence of a target protein in a sample include:
Enzyme linked immunosorbent assay (ELISA): This method involves fixation of a sample, for example saliva, containing a target protein, peptide or fragment thereof to a surface such as a well of a microtiter plate. A target protein specific antibody coupled to an enzyme is applied and allowed to bind to the target protein, peptide or fragment thereof. Presence of the antibody is then detected and quantitated by a colorimetric reaction employing the enzyme coupled to the antibody. Enzymes commonly employed in this method include horseradish peroxidase and alkaline phosphatase. If well calibrated and within the linear range of response, the amount of target protein, peptide or fragment thereof present in the sample is proportional to the amount of color produced. A target protein, peptide or fragment thereof standard is generally employed to improve quantitative accuracy.
Western blot: This method involves separation of a target protein, peptide or fragment thereof from other protein by means of an acrylamide gel followed by transfer of the protein, peptide or fragment thereof to a membrane (e.g., nylon or PVDF). Presence of the target protein, peptide or fragment thereof is then detected by antibodies specific to the target protein, peptide or fragment thereof, which are in turn detected by antibody binding reagents. Antibody binding reagents may be, for example, protein A, or other antibodies. Antibody binding reagents may be radiolabeled or enzyme linked as described hereinabove. Detection may be by autoradiography, colorimetric reaction or chemiluminescence. This method allows both quantitation of an amount of target protein, peptide or fragment thereof and determination of its identity by a relative position on the membrane which is indicative of a migration distance in the acrylamide gel during electrophoresis.
Radio-immunoassay (RIA): In one version, this method involves precipitation of the desired target protein, peptide or fragment thereof with a specific antibody and radiolabeled antibody binding protein (e.g., protein A labelled with I125) immobilized on a precipitable carrier such as agarose beads. The number of counts in the precipitated pellet is proportional to the amount of target protein, peptide or fragment thereof.
In an alternate version of the RIA, a labelled target protein, peptide or fragment thereof and an unlabelled antibody binding protein are employed. A sample containing an unknown amount of a target protein, peptide or fragment thereof is added in varying amounts. The decrease in precipitated counts from the labelled target protein, peptide or fragment thereof is proportional to the amount of target protein, peptide or fragment thereof in the added sample.
Fluorescence activated cell sorting (FACS): This method involves detection of a target protein, peptide or fragment thereof in situ in cells by target protein, peptide or fragment thereof specific antibodies. The target protein, peptide or fragment thereof specific antibodies are linked to fluorophores. Detection is by means of a cell sorting machine which reads the wavelength of light emitted from each cell as it passes through a light beam. This method may employ two or more antibodies simultaneously.
lmmunohistochemical analysis: This method involves detection of a target protein, peptide or fragment thereof in situ in fixed cells by target protein, peptide or fragment thereof specific antibodies. The target protein, peptide or fragment thereof specific antibodies may be enzyme linked or linked to fluorophores. Detection is by microscopy and subjective or automatic evaluation. If enzyme linked antibodies are employed, a colorimetric reaction may be required. It will be appreciated that immunohistochemistry is often followed by counterstaining of the cell nuclei using for example Hematoxyline or Giemsa stain.
In situ activity assay: According to this method, a chromogenic substrate is applied on the cells containing an active enzyme and the enzyme catalyzes a reaction in which the substrate is decomposed to produce a chromogenic product visible by a light or a fluorescent microscope.
In vitro activity assays: In these methods the activity of a particular enzyme is measured in a protein mixture extracted from the cells. The activity can be measured in a spectrophotometer well using colorimetric methods or can be measured in a non- denaturing acrylamide gel (i.e., activity gel). Following electrophoresis the gel is soaked in a solution containing a substrate and colorimetric reagents. The resulting stained band corresponds to the enzymatic activity of the protein of interest. If well calibrated and within the linear range of response, the amount of enzyme present in the sample is proportional to the amount of colour produced. An enzyme standard is generally employed to improve quantitative accuracy.
In a preferred embodiment, the assay is a point-of-care or point-of-use diagnostic test. Point-of-care testing (POCT) is defined as diagnostic testing at or near the site of patient care or tissue or body fluid or plaque sampling. The motivation behind POCT is to bring the test conveniently and immediately to the patient, which in turn increases the likelihood that the patient will receive the results in a timely manner. Therefore, treatment can immediately follow diagnosis.
A non-limiting example of a point-of-care test is a lateral flow test. Lateral flow tests, also known as lateral flow immunochromatographic assays are a simple device intended to detect the presence (or absence) of a target protein, peptide or fragment thereof in sample. Lateral flow tests are a form of immunoassay in which the test sample flows along a solid substrate, for example paper strip, via capillary action. After the sample is applied to the test it encounters a coloured reagent which mixes with the sample and transits the substrate encountering lines or zones which have been pretreated with an antibody or antigen. Depending upon the analytes present in the sample the coloured reagent can become bound at the test line or zone. One result of the antibody-antigen binding is that it can release some material that has been pre- bound to the antibody, such as gold or colloid nanoparticles. The colloid in turn may produce a visible line on the substrate which can be detected either by the naked eye or imaging device such as silicon photodiode or CCD device. Lateral flow tests can operate as either competitive or sandwich assays. In principle any coloured particle can be used, however in a preferred embodiment either latex (blue colour) or nanometer sized particles of gold (red colour) are used. The gold particles are red in colour due to localised surface plasmon resonance. Fluorescent or magnetic labelled particles can also be used in combination with an electronic reader to assess the test result. In the case of sandwich assay format the sample first encounters coloured particles which are labelled with antibodies raised to the target protein, peptide or fragment thereof. The test line will also contain antibodies to the same target, although it may bind to a different epitope on the target protein, peptide or fragment thereof. The test line will show as a coloured band in positive samples. However, for competitive assays the sample first encounters coloured particles which are labelled with the target protein, peptide or fragment thereof or an analogue. The test line contains antibodies to the target/its analogue. Unlabelled target protein, peptide or fragment thereof in the sample will block the binding sites on the antibodies preventing uptake of the coloured particles and the test line will show as a coloured band in negative samples.
Different versions of the methods and kits of the present invention can be used for various applications.
For example, one version could be used to assess the risk of future periodontal disease development in an individual (e.g., high, medium or low risk for future periodontal disease development).
Another test version could be used to quantitate periodontal disease risk leading to the prediction of periodontal disease experience at subsequent ages. This test might be administered in a dentist's office where appropriate countermeasures could be initiated. Yet another test version would be diagnostic and used with medically compromised patients, such as those suffering from diabetes or AIDS. Still another test version would feature multiple sample, high throughput characteristics. The use of this test version would be targeted to screening populations of samples, for example saliva samples, such as those used for epidemiological surveys. It will be appreciated that the tools necessary for detecting the presence of a target protein, peptide or fragment thereof in a sample from an individual may be provided as a kit, which may contain one or more unit dosage form containing an active ingredient for detection of the target protein, peptide or fragment thereof.
Alternatively, the kit may comprise means for collecting the sample and specific detection means packaged separately. The kit may be accompanied by instructions for use.
For example, the kit may include devices such as a dipstick or a cartridge, (optionally comprised in a housing) which the individual or clinician places into the oral cavity or sample obtained from the individual. The device may comprise any agent capable of specifically detecting the target proteins, peptides or fragments thereof. For example, the device may comprise one or a combination of monoclonal and polyclonal antibody reagents or fragments thereof and an indicator for detecting binding. Antibody supports are known in the art. In an embodiment of this invention, antibody supports are absorbent pads to which the antibodies are removably or fixedly attached.
According to a preferred embodiment, the device of the invention is a lateral flow device comprising an inlet means for flowing a fluid, for example body fluid, into contact with one or more agents, for example a polyclonal or monoclonal antibody or fragment thereof, capable of detecting the proteins, peptides or fragments thereof of the present invention. The test device can also include a flow control means for assuring that the test is properly operating. Such flow control means can include control proteins, peptides or fragments thereof bound to a support which capture detection antibodies as a means of confirming proper flow of sample fluid through the test device. Alternatively, the flow control means can include capture antibodies in the control region which capture the detection antibodies, again indicating that proper flow is taking place within the device. In one embodiment, the kit comprises a monoclonal target protein, peptide or fragment thereof coloured conjugate and polyclonal anti-target protein coated on a membrane test area. By capillary action, the sample migrates over the test area and reacts with the impregnated reagents to form visible coloured bands in the test window. The presence of the target protein, peptide or fragment thereof in concentrations above normal will result in the formation of a distinct coloured band in the test area thus indicating a positive result for a Tannerella infection or risk of progression of periodontal disease or any other outcome described herein. Conversely, if no line appears in the test area, the test is negative.
It will be understood that in certain embodiments the bacteria may be a species or subspecies of Tannerella including Tannerella forsythia. Preferably the Tannerella is of the species forsythia (Tannerella forsythia).
In certain embodiments, the test for a Tannerella, particularly T. forsythia, may be used in combination with a method detection of other bacteria known to effect periodontal disease initiation or progression. For example, the bacteria may be Porphyromonas gingivalis or Treponema denticola.
The methods of the present invention may be performed at the same time as analysis of clinical parameters. Such clinical parameters include modified gingival index (Lobene et al. Clin Rev Dent 1986: 8:3-6), plaque index (Silness et al. Acta Odontol Scand 1964: 22 121-135), pocket depth, recession, clinical attachment level, bleeding on probing and suppuration.
In certain embodiments, the method may be useful for assessing a response of an individual to administration of a protein or substance representing part or all of a Tannerella. In these embodiments, the protein or substance is administered to an individual and the expression of a target protein described in Table 1 or 2 herein is assessed to determine a response to the Tannerella protein or substance.
In a particular embodiment, the individual is a human. The human may be either male or female. The age of the human can be between 18 and 35 years old, between 2 and 45 years old; between 2 and 80 years old or above; or between 15 and 60 years old or above. It is not intended that the methods of the invention are limited to determining Tannerella infection in individuals within a particular age group.
In another embodiment, the individual may be an animal including a domestic animal. Preferably the animal is a cat, dog, sheep, cow or horse. In one embodiment the invention provides a substantially non-glycosylated protein, peptide or fragment thereof described in Table 1 or 2 herein. Preferably, the protein, peptide or fragment thereof described in Table 1 or 2 herein does not contain any glycosylated amino acids. Preferably, the substantially non-glycosylated protein is a non-glycosylated form of TF1342, TF0090, TF0091 , TF1331 , TF2123. The amino acid sequences of these proteins, and others, of the invention are listed in Table 6 and referenced by the SEQ ID NOs: 1 to 5 shown therein.
In one embodiment the invention provides an antigenic region of a substantially non- glycosylated protein of the invention. An antigenic region of a protein may be determined using various algorithms including EMBOSS Antigenic (Kolaskar.AS and Tongaonkar.PC (1990). A semi-empirical method for prediction of antigenic determinants on protein antigens. FEBS Letters 276: 172-174) or Antigenicity Plot (Hopp.T.P. and Woods.K.R. (1981 ) Prediction of protein antigenic determinants from amino acid sequences. Proc Natl Acad Sci USA 86:152-156).
In one embodiment, the fragment of a substantially non-glycosylated protein of the invention is a region of the protein that is exposed on the surface of the bacteria when the protein is in its native conformation in the bacteria.
Tannerella have an outer membrane and outer membrane proteins are those which are embedded in or protrude from the outer membrane.
In other embodiments, a protein, peptide or fragment thereof of the invention has a sequence that has 60, 70, 80, 90, 91 , 92, 93, 94, 95, 96, 97, 98 or 99% amino acid identity to a protein, peptide or fragment thereof described in Table 1 or 2 herein. In certain embodiments, the protein, peptide or fragment thereof is substantially non- glycosylated.
In one embodiment, a protein, peptide or fragment thereof of the invention has a sequence that has 60, 70, 80, 90, 91 , 92, 93, 94, 95, 96, 97, 98 or 99% amino acid identity to TF1342, TF0090, TF0091 , TF1331 TF2123. In certain embodiments, the protein, peptide or fragment thereof is substantially non-glycosylated. Typically, the protein, peptide or fragment thereof is in a composition. In one embodiment, substantially all of the protein in a composition of the invention is substantially non-glycosylated. In another embodiment, all of the protein in a composition of the invention is substantially non-glycosylated.
In one embodiment the composition consists solely of protein. In another embodiment, the composition of the invention may include, further to protein, other cellular components such as fragments or parts of a membrane or cell wall. In other words, the composition may include lipids, carbohydrates and nucleic acids.
A composition of the invention may be used to generate antibodies in an animal, for example a mouse, rat, rabbit, sheep or human. The composition may be used to generate polyclonal or monoclonal antibodies. The antibodies have a number of utilities including detection of an immune response in an individual generated against a Tannerella or other in vitro or in vivo applications.
A protein or composition of the invention may also be used to detect an immune response in an individual generated against a Tannerella.
The composition further comprises a carrier, diluent preservative or other component that could be used to modify the immune response in an animal having received the composition. The carrier, diluent, preservative or other component is particularly useful for increasing the likelihood of generating an antibody response in an animal to a protein, peptide or fragment thereof or composition of the invention.
In another embodiment, a composition of the invention comprises a substantially non- glycosylated protein, peptide or fragment thereof differing from a protein, peptide or fragment thereof described in Table 1 or 2 by conservative amino acid substitutions.
Whilst the concept of conservative substitution is well understood by the person skilled in the art, for the sake of clarity conservative substitutions are those set out below.
GIy, Ala, VaI, lie, Leu, Met;
Asp, GIu, Ser; Asn, GIn ;
Ser, Thr;
Lys, Arg, His;
Phe, Tyr, Trp, His; and
Pro, Nα-alkalamino acids.
Accordingly, without being bound by any theory, or mode of action, it is believed that a non-glycosylated protein, peptide or fragment thereof described in Table 1 or 2 herein when used to generate antibodies will only generate antibodies to the protein, peptide or fragment thereof and not to any glycan moiety.
"Substantially non-glycosylated" with respect to a protein, peptide or fragment thereof described in Table 1 or 2 herein, is defined as less than half the maximum level of glycosylation of the protein, peptide or fragment thereof predicted by any available computer software anaylsis, for example on http://www.expasy.ch/tools/ or http://www.cbs.dtu.dk/services/. "Substantially non-glycosylated" in certain embodiments is also defined as less than half the maximum level of glycosylation of the protein, peptide or fragment thereof as determined experimentally. "Substantially non- glycosylated" may refer to a reduced number of amino acids per protein, peptide or fragment thereof that are glycosylated or to reduced number of saccharides on each glycosylated amino acid. "Substantially non-glycosylated" may also refer to a protein, peptide or fragment thereof that has been treated with one or more glycosidases or may refer to a protein, peptide or fragment that has a glycosylation profile that is the same or similar to the glycosylation profile of a protein, peptide or fragment thereof that has been treated with a glycosidase.
"Percent (%) amino acid identity" or " percent (%) identical" with respect to a protein, peptide or polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the specific protein, peptide or polypeptide sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity.
Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms (non-limiting examples described below) needed to achieve maximal alignment over the full-length of the sequences being compared. When amino acid sequences are aligned, the percent amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (which can alternatively be phrased as a given amino acid sequence A that has or comprises a certain percent amino acid sequence identity to, with, or against a given amino acid sequence B) can be calculated as: percent amino acid sequence identity = X/Y*100, where X is the number of amino acid residues scored as identical matches by the sequence alignment program's or algorithm's alignment of A and B and Y is the total number of amino acid residues in B. If the length of amino acid sequence A is not equal to the length of amino acid sequence B, the percent amino acid sequence identity of A to B will not equal the percent amino acid sequence identity of B to A.
In calculating percent identity, typically exact matches are counted. The determination of percent identity between two sequences can be accomplished using a mathematical algorithm. A nonlimiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264, modified as in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877. Such an algorithm is incorporated into the BLASTN and BLASTX programs of Altschul et al. (1990) J. MoI. Biol. 215:403. To obtain gapped alignments for comparison purposes, Gapped BLAST (in BLAST 2.0) can be utilized as described in Altschul et al. (1997) Nucleic Acids Res. 25:3389. Alternatively, PSI-Blast can be used to perform an iterated search that detects distant relationships between molecules. See Altschul et al. (1997) supra. When utilizing BLAST, Gapped BLAST, and PSI-Blast programs, the default parameters of the respective programs (e.g., BLASTX and BLASTN) can be used. Alignment may also be performed manually by inspection. Another non- limiting example of a mathematical algorithm utilized for the comparison of sequences is the ClustalW algorithm (Higgins et al. (1994) Nucleic Acids Res. 22:4673- 4680). ClustalW compares sequences and aligns the entirety of the amino acid or DNA sequence, and thus can provide data about the sequence conservation of the entire amino acid sequence. The ClustalW algorithm is used in several commercially available DNA/amino acid analysis software packages, such as the ALIGNX module of the Vector NTI Program Suite (Invitrogen Corporation, Carlsbad, CA). After alignment of amino acid sequences with ClustalW, the percent amino acid identity can be assessed. A non- limiting examples of a software program useful for analysis of ClustalW alignments is GENEDOC™ or JalView (http://www.jalview.org/). GENEDOC™ allows assessment of amino acid (or DNA) similarity and identity between multiple proteins. Another non- limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller (1988) CABIOS 4:11-17. Such an algorithm is incorporated into the ALIGN program (version 2.0), which is part of the GCG Wisconsin Genetics Software Package, Version 10 (available from Accelrys, Inc., 9685 Scranton Rd., San Diego, CA, USA). When utilizing the ALIGN program for comparing amino acid sequences, a PAM 120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used.
In one embodiment the invention provides a system for determining whether an individual has a Tannerella infection including:
- selecting an individual;
-detecting whether the selected individual contains a protein, peptide or fragment thereof described in Table 1 or 2 herein, wherein detection of said protein, peptide or fragment thereof determines that the individual has a Tannerella infection,
thereby determining whether the individual has a Tannerella infection.
The invention is further illustrated by the following Examples which are included by way of exemplification and not limitation of the invention.
Examples
The outer membrane (OM) of T. forsythia was separated by 2D-PAGE and the spots were identified by MALDI-TOF/TOF MS. The sixty-nine putative outer membrane proteins (Omps) that were identified represent a sub-proteome that includes many unique features that are compared and contrasted to Omps identified from the related Porphyromonas and Bacteroides genuses.
Example 1 - Materials and Methods
Growth of Tannerella forsythia
T. forsythia strain ATCC43037 was grown anaerobically in brain heart infusion media supplemented with 5 μg/ml hemin, 0.5 μg/ml menadione, 0.001% w/w N-acetyl muramic acid and 5% fetal bovine serum. The conditions for the cultivation of T. forsythia mimic the environment of a site affected by periodontal disease in an oral cavity.
Outer Membrane Preparation: T. forsythia outer membrane was prepared by the sarkosyl method (Filip, C et al. (1973). Solubilization of the cytoplasmic membrane of Escherichia coli by the ionic detergent sodium-lauryl sarcosinate. J Bacteriol 115, 717- 722). Briefly, harvested cells were resuspended in 35 ml buffer (50 mM Tris pH 8.0, 150 mM NaCI, 5 mM MgCI2), and sonicated on ice for 15 min at 50% power and a duty cycle of 5 in a Branson Sonifier. The membranes were collected by centrifugation at 40,000 g, and washed twice with buffer. The membranes were then resuspended in an equal volume of buffer and 2% sodium-lauryl sarcosinate (aq) and incubated at 370C for 1 h to facilitate inner membrane solubilisation. The OM was pelleted at 40,000 g, washed with buffer and stored at -7O0C.
TF1331 enrichment: OM sample from above was incubated in solubilizer 2 (Invitrogen) at 50° C for 10 min and centrifuged at 50,000 g for 15 min. The pellet was resuspended in solubilizer 2, centrifuged again and the supernatant discarded. The pellet containing mainly TF1331 was analysed by SDS-PAGE using a precast NuPAGE Bis-Tris mini gel with MOPS running buffer (Invitrogen).
Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE): 2D-PAGE was performed using pH4-7 IPG strips in the first dimension and 4-12% Bis-Tris Zoom gels in the second dimension according to the manufacturer's instructions (Invitrogen). Solubiliser 2 (Invitrogen) was used to solubilise the outer membrane samples, and IPG strips were reduced with DTT and alkylated with iodoacetamide before the second dimension. Gels were stained for protein with colloidal Coomassie Blue (Molloy, M. P., et al. (2000). Proteomic analysis of the Escherichia coli outer membrane. Eur J Biochem 267, 2871-2881.) or for carbohydrate with Pro-Q Emerald 300 fluorescent stain according to the manufacturer's protocol (Invitrogen) and imaged with a LAS 3000 (Fuji).
Spot Picking: After thorough equilibration of the gel in water, the Coomassie stained gel was placed in an SP gel frame of cut-out size 76x70 mm and placed onto the scanner of the Proteineer SP spot picker (Bruker Daltonics) where it was submerged under water. The gel was scanned and analyzed using Proteomweaver software (Biorad) to enable spot detection and gel calibration with respect to pi and MW. The spotlist was transferred to the spot picker and spot picking was performed using a 1mm diameter cutting tool and the preset "pick pick" method for non-backed gels. The gel plugs were transferred to 96 well digest adaptors (Bruker Daltonics).
Trypsin digestion and MALDI target preparation: This procedure was performed in a Class III cabinet (Laminar flow is sufficient) to minimize keratin contamination. After spot picking, the 96 well plates containing the gel plugs were removed from the digest adaptors and blotted onto lint-free tissue and alternately washed with 30 μl per well of solution A (8% CH3CN in 20 mM NH4HCO3) and 30 μl per well of solution B (50% CH3CN in 20 mM NH4HCO3) and finally dehydrated in solution C (100% CH3CN) for a total of eight washes (ABABABCC). Each wash was for 5 min duration after which the wash solution was removed by blotting. Immediately after the second acetonitrile wash was removed by blotting, 1.8 μl trypsin solution (10 ng/μl sequencing grade modified porcine trypsin (Promega), 20 mM NH4HCO3, 1 mM CaCb) was added to each gel plug with a 10 μl multichannel pipette, carefully ensuring contact between each gel plug and the trypsin solution. After 5 min reswelling at room temperature, the gel plugs were rewetted with 5 μl solution A, and incubated at 30° C in the digest adaptors for 4 hours. Peptides were extracted with 4 μl 1 % TFA v/v at 2O0C for a minimum of 30 min.
A 600 μm anchorchip MALDI target was manually prepared with HCCA matrix using the thinlayer technique according to the anchorchip manual (Bruker Daltonics) just prior to sample deposition. Digested peptides (4 μl) were deposited onto the target and after 10 min adsorption the samples were collectively washed by the rapid pouring of at least 100 ml of 0.1 % TFA over the plate. Automated TOF/TOF MS
All MS related hardware and software in this section are from Bruker Daltonics. Automated TOF/TOF was performed an Ultraflex TOF/TOF (Suckau, D. et al. (2003). A novel MALDI LIFT-TOF/TOF mass spectrometer for proteomics. Analytical and bioanalytical chemistry 376, 952-965) upgraded to a PAN source and Lift II, and controlled by FlexControl v2.4 software. MS was performed using a 25 kV positive reflectron method which was calibrated using a peptide mix applied to the centre of the target prior to automated analysis. MS spectra were then automatically acquired for each sample with the laser power set to a narrow range and under fuzzy logic control. 300 spectra were accumulated for each sample in sets of 15 spectra, with each set having to exceed a S/N of 2 and resolution of 5000 in order to be included. Peaks within a mass range of 1200-2500 were used for this evaluation. At most, 75 spectra were recorded at each raster position. Peak detection and subsequent export to the ProteinScape database were achieved using FlexAnalysis v2.4. Monoisotopic peaks were automatically labeled using the SNAP algorithm within the range 800-4000 Da with a S/N threshold of 6, a quality factor of 50 and a maximum of 100 peaks. MS/MS spectra were acquired using the Lift method on parent ions selected by ProteinScape (see below). A single set of 50 spectra were accumulated for the parent ion with a S/N threshold of 3 at a fixed laser power followed by 14 sets of 50 fragmentation spectra at a laser power elevated by 30%, each set being acquired at a different raster position. The MS/MS spectra were smoothed, baseline subtracted and peaks were detected as above except that the quality factor threshold was reduced to 30. Peaklists were automatically sent to ProteinScape as above.
Protein identification
ProteinScape v1.2 was used for internal calibration of MS spectra, removal of calibrants and contaminants from the MS peaklists and submission of peaklists to Mascot 2.1 (Matrix Science) for both PMF and MS/MS ions searches (Chamrad, D. C, Koerting, G., Gobom, J., Thiele, H., Klose, J., Meyer, H. E. & Blueggel, M. (2003). Interpretation of mass spectrometry data for high-throughput proteomics. Analytical and bioanalytical chemistry 376, 1014-1022. Interpretation of mass spectrometry data for high-throughput proteomics. Analytical and bioanalytical chemistry 376, 1014-1022). All searches were against the T. forsythia sequence database obtained from LANL (http://www.oralgen.lanl.gov) in May 2006 and limited to fully tryptic peptides with carbamidomethyl-Cys and Met-Oxidation set as fixed and variable modifications, respectively. Two PMF searches were conducted for each MS spectrum, the first with a large mass tolerance of 300 ppm and zero partial cleavages allowed, and the second with 150 ppm and one partial cleavage allowed. Internal calibration using trypsin autolysis products and common contaminants such as keratins was applied for the second PMF search only. A third PMF search was conducted on positively identified MS spectra to enable further proteins to be identified. In this case internal calibration was applied using the peptides already identified and a mass tolerance of 20 ppm and zero partial cleavages were allowed. After the first two PMF searches, data dependent MS/MS acquisition was triggered by ProteinScape. Providing that peaks had a "goodness for MS/MS" value greater than 200, up to two peaks were chosen to verify PMF results, a further two to identify additional proteins and two if no protein was identified from PMF. MS/MS ions searches were conducted with a mass tolerance of 300 ppm on the parent and 0.8 Da on fragments. One missed cleavage was allowed. PMF and MS/MS searches were deemed correct if Mascot score was greater than 60 (p<0.005) or 25 (p<0.01 ) respectively.
Formalin killing of bacteria
Bacterial cell cultures were grown to a cell density of ~7 x 108 cells/mL and harvested by centrifugation (7,000 g, 20 min, 40C). The cells were then washed once in TC150 (50 mM Tris, 150 mM NaCI, 5 mM CaCI2, pH 7.4) and suspended in a 2-5% vol. of formal saline (0.5% v/v formaldehyde in saline, 150 mM NaCI). Cells were incubated overnight at room temperature under agitation, then washed and suspended in TC150. After preparation, formalin-killed cells were stored at 40C.
Preparation of antisera
25 BALB/c female mice (6-8 weeks old) were immunized twice, one week apart with 2 x
109 formalin-killed T. forsythia cells or an equivalent amount of outer membrane sample per experimental animal in a total volume of 100 μL (50 μL of adjuvant); the first was an intraperiotoneal injection with Complete Freund's Adjuvant (CFA) and the second a subcutaneous injection with Incomplete Freund's Adjuvant 30 days later. Sera were collected by eye bleed two weeks after the second immunization and immediately stored at -2O0C. Experiments were approved by the University of Melbourne Ethics Committee for Animal Experimentation.
Electrophoretic transfer onto PVDF membranes
Immediately after the second dimension of the 2D gel electrophoresis procedure, the gels were equilibrated for 15 min in transfer buffer (25 mM Tris, 192 mM glycine pH 8.3, 2% v/v methanol) before being electroblotted onto a PVDF membrane prewetted with methanol. Electroblotting was conducted for 2 h at 70 V using a Transblott Cell (Bio- Rad) filled with transfer buffer at 40C.
Western blotting
Western blots were performed using a transblot cell (Biorad). The primary antibody used was pooled sera collected from mice as described above at a 1/100 dilution, and goat anti-mouse horseradish peroxidase conjugate was used as a secondary antibody at a dilution of 1/10,000. The membranes were developed using SuperSignal® West Pico Chemiluminescent Substrate (Pierce). The antigenic proteins determined by Western blot analysis were compared with the corresponding 2D gel by overlapping the images using Photoshop software. The images were aligned by linear stretches of one image with respect to the other image until the best fit was obtained.
Example 2 - 2D gel analysis
Outer membrane preparations of T. forsythia were separated by 2D-PAGE in the pi 4-7 range resulting in a reproducible pattern of spots in both technical and biological replicates (Fig 1 ). Most striking was the presence of several groups of very intense spots in the 100- 200+ kDa range. Also of note were several intense discrete spots between 30-70 kDa in the center of the gel, and some very intense spots that were usually poorly resolved around 40 kDa at the basic end of the gel. There was a surprising lack of spot intensity between 70-100 kDa where TonB-dependent receptors (TDRs) are usually found. Gel spots were excised from the gels with a robot, manually digested and analysed automatically using a MALDI-TOF/TOF mass spectrometer. In all, 56 non-redundant proteins were reproducibly identified and mapped to one or more spots, as shown on the master 2D gel and table (Fig 1 , Table 2). For simplicity, multiple spots matching the same protein were labelled with the same number. To be included in this list, each spot had to be identified from at least two 2D gels. A further eleven proteins were identified from a single 2D gel only (Table 2) and an additional ten proteins were identified only from 1D gels (Table 3). In total, 77 non-redundant proteins were identified by Mascot using either PMF (p<0.002) or MS/MS ions search (p<0.03). At this level, the rate of false positive detection against random (PMF) and reverse (MS/MS) databases was undetectable and 0.2% respectively.
All proteins except six exhibited an N-terminal signal sequence suggesting that the sample was a relatively clean membrane preparation with only minor cytoplasmic contamination. The identified proteins were grouped into categories, namely: CTD family proteins, putative lipoproteins (LP), putative Omps and TDRs, putative IMPs and proteins with no apparent N-terminal signal (Sig-) (Table 2 and 3).
CTD family proteins
All of the high molecular weight proteins identified from 2D gels (MW>80kDa) were found to share sequence similarity over approximately 60 amino acid residues at their extreme C-terminal end, which we designate the C-terminal domain (CTD) due to its similarity to the CTD of a similar family of proteins in P. gingivalis and certain other members of the Bacteroidetes. In P. gingivalis, the presence of the CTD has been demonstrated to be required for proper maturation of the CTD-containing proteinase, RgpB and also for proper secretion and attachment of RgpB to the cell surface. The CTD may therefore be a secretion signal for a novel Bacteroidetes secretion pathway. A search within the T. forsythia database for further T. forsythia proteins with this domain revealed the presence of 26 CTD family proteins, of which ten were identified in this study. The ten include surface-layer protein A (TfsA) and surface-layer protein B (TfsB), BspA and the remainder are annotated as hypothetical proteins. Of these, TF2339, TF1741 and its homolog, TF2592 were particularly abundant (Fig 1 ). The observed MW of eight CTD family proteins was higher than the theoretical mass raising the possibility that they may be glycosylated (Table 4). Therefore, replicate 2D gels were stained with a carbohydrate-specific flourescent dye (Fig 2). The spots which reacted strongly with the dye were indeed mostly the CTD family proteins, especially TF2646. The observed MW of this protein was 114 kDa as compared to the expected value of only 48 kDa, consistent with a high level of glycosylation. Furthermore, despite the significant intensity of this protein on 2D gels, only 5 different peptides could be recovered (Table 4). Non-CTD family proteins TF1342, TF2804 and TF2414 also appeared to be glycosylated. The only CTD family proteins not to have a MW greater than that predicted were TF3080 and the BspA proteins TF1843 and TF2998 (Table 4). Each of these had an observed MW considerably less than expected suggesting proteolysis rather than non-glycosylation. The identification of the BspA proteins could not be distinguished, as their identification is based on a single peptide that is identical in both proteins.
To assess whether any of the other CTD family proteins might be N- or C-terminally truncated, peptides close to the N- or C-termini in several of the CTD family proteins were specifically analysed by MS/MS to confirm their identity (Table 4). In this way, the mature N-terminal peptides containing pyroglutamine of three proteins, TF1259, TF2339 and surface layer protein B were identified indicating that the N-terminus of these proteins was intact. Besides TF3080 and the BspA proteins (see above), there was no indication that any of the other CTD proteins had undergone significant N-terminal truncation. Towards the C-terminus however, there was a consistent lack of sequence coverage in the CTD region, with no peptides being found within the last 78 residues. Two peptides were found adjacent to this area with strong Mascot scores in TfsB and TF1741 (Table 4). A lack of sequence coverage in the CTD was also observed in P. gingivalis. The CTD family proteins identified in P. gingivalis tended to be smaller, and diffused over a broad MW in SDS-PAGE or 2D-PAGE. This was not observed in T. forsythia however, even for C-terminal fragments of TfsB which still maintained sharp spots despite a higher than expected MW. This may suggest that the modification in T. forsythia has different properties or is less heterogeneous to that in P. gingivalis. As with P. gingivalis, the absence of CTD domain peptides suggests that the CTD is heavily glycosylated or that part or all of the CTD is removed by proteolytic processing. Comparison of the CTD sequences of T. forsythia compared to P. gingivalis reveals interesting differences (Fig 3). Motif E, consisting of an invariant Lys residue followed by hydrophobics is essentially the same in each species. Likewise, motif D characterized by an invariant GxY motif in P. gingivalis is also shared by T. forsythia with the notable exception of the surface layer proteins (TF2661-2, TF2663) which have VaI in place of Tyr. Motif B also has a very similar character with invariant GIy and conserved hydrophobic, polar and aromatic residues, again with the notable exception that the surface layer proteins lack conservation of the aromatic residue. The main difference in motif B is that in place of the invariant Asp in the P. gingivalis CTD, T. forsythia appears to prefer neutral hydrophilic residues such as Asn, Thr or Ser. The overall length of the T. forsythia CTD is similar to the average length in P. gingivalis, however in P. gingivalis the spacing of the motifs is more heterogeneous. Interestingly, the less well conserved N-terminal region of the CTD has a well conserved length between motif B and the most C-terminal peptide identified between the two species (Fig 3). In the apparent absence of a highly conserved motif in this region it is likely that its overall character and positioning allows it to be post-translationally modified and or proteolytically processed.
Putative Lipoproteins
A total of 42 putative lipoproteins were identified based on the presence of a signal sequence suitable for cleavage by lipoprotein signal peptidase. The majority of the lipoproteins are currently annotated as hypothetical, or conserved hypothetical indicating that their function is as yet unknown. The signal sequences of these proteins contained preferably serine, or alternatively glycine or alanine in the -1 position relative to the predicted cleavage site . Lipoproteins identified that have a functional name were a peptidyl-prolyl cis-trans isomerase (TF0304), proteinases (TF2531 , TF0749, TF1033), a TPR domain protein (TF1940), a thiol:disulfide interchange protein (TF3165) and an exo-alpha-sialidase (or neuraminidase, TF2207). A basic protein (TF0447) with a predicted mass of 10.7 kDa (Table 3) was identified in 1 D gel bands corresponding to a mass of about 120 kDa suggesting this predicted lipoprotein is part of an SDS-resistant complex, perhaps with itself. The genes for 18 lipoproteins were found to be located adjacent to predicted TDR genes, suggesting that their products have a role in the transport of solutes together with their respective TDRs (see below). As shown in Fig 1 , these TDR-associated lipoproteins (LP-Ts) represent some of the most intense spots on the gel, particularly within the 39-64 kDa range.
TonB-dependent transport
As there is a very large number (>60) of predicted TDRs in the TF database, and only fragments of three were identified from the 2D gel, the MW and isoelectric points was sort of the TDRs whose genes are adjacent to the major LP-Ts that were identified (see above). It was found that the TDRs typically exhibited a MW of 80-120 kDa and a basic pi, demonstrating why they were not observed on a pH 4-7 range. Attempts to resolve the OM samples using a pH 7-11 range were unsuccessful, and therefore instead sample separated by 1 D SDS-PAGE in the appropriate MW range was analysed. In this way, after in-gel digestion and MALDI-TOF/TOF analysis, a total of ten TDRs were identified with predicted isoelectric points ranging from 7.0 to 9.5 (Table 2 & 3).
Comparing the ten identified TDRs with the 18 identified LP-Ts revealed that a total of six TDR/ LP-T pairs were found and a total of 17 TDR loci were identified (Fig 4). Only one of these, the susB operon has been functionally annotated. The susB operon which comprises susB through to susG in Bacteroides thetaiotaomicron is involved in starch utilization. SusB and SusG are glycosidic enzymes, SusC is a TDR, and SusD, SusE and SusF are outer membrane lipoproteins. SusC and SusD are reported to form a complex that is essential for binding of cells to starch. SusE and SusF also appeared to interact with each other and the SusC-D complex, however they were not essential for binding to starch. The susB operon in T. forsythia appears to be homologous to the S. thetaiotaomicron system except that the susG gene is absent. Interestingly, SusD (TF0092) was only identified from 1 D gels, which may be consistent with it forming a strong complex with SusC (TF0093) and therefore due to the high pi of SusC not be resolved on the pi 4-7 2D gels. SusD exhibits sequence similarity to all of the other LP- Ts identified whose genes are directly downstream of their respective TDR, except TF2606 (Fig 4), however the additional LP-Ts whose genes are not directly downstream of a TDR are not well conserved. These results suggest that the primary LP-Ts identified all have a similar solute binding function associated with their respective TDRs. An overrepresentation of TDR genes was also reported for the B. thetaiotaomicron genome which encodes 106 predicted TDRs, and 57 LP-Ts (SusD paralogs). The majority of these are part of loci containing polysaccharide degrading enzymes and therefore the TDR's and LP-Ts of such loci are likely to be involved in binding to and transporting polysaccharides into the periplasm. T. forsythia however does not appear to share the same plethora of polysaccharide utilization enzymes and most TDR loci are devoid of them. Overrepresentation of TDR genes is also observed in various Proteobacteria, specifically those that share the ability to degrade a wide range of complex carbohydrates. The difference between TDR systems found in Proteobacteria compared to Bacteroidetes, is that the former do not contain SusD homologs.
Other Om ps
Other proteins identified that are predicted to be integral to the OM include the P. gingivalis homologs, Omp41 (TF1331 ), P40 (TF2852) and P58 (TF1444), and a ToIC homolog (TF0773). Due to the finding in P. gingivalis that Omp41 and its homolog Omp40 were heterodimeric under non-reducing conditions, held together by two disulfide bridges, and also exhibited heat-modifiability similar to OmpA, TF1331 was partially purified and examined by SDS-PAGE in both reducing and non-reducing conditions and at both 5O0C and 1000C (Fig 5). Under reducing conditions, TF1331 migrated at 42 kDa when fully denatured, or 33.5 kDa when partially denatured by heating at 5O0C, consistent with the heat modifiability demonstrated for Omp40/41 of P. gingivalis and of OmpA. Under non-reducing conditions, TF1331 migrated to a MW of 99 kDa when fully denatured, or 72 kDa at 5O0C which is very similar to the MW estimated for heterodimeric Omp40/41 of P. gingivalis suggesting that the non-reduced form of TF1331 is a homodimer held together by disulfide bridges. To confirm this, the mass spectra of digests of the non-reduced forms were compared to digests from the reduced forms and also to the predicted masses of disulfide bonded tryptic peptides. A peak at m/z 3516.63 was found to be specific to the non-reduced TF1331 and its mass corresponded to the tryptic peptide 244RPEFCPECPKCPEVK258 triply disulfide bonded to itself to form a dimer of theoretical m/z 3516.57. To confirm this assignment, the digest was reduced with 10 mM DTT and re-analysed by MS. Reduction caused the disappearance of the 3516.57 peak, and instead, a new peak at 1761.84 corresponding to the reduced peptide appeared (data not shown). MS/MS of this peak confirmed the assignment with a Mascot score of 57. MS/MS was also attempted on the m/z 3516.63 to directly confirm its identity. The MS/MS spectrum (Fig 6), was dominated by a cluster of symmetrical peaks centred at half the MW of the parent ion. The spacing between the isotopes was 1 Da, discounting the possibility of it representing a doubly-charged parent ion. The central peak appeared to consist of three distinct peaks that correspond to the monomer peptides with each cysteine reduced (m/z 1761.8), and the presence of dehydroalanine and dithiocysteine (m/z 1759.8) and the presence of dehydroalanine, dithiocysteine and thioaldehyde (m/z 1757.8) representing LIFT-TOF/TOF fragmentation patterns that have been previously reported for di-sulfide bonded peptides. The groups of peaks either side of the central peak correspond to a different mix of reduced cysteines, dithiocysteines, dehydroalanines and thioaldehydes producing a difference of 32 Da (a sulfur atom) between clusters (Fig 6). Small peaks at approximately -17 Da relative to the large peaks is probably due to the loss of ammonia from the N-terminus. The intensity of other fragments was generally low, however by comparing the fragmentation pattern with that of the reduced peptide it was possible to assign a significant series of a- and b-ions (Fig 6). The b-5 ion was 2 Da less than the b-5 ion of the reduced peptide, possibly due to the formation of a C=S bond, and also appeared to lose sulfur. An analogous phenomenon appeared to happen for the b-8 ion (Fig 6). Taken together, the MS/MS data provides substantial evidence that the m/z 3516.63 peak corresponds to a disulfide-bonded dimer of the 244RPEFCPECPKCPEVK258 peptide.
It is not certain whether the main role of OmpA-like proteins such as TF1331 is simply to form a structural link between the OM and peptidoglycan wall, or whether the ability of some to form a diffusion pore is biologically important.
Antigenic Proteins
Two 2D gels prepared in an identical manner and with the same sample to that shown in Fig 1 were prepared and subjected to Western Blot analysis using antisera raised to formalin-killed whole cells and outer membranes (Fig 7). The Western blot images were overlayed with images from Coomassie Blue stained gels in order to identify the antigenic proteins (Fig 7, Table 5). All of the abundant CTD family proteins were easily identified in the Western images due to their characteristic spot patterns. As these are also glycosylated it is uncertain whether the protein or the carbohydrate is being recognized by the antisera. The three non-CTD family proteins that appeared to be glycosylated, TF1342, TF2804 and TF2414 were also strongly antigenic. Six further antigens that do not appear to be glycosylated were identified of which four are predicted lipoproteins (Table 5). Except for the surface layer proteins none of the other antigenic proteins have been previously identified in T. forsythia. TF1331 is homologous to the Omp40 and Omp41 antigens (PG33, PG32) from P. gingivalis, and TF0090 and TF0091 are homologous to starch binding proteins in B. thetaiotaomicron .
This study defines and maps the major Omps of T. forsythia. The OM proteome is dominated by CTD family proteins, proteins involved with TonB dependent transport and the OmpA-like protein, TF1331. TF1331 is a novel di-sulfide bonded homodimer that shares the OmpA-like properties of heat modifiability and high copy number. The CTD family members include the surface layer proteins which are very abundant and also the more weakly expressed BspA protein that exhibits multiple virulence properties. Several other CTD-family proteins of very high abundance (e.g TF2339 & TF1741 ) are implicated as being present on the surface of the cell. Seventeen TDR loci were identified including ten TDRs and 18 lipoproteins, most of which were very abundant. As there are more than 60 TDRs encoded in the T. forsythia genome, proteomic studies are essential to determine which systems are abundant, or present under given growth conditions.
Example 3
The following example shows the characterisation of T. forsythia outer membrane using 1 D SDS-PAGE and LC-MALDI analysis. The results are reported here in the context of those in Example 2.
SDS-PAGE and LC-MALDI Analysis
Polyacrylamide gel electrophoresis (PAGE): For SDS-PAGE 30 μg of T. forsythia OM was reduced with 50 mM DTT in LDS sample buffer at 1000C for 5 minutes, centrifuged to remove insoluble particles and loaded onto a 10% NuPAGE Bis-Tris gel and SDS- PAGE performed at 150 V for 50-60 min with NuPAGE MOPS SDS Running Buffer and using the XCeII SureLock™ Mini-cell system (Invitrogen, NSW1 Australia). For 2D- PAGE the ZOOM® IPGRunner™ system was used and the isoelectric focusing and SDS-PAGE performed as per the manufacturer's instructions (Invitrogen, NSW, Australia). Briefly, 50 μg of T. forsythia OM was solubilised with 2D protein solubilizer 2 containing 50 mM DTT, 2% v/v Zoom carrier ampholytes pH 4-7 and a trace amount of bromophenol blue. IPG strips (pH 4-7) were rehydrated with the solubilised T. forsythia OM (25°C, 18 hrs). After rehydration the IPG strips were assembled in the IPGRunner Min-Cell system and isoelectric focusing (IEF) performed (175 V, 15 min, 175-2000 V ramp for 45 min, 2000 V for 120 min). After, IEF the IPG strips were either used directly for SDS-PAGE for Western blotting and carbohydrate staining or were alkylated using 125 mM iodoacetamide in NuPAGE LDS sample buffer (15 min, 25°C) for protein identification by MALDI-TOF. The IPG strips were set by 0.5% w/v agarose in running buffer on top of the ZOOM gel and SDS-PAGE performed as described above. Gels were stained for protein with colloidal Coomassie Blue 19 or for carbohydrate with Pro- Q Emerald 300 fluorescent stain (Invitrogen, NSW, Australia) according to the manufacturer's protocol and gels imaged with a LAS 3000 imaging system (Fuji, Tokyo, Japan).
LC- MALDI TOF/TOF of SDS-PAGE bands.
Fifty-five bands were excised from the SDS-PAGE gel from each of four identical lanes and pooled together. Each sample was cut into approximately 1 mm3 cubes and washed, alkylated and digested according to Mortz, E.; Krogh, T. N.; Vorum, H.; Gorg, A., Improved silver staining protocols for high sensitivity protein identification using matrix-assisted laser desorption/ionization-time of flight analysis. Proteomics 2001 , 1 , 1359-1363. The peptides were further extracted once with water and once with CH3CN, pooled with the original digest, dried in a vacuum centrifuge and stored at -20° C prior to LC-MALDI TOF/TOF analysis. HPLC of peptide extracts together with their deposition onto PAC target plates (Bruker Daltonics) was performed according to Ang et. al. 23 with the following modifications. Separation was achieved using a RP column (C18 Acclaim PepmaplOO , 75μm id * 15 cm, 5 μm, 100A, Dionex) and eluted with 0.1% TFA and a gradient of 0-64% CH3CN over 40 min followed by 64-80% CH3CN over 5 min. The flow rate was 6 μL/min through the column. Fractions were collected every 12 s from approximately 20% to 74% CH3CN. The target plate was analyzed with Bruker Ultraflex III MALDI TOF/TOF. All spectra acquisition was performed automatically using FlexControl version 3.0.151 and WARP- LC version 1.1 software. MS analysis was carried in reflectron mode measuring from 700 to 4000 Da using an accelerating voltage of 25 kV. All MS spectra were produced from five sets of 100 laser shots using random movement. Calibration of the instrument was performed externally with ions of pre-spotted internal standards. MSMS analysis was carried in LIFT mode in which the ions were accelerated to 8 kV and subsequently "lifted" to 19 kV in the LIFT cell. MSMS spectra were produced from 750 laser shots using random movement.
MS peak lists were generated by FlexAnalysis version 3.0.90 using SNAP algorithm, with S/N threshold 4. The peak list was filtered to remove common contaminants such as Coomassie Blue and keratin peaks. Selection of parent precursors was determined using WARP-LC software. The compounds separated by less than six fractions were considered the same and were selected as parent precursors if the SN was > 25. The MSMS peak list was also generated using SNAP algorithm after the spectra were smoothed using Savitsky-Golay algorithm (width 0.2 m/z) and baseline subtraction using TopHat algorithm. WARP-LC generated a combined MS/MS peak list which was searched using MASCOT version 2.2.04 (Matrix Science) via BioTools 3.1.0 software. MS/MS ions searches against the T. forsythia database (described above) were conducted with a mass tolerance of 100 ppm on the parent and 0.5 Da on fragments. One missed cleavage was considered with carbamidomethyl (C) as fixed modification, and oxidation (MHW) as variable modifications. Decoy search was done automatically by MASCOT on randomized database of equal composition and size.
All of the results for each band were pooled together and all peptide assignments with scores less than 15 or E-values > 0.2 were deleted. A protein assignment was accepted if it had two or more peptides assigned with a score greater than the Mascot identity threshold which ranged from 15 to 22 (p<0.05, false positive rate measured to be
0.047). Proteins identified from a single peptide were accepted only if the peptide score was greater than 25 (p<0.02, false positive rate measured to be 0.047). Peptides with a score less than identity threshold were still included for unique peptide count.
Identification of proteins by SDS-PAGE and LC-MALDI. Due to inherent limitations of 2D-PAGE and the fact that we only analyzed proteins within a pi range of 4-7, we also separated the OM sample by SDS-PAGE (Figure 8). Interestingly, in addition to having strong bands at positions consistent with the 2D gel profile (Figure 1 ), the 1 D gel (Figure 8) also exhibited significant band density in the 70- 100 kDa region. Fifty-five continuous gel segments were manually excised from the gel, in-gel digested with trypsin and subjected to LC-MALDI TOF/TOF analysis. A total of 2,473 peptides were identified that were above threshold (p<0.05) and had a minimum Mascot score of 15. Of these, 1 ,163 peptides were non-redundant corresponding to 210 different proteins of which 134 were identified with at least two peptides above threshold (p<0.05), and 76 were identified on the basis of having a single high scoring peptide (p<0.02) (Table 1 ).
Considering proteins identified from both techniques, all except 24 were predicted to be localized to the inner membrane, periplasm, OM or beyond on the basis of sequence similarity to proteins of known subcellular location, by the presence of predicted transmembrane helices or by use of the prediction program CELLO 25 (Table 1). The high proportion of cell envelope proteins identified suggests that the sample was a relatively clean membrane preparation with only minor cytoplasmic contamination. Of the 130 proteins with the annotation "outer membrane" in their definition, 71 were identified in this study. Similarly, 13 out of the 26 CTD family proteins were identified (see below) suggesting that approximately 50% of the theoretical outer membrane proteome was identified. The identified proteins were grouped into categories, namely: CTD family proteins, putative lipoproteins (LP), outer membrane located proteins (OM) and TDRs, putative inner membrane proteins (IMP), putative periplasmic proteins (PP) and proteins with neither N-terminal signal nor trans-membrane helices and therefore likely to be located in the cytoplasm (Cyt) (Table 1 ). The gel spots are colour coded according to these categories (Figure 1 ).
CTD family proteins. All of the high molecular weight proteins identified from 2D gels (MW>80 kDa) were found to share sequence similarity over approximately 60 amino acid residues at their extreme C-terminal end, which we designate the C-terminal domain (CTD) due to its similarity to the CTD of a family of proteins in P. gingivalis and certain other members of the Bacteroidetes. In P. gingivalis, the presence of the CTD has been demonstrated to be required for proper maturation of the CTD-containing proteinase, RgpB together with its correct secretion and attachment to the cell surface. The CTD may therefore be a secretion signal for a novel Bacteroidetes secretion pathway. A search within the T. forsythia database for further T. forsythia proteins with this domain revealed the presence of 26 CTD family proteins of which ten were identified from 2D gels in this study, and a further three by LC-MALDI. The thirteen include surface-layer protein A (TfsA) and surface-layer protein B (TfsB), BspA, and a possible internalin-related protein (TF1032) while the remainder are annotated as hypothetical proteins. Of these, TF2339, TF1741 and its homolog, TF2592 were particularly abundant (Figure 1 ). BspA, as previously sequenced, corresponds most closely to TF2998 with 97% sequence identity. The C-terminal -300 residues of BspA are almost identical to TF1843, which is also annotated as BspA. Five other proteins in the database but not identified in this study are also annotated as BspA due to extensive sequence similarity to the original BspA. As mutants lacking a functional BspA gene (presumably TF2998) are highly attenuated with respect to binding to, and invading KB epithelial cells, it appears that the other BspA-like proteins are unable to compensate for the lack of TF2998 suggesting they have a different function. Alternatively, TF2998 may be the only BspA protein produced at a sufficiently high enough level to allow binding and invasion to be reliably detected. The surface layer of T. forsythia has been shown to be composed primarily of TfsA and TfsB. As these proteins have a CTD, it is likely that the secretion of these proteins and hence the production of the surface layer is dependent on the presence of the CTD and the CTD secretion system.
The observed 2D gel MW of eight CTD family proteins was higher than the theoretical mass raising the possibility that they may be glycosylated (Table 4). Therefore, replicate 2D gels were stained with a carbohydrate-specific flourescent dye (Figure 2). The spots which reacted strongly with the dye were indeed mostly the CTD family proteins, especially TF2646 (Table 4). The observed MW of this protein was 114 kDa as compared to the expected value of only 48 kDa, consistent with a high level of glycosylation. Furthermore, despite the significant intensity of this protein on 2D gels, only 5 different peptides could be recovered (Table 4). Non-CTD family proteins TF1342, TF2804 and TF2414 also appeared to be glycosylated (Table 4). The only CTD family proteins not to have a MW greater than that predicted were TF3080 and the BspA proteins TF1843 and TF2998 (Table 4). Each of these had an observed MW considerably less than expected suggesting proteolysis rather than non-glycosylation. The individual BspA proteins could not be distinguished, as their identification was based on a single peptide that is identical in both proteins.
To assess whether any of the other CTD family proteins might be N- or C-terminally truncated, peptides close to the N- or C-termini in several of the CTD family proteins were specifically analyzed by MS/MS to confirm their identity (Table 4). In this way, the mature N-terminus of four proteins (TF1259, TF2339, TF2661-2, and TF2663) was determined from peptides identified by MS/MS that correspond to the predicted N- terminus, and were non-tryptic at the N-terminal side. Three of the N-termini were found to be modified to pyroglutamine (Table 4). Besides TF3080 and the BspA proteins (see above), there was no indication that any of the other CTD proteins had undergone significant N-terminal truncation. Towards the C-terminus however, there was a consistent lack of sequence coverage in the CTD region, with no peptides being found within the last 77 residues. Four peptides were found adjacent to this area with strong Mascot scores in TF2661-2, TF2663, TF2339 and TF1741 (Table 4). A lack of sequence coverage in the CTD was also observed in P. gingivalis. The CTD family proteins identified in P. gingivalis tended to be smaller, and diffused over a broad MW in SDS-PAGE or 2D-PAGE. This was not observed in T. forsythia however, even for C- terminal fragments of surface layer protein B which still maintained sharp spots despite a higher than expected MW (Figure 1 ). This may suggest that the modification in T. forsythia has different properties or is less heterogeneous to that in P. gingivalis. As with P. gingivalis, the absence of CTD domain peptides suggests that the CTD is heavily glycosylated or that part or all of the CTD is removed by proteolytic processing.
Comparison of the CTD sequences of T. forsythia to P. gingivalis reveals interesting differences (Figure 3). Motif E, consisting of an invariant Lys residue followed by hydrophobic residues is essentially the same in each species. Likewise, motif D characterized by an invariant GxY motif in P. gingivalis is also shared by T. forsythia with the notable exception of the surface layer proteins (TF2661-2, TF2663) which have
VaI in place of Tyr. Motif B also has a very similar character with invariant GIy and conserved hydrophobic, polar and aromatic residues, again with the notable exception that the surface layer proteins lack conservation of the aromatic residue. The main difference in motif B is that in place of the highly conserved Asp in the P. gingivalis CTD, T. forsythia appears to prefer neutral hydrophilic residues such as Asn, Thr or Ser. The overall length of the T. forsythia CTD is similar to the average length in P. gingivalis, however in P. gingivalis the spacing of the motifs is more heterogeneous. Interestingly, the less well conserved N-terminal region of the CTD has a well conserved length between motif B and the most C-terminal peptide identified between the two species (Figure 3). In the apparent absence of a highly conserved motif in this region it is likely that its overall character and positioning allows it to be post-translationally modified and or proteolytically processed.
Antigenic Proteins. Two 2D gels prepared in an identical manner and with the same sample to that shown in Figure 1 were prepared and subjected to Western Blot analysis using antisera raised to T. forsythia formalin-killed whole cells and outer membranes (Figure 7). The Western blot images were overlaid with images from Coomassie Blue stained gels in order to identify the antigenic proteins (Figure 7, Tables 5). Sixteen antigenic proteins were identified (Figure 7, Tables 5). All of the abundant CTD family proteins were easily identified in the Western images due to their characteristic spot patterns (Table 4). As these are also glycosylated it is uncertain whether the protein or the carbohydrate is being recognized by the antisera. The three non-CTD family proteins that appeared to be glycosylated, TF1342, TF2804 and TF2414 were also strongly antigenic (Table 5). Six further antigens that do not appear to be glycosylated were identified of which four are predicted lipoproteins (Table 5). Except for the surface layer proteins none of the other antigenic proteins have been previously identified in T. forsythia. TF1331 is similar to the Omp40 and Omp41 antigens (PG33, PG32) from P. gingivalis, and TF0090 and TF0091 are similar to TDR-associated starch binding proteins in B. thetaiotaomicron. Apart from TF1331 , all of the identified antigens were reactive to sera raised against formalin killed whole T. forsythia cells indicating their likely cell-surface exposure.
There is a considerable number of unidentified antigenic spots in the >65 kDa region, particularly in the Western probed with anti-OM sera. The spots that were identified in this region were primarily CTD proteins and CTD protein fragments. As all the identified CTD proteins were antigenic, it is likely that many of these unidentified antigens also correspond to CTD proteins or fragments thereof. There are also major antigenic spots close to spot #22 that could not be positively assigned to a Coomassie-stained spot. Putative Lipoproteins. A total of 75 putative lipoproteins were identified based on the presence of a signal sequence suitable for cleavage by lipoprotein signal peptidase (Table 1). Many of the lipoproteins were annotated as hypothetical, or conserved hypothetical indicating that their function is as yet unknown. The signal sequences of the putative lipoproteins contained preferably serine, or alternatively glycine or alanine in the -1 position relative to the predicted cleavage site. Lipoproteins identified that have functional names include peptidyl-prolyl cis-trans isomerases (TF2214, TF0304, TF0305), proteinases (TF0749, TF1033, TF1755, TF2531 , TF3024), a TPR domain protein (TF1940), a thiol:disulfide interchange protein (TF3165), a previously described beta-N-acetylglucosaminidase (TF2925) and an exo-alpha-sialidase (or neuraminidase, TF2207). A basic protein (TF0447) with a predicted mass of 10.8 kDa (Table 1 ) was identified in almost every 1 D gel band from 49-160 kDa, being most abundant in the 110 kDa band suggesting this predicted lipoprotein forms SDS-resistant complexes, with itself and or other proteins. The genes encoding 28 putative lipoproteins were found to be located adjacent to predicted TDR genes, suggesting that their products have a role in the transport of solutes together with their respective TDRs (see below). As shown in green in Figure 1 , these TDR-associated lipoproteins (LP-Ts) represent some of the most intense spots on the 2D gel, particularly within the 39-64 kDa range.
TonB-dependent transport. As there is a very large number (>60) of predicted TDRs in the T. forsythia database, and we could only identify fragments of three from the 2D gel, we obtained the theoretical MW and isoelectric points of the TDRs whose genes are adjacent to the major LP-Ts that were identified (see above). These TDR sequences typically predict a MW of 80-120 kDa and a basic pi, demonstrating why they were not observed in the pH 4-7 range used. LC-MALDI of 1 D gel bands however resulted in the identification of 46 putative TDRs. Comparing the 46 identified TDRs with the 28 identified LP-Ts revealed that a total of 26 TDR/ LP-T pairs were identified, 11 'lone' TDRs were identified that did not have an adjacent LP-T, nine TDRs were identified whose genes were adjacent to unidentified LP-Ts and two LP-Ts were identified whose genes were adjacent to unidentified TDRs (Table 1 ). The 37 TDR/LP- T pairs (including unidentified proteins) are presented together in Table 1 below the 'lone' TDRs. Interestingly, multiple sequence alignment of all identified TDRs revealed two major groupings (sequence alignment not shown). The larger group consists of large interrelated TDRs (108-140 kDa) with adjacent and interrelated LP-Ts. In contrast, the remaining TDRs exhibit poorer sequence conservation, are smaller (69- 121 kDa) and are mostly 'lone' TDRs. The exceptions are TF1535, TF0682, TF2597, TF0045 and the unidentified TDR, TF2606. Each of these has a downstream LP that is not significantly related to the other LP-Ts identified.
A small number of TDR loci such as TF0094 - TF0090 which is similar to the susB operon of Bacteroides thetaiotaomicron contain multiple downstream genes that encode lipoproteins. The susB operon which comprises susB through to susG is involved in starch utilization. SusB and SusG are glycosidic enzymes, SusC is a TDR, and SusD, SusE and SusF are OM lipoproteins. SusC and SusD are reported to form a complex that is essential for binding of cells to starch. SusE and SusF also appeared to interact with each other and the SusC-D complex, however they were not essential for binding to starch. The susB operon in T. forsythia appears to be homologous to the S. thetaiotaomicron system except that the susG gene is absent. Interestingly, SusD (TF0092) was only identified from 1 D gels in this study, which may be consistent with it forming a strong complex with SusC (TF0093) and therefore due to the high pi of SusC was not resolved on the pi 4-7 2D gels. SusD exhibits sequence similarity to many of the other LP-Ts identified whose genes are directly downstream of their respective TDR, however the additional lipoproteins such as TF0090 and TF0091 whose genes are not directly downstream of a TDR are not well conserved. These results suggest that the primary LP-Ts identified have a similar solute binding function associated with their respective TDRs.
Most sequenced Gram-negative bacteria have up to nine TDRs encoded in their genomes and more than 80% have less than 25 TDRs and only 4% have more than 60. The 60 or more present in T. forsythia therefore is unusual. An overrepresentation of TDR genes was also reported for the B. thetaiotaomicron genome which encodes 106 predicted TDRs, and 57 LP-Ts (SusD paralogs). The majority of these are part of loci containing polysaccharide degrading enzymes and therefore the TDRs and LP-Ts of such loci are likely to be involved in binding to and transporting polysaccharides into the periplasm. T. forsythia however does not appear to share the same plethora of polysaccharide utilization enzymes and most TDR loci are devoid of them. Overrepresentation of TDR genes is also observed in various Proteobacteria, specifically those that share the ability to degrade a wide range of complex carbohydrates. The difference between TDR systems found in Proteobacteria compared to Bacteroidetes, is that the former do not contain SusD homologs. This is the first time to our knowledge that TDR overrepresentation has been demonstrated at the protein level. As proteomic techniques are currently unable to detect all proteins expressed within a proteome, the results imply that T. forsythia prefers to express the majority of its TDRs at moderate to high levels rather than expressing a small range of receptors to suit the availability of specific nutrients.
Other Omps. Other proteins identified that are predicted to be integral to the OM include the P. gingivalis homologs, Omp41 (TF1331), P40 (TF2852) and P58 (TF1444), and the OM efflux proteins TF0773, TF0810, TF1409, TF1476, TF1822 which are related to ToIC. Due to the finding in P. gingivalis that Omp41 and its homolog Omp40 were heterodimeric under non-reducing conditions, held together by two disulfide bridges, and also exhibited heat-mod if iability similar to OmpA, TF1331 was partially purified and examined by SDS-PAGE in both reducing and non-reducing conditions and at both 50° C and 100° C (Figure 5). Under reducing conditions, TF1331 migrated at 42 kDa when fully denatured, or 33.5 kDa when partially denatured by heating at 50° C, consistent with the heat modifiability demonstrated for Omp40/41 of P. gingivalis and of OmpA. Under non-reducing conditions, TF1331 migrated to a MW of 99 kDa when fully denatured, or 72 kDa at 50° C which is very similar to the MW estimated for heterodimeric Omp40/41 of P. gingivalis suggesting that the non-reduced form of TF1331 is a homodimer held together by disulfide bridges. To confirm this, the mass spectra of digests of the non-reduced forms were compared to digests from the reduced forms and also to the predicted masses of disulfide bonded tryptic peptides. A peak at m/z 3516.63 was found to be specific to the non-reduced TF1331 and its mass corresponded to the tryptic peptide 244RPEFCPECPKCPEVK258 triply disulfide bonded to itself to form a dimer of theoretical m/z 3516.57. To confirm this assignment, the digest was reduced with 10 mM DTT and re-analyzed by MS. Reduction caused the disappearance of the 3516.57 peak, and instead, a new peak at 1761.84 corresponding to the reduced peptide appeared (data not shown). MS/MS of this peak confirmed the assignment with a Mascot score of 57. MS/MS was also attempted on the m/z 3516.63 to directly confirm its identity. The MS/MS spectrum (Figure 6), was dominated by a cluster of symmetrical peaks centred at half the MW of the parent ion. The spacing between the isotopes was 1 Da, discounting the possibility of it representing a doubly- charged parent ion. The central peak appeared to consist of three distinct peaks that correspond to the monomer peptides with each cysteine reduced (m/z 1761.8), and the presence of dehydroalanine and dithiocysteine (m/z 1759.8) and the presence of dehydroalanine, dithiocysteine and thioaldehyde (m/z 1757.8) representing LIFT- TOF/TOF fragmentation patterns that have been previously reported for disulfide bonded peptides. The groups of peaks either side of the central peak correspond to a different mix of reduced cysteines, dithiocysteines, dehydroalanines and thioaldehydes producing a difference of 32 Da (a sulfur atom) between clusters (Figure 6). Small peaks at approximately -17 Da relative to the large peaks is probably due to the loss of ammonia from the N-terminus. The intensity of other fragments was generally low, however by comparing the fragmentation pattern with that of the reduced peptide it was possible to assign a significant series of a- and b-ions (Figure 6). The b-5 ion was 2 Da less than the b-5 ion of the reduced peptide, possibly due to the formation of a C=S bond, and also appeared to lose sulfur. An analogous phenomenon appeared to happen for the b-8 ion (Figure 6). Taken together, the MS/MS data provides substantial evidence that the m/z 3516.63 peak corresponds to a disulfide-bonded dimer of the 244RPEFCPECPKCPEVK258 peptide.
It is not certain whether the main role of OmpA-like proteins such as TF1331 is simply to form a structural link between the OM and peptidoglycan wall, or whether the ability of some to form a diffusion pore is biologically important.
This study defines and maps the major Omps of T. forsythia. The OM proteome is dominated by CTD family proteins, proteins involved with TonB dependent transport and the OmpA-like protein, TF1331. TF1331 is a novel disulfide bonded homodimer that shares the OmpA-like properties of heat modifiability and high copy number. The CTD family members include the surface layer proteins which are very abundant and also the more weakly expressed BspA protein that exhibits multiple virulence properties. Several other CTD-family proteins of very high abundance (e.g TF2339 & TF1741 ) are implicated as being present on the surface of the cell. Forty-eight TDR loci were identified including 46 TDRs and 28 lipoproteins, most of which were very abundant. Fifteen proteins were found to be antigenic, and these could be useful for developing diagnostics and therapeutics against T. forsythia infection. As used herein, except where the context requires otherwise, the term "comprise" and variations of the term, such as "comprising", "comprises" and "comprised", are not intended to exclude further additives, components, integers or steps.
Table 1: Identification data for all proteins identified. The 2D gel data and LC- MALDI data, Example 2 and 3, is merged and sorted according to Category, and Accession
Band # Protein Description MWcalc PMF Best Protein Unique b (Spot #) Accession abbreviated Category0 (kDa) Score MS/MS' Score Peptides*
Figure imgf000056_0001

Figure imgf000057_0001
Figure imgf000058_0001
Figure imgf000059_0001
protein Impl 40.9 41
Figure imgf000060_0001
dehydrogenase/N AD(P)H (-) TF0841 nitroreductase Cyt 30.0 66 65 195 glycosyl transferase,
29 TFl 193 group 1 family
13!
DNA-binding response
Figure imgf000061_0001
succinate dehydrogenase,
Figure imgf000061_0002
Accession numbers and protein descriptions are from the Oralgen website (www.oralgen.lanl.gov) Hyphenated accession numbers are where two adjacent genes in the database correspond to a single protein as indicated by both proteomics and homology data. Abbreviations used are: HP, hypothetical protein; HP-C, conserved HP; and others as already defined
Cellular location or protein type as explained in the text. The number of predicted transmembrane helices is provided for IMPs (from Oralgen), and a localization score from CELLO (http://cello.life.nctu.edu.twΛ is provided for many other proteins The PMF Mascot score provided is the highest obtained for the protein/2D gel spot combination indicated.
The MS/MS Mascot score provided is the best single peptide score obtained for the protein/2D gel spot combination indicated
The protein score is the best total MS/MS score obtained for the indicated protein by LC- MALDI of SDS-PAGE bands g
The number of unique peptides identified by MS/MS is from the LC-MALDI data except for proteins identified from 2D gels only. Overlap of identical peptides is provided for TF0063 & TF0064, TF1053 & TF1057, TF2403 & TF2412, TF1259 & TF2339, TF1741 & TF2592, and TF2661-2 & TF2663. The protein with accession TF extra was identified from DNA sequence obtained from TIGR (www.tigr.org) and has not yet been annotated in the Oralgen database
Proteins identified from 2D gel spot numbers labeled (-) were only identified from a single 2D gel, and are not included in Fig 1 The BspA proteins TF 1843 and TF2998 could not be distinguished as their identification was based on a single overlapping peptide.
Table 2: Identification data for proteins identified from 2D gels in Example 2.
Master Accession Annotation category gel calc best best #MS/MS
Spot # MW MW PMF MS/MS (unique)
(kDa) (kDa)
Figure imgf000063_0001
Figure imgf000064_0001
subunit Signal
Table 3: Identification data for selected SDS-PAGE separated proteins.
Figure imgf000065_0001
Table 4: Additional identification data for CTD family proteins
Figure imgf000066_0001
Table 5: Antigenic proteins identified from Fig 7
Figure imgf000067_0001
TF2123 HP-C; TPR- Contains approx 8 TPR repeats. No repeat functional hits. Highly conserved protein
TF0091 outer SusE (BT) Id = 157/386 (40%), Pos = accessory LP membrane 218/386 (56%) to TDR. Part protein of S us operon
TF0945 HP-C; highly conserved. Surface protein accessory LP possible involved in nutrient binding. to TDR. surface protein
TF1331 outer Omp40, Id = 159/371 (42%), Pos = OmpA-like membrane 231/371 (62%) protein protein Omp41 , Id = 162/399 (40%), Pos = (similar to 238/399 (59%) Omp40/41 )
Table 6: Amino acid sequences
SEQ Gene ID Protein Sequence IDNO:
1 TF1342 Lipoprotein MKIKHAAALLLLGGISLFSGCNDDLTLVGSTIQPQGDRNTVYTDTFRMTAS
TVKRDSLYAKTTRGLLG El YDPLYGHLKSDYLCQFYCAEN YRFPHKPHQG
EVDSVKLNIRFYFWNGDGQALMRARAYAVNKPIEKNYYTNLNPADYCDM
QNVFGTQVYTTSGCFSDSVSTGRKDQKTNREIKQARYALKIALPREWGT
RFYNETVNNPSSFATQEAFNRFFPGLYITTDYGSGNILNVSDTQLYFYYR
RPKSSKNDTLVRDSVMFQVTKEVIQHSRFLHTDEEKLLTPNDQYAYVKTP
AGIYPRLVIPSREIAKTIKGRFTNNLMLSLKYDPQENWKYAMSPPPYLMLM
PEDSLRTFFENRSIENNATTFLSRDNTRVSTQYGYDPATRTYYFSNIINLL
NIHIKEKPDEDLRLLIVPVERDFATTTDRFGNVTGYYTRAIHNYLMLSGVK
FPIDQEHMKIVVVSNKYTGK
TF0090 conserved MKKWSIYRYMMLLCPVLILTACTEDFNGNVAAPQQWEQETAKTIGFKAEK hypothetic VGTIDLNTVASEKVKVCSITKPALTAGRVKGYVLYLNEKVTLEVDSLGQVK
TGALQDSIVSIYGKRPELRTLSGRLYALVALGEQTLRSDSVALTLAVKPKA al protein PFIDKAYYLIGNMNDWKADDVSKLIKLNQSGDVYENPVFSMILKVPENCY
WKVIPQTRVDAFTKGEAENVWGEGVLGCAVDGDESASGKLTVNGNAMKI
KNGGWTKIELNMLDYTYKVTVLGNVSPFLYVPGNHQGWSPATAPFVHST
DFMNYGGFVSLDGEFKFTSEKSWDGVNYGAGAKDGALSTDGGAGNLKA
EKGFYLLKANISSLTWSAVLIQTFGLIGSATDGGWDTSTPMTFDAAKSEY
SITAALKDGELKFRANDVWDVNLGGDPEHLTFGGANIAVKAGTYKITLSLS
DAQKYTCTIAKP
TF0091 outer MMKKNRNKIAVMLLAAGLSFVFASCEDDRNSNPTLQTPTKFVLNTPAYAG membrane SAVYDLENSSSVELTCSQPDYGFTAATVYSVQVSLDNDFTTEGKFTTLAT
TYTTAKMAIDATEIAVAQTTLALEKGITEDRFPLTSKLYVRLKAALTNGKGE protein IFSNTVTLRMRTKFALSPIVLPKTMNIVGSTIGNWKWEDCMEMVPTVAND
GTFWRILYFEKDAKMKFNIEKGWDGREFGGSATLEDHAGAGLSDAGGNL
RVAKAGWYLVVIRTAIEGRNLKYTVAFEKPNVYVVGHTMNGNWDTTDEK
RFTVPADAKGDFVSPAFLSTGEVRMCVKLDDADWWHSEFIVYADGKIAY
RGSGDDQERYTQPAGKRAYLNFMTGKGRYE
TF1331 outer MKTKVLLLAMLFGAALSVSAQQYQPQVGFSTENGSKTNFKKNKATDNMF membrane ISLAGGGNILFGDLNGNADFADRIAPSGAISIGKWYNPYMAFRLQVNGGK
MKNYSYVKDYKSDNAQDFWWINPHVDIMWDVTNFWAPYKESKVFRFIPF protein VGLGYALRPGYSDKNNNSFPRAESASINGGVQFMFRLGKRVDLFLEGQY
TLLGEHWNWDSHARPRYDRPVQAMLGLNFNLGRKEFEVLEPMDYDLLN
DLNSQI NALRAENAELSKRPEFCPECPKCPEVKEPRENLQNVVYF RLNSA
RIDKHQEVSIFNTAEYAKKHSLPIKLVGYADRKTGNPDYNKGISERRARAV
AKQLIDKYGISSDNISIEWMGDTVQPYAENAWNRVVIMNTDDK
TF2123 TPR- MKKIVISLFLMTGCCLTYGQRTHQFESPERLFNEGKELFHLRNYPGCSDK
LNAYKAQSTNRDLIQEADYMLACVAFGQDHPAAIEILENYLTTYPDTRHG repeat DEICFMLGSTSFAQENYQAAIEWFNRSEIDYLDEEQQEAYAFRMAYALLQ protein TDDLRTARNYFARIQQVGHKYKEASGYYLAYIDYATGNYEKALTGFNRLK
NSLTYREQALYYITQINFIENRYDRVIADGEELLRAYPNSANNSEIYRLLGN
AYYRNGEPSKAIDRLEKYVAHTDSVLRGDMYILGVCHYNQGNYDQAIEAL
TEAIDEEDALTQNAYLYLGQSYLKTNDKNKARMAFEMATTSEFDKQVQET
AMYNYALLIHETSFSGFGESVTIFEDFLNRFPDSKYTDKVNDYLAEVY
LTTKNYEAALASIEKIRQPGAKIQAARQNVLFRLGTQAFANQQPEKAVDY FNSAIALGNYDKEVYSDAYFWRGEAYYRQHNFSDAASDFRAFAANTPDR SEQ Gene ID Protein Sequence
ID NO:
ASDA YALAH YNLG YCYFKQKN YEAAHS AFRQYVDLEKNTSAISLADAYNR
IGDCLFHNRQFTSAEEQYTRAASLQPSSGDYALYQKGFLLGLQKDYKGKI
SLMDRVIREYPESPYADDALYEKGRSYVLMENYDLAADAFKELQQRFPQ
SSLARKSGLQLGLLYFNNNQPERSVEAYKKVISDYPGSEEAMTAVQDLKS
VYVDMNDVASYASYVNSLGNTSTRLGASEQDSLTYFAAEKLFMRGDNEG
AFRSLNNYLQQFPQGAFSPNANYYLAQIAFNQKNYDEARARYTAVAESG
NTKFLEESVARKAEVEYLQKDCAEAIKSFKWLAVIAETAHNREAARLGI M
RCARQTGQQTEALLAADELLKSSKLRPEVEAEARYLRAQSYLGLGEENK
ARVDLQALSKDTHTVYGAEAKYLLAQSYYDGNELDKAEKELLNFIEKGTT
HRYWLARGFVLLSDVYVRKGDKFQARQYLTSLQKNYKGNDDIAGMITER
LAKLK

Claims

THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS:
1. A method for determining whether an individual or periodontal site has a Tannerella infection including:
- selecting an individual or periodontal site;
-detecting whether the selected individual or site contains a protein, peptide or fragment thereof described in Table 1 or 2 herein, wherein detection of said protein, peptide or fragment thereof determines that the individual or site has a Tannerella infection,
thereby determining whether the individual or site has a Tannerella infection.
2. A method for determining risk of periodontal disease progression in an individual or periodontal site including:
- selecting an individual or periodontal site;
-detecting whether the selected individual or site contains a protein, peptide or fragment thereof described in Table 1 or 2 herein, wherein detection of said protein, peptide or fragment thereof determines the risk of periodontal disease progression in an individual or site;
thereby determining the risk of periodontal disease progression in an individual or site.
3. A method according to claim 1 or 2, wherein the protein, peptide or fragment thereof is CTD family protein, lipoprotein, outer membrane protein or involved in TonB- dependent transport.
4. A method according to claim 1 to 3, wherein the protein, peptide or fragment thereof is an outer membrane protein.
5. A method according to claim 1 to 3, wherein the protein, peptide or fragment thereof is a lipoprotein.
6. A method according to claim 1 or 2, wherein the protein, peptide or fragment thereof is selected from the group consisting of TF1342, TF0090, TF0091 , TF1331 TF2123.
7. A method according to any one of claims 1 to 6, wherein the Tannerella is Tannerella forsythia.
8. Use of a method of any one of claims 1 to 7 in any one of the following applications:
- determining whether an individual or periodontal site is susceptible to periodontal disease;
-determining whether an individual or periodontal site is likely to develop periodontal disease;
- screening for early stage periodontal disease;
- monitoring Tannerella infection in an individual or periodontal site receiving treatment for periodontal disease; or
- determining the risk of an individual or periodontal site developing periodontal disease.
9. A substantially non-glycosylated protein, peptide or fragment thereof described in Table 1 or 2 herein.
10. A substantially non-glycosylated protein, peptide or fragment according to claim
9. wherein the protein, peptide or fragment thereof is selected from the group consisting of TF1342, TF0090, TF0091 , TF1331 TF2123.
11. A composition comprising a substantially non-glycosylated protein, peptide or fragment thereof described in Table 1 or 2 herein and a carrier.
12. A composition according to claim 11 , wherein the protein, peptide or fragment thereof is selected from the group consisting of TF1342, TF0090, TF0091 , TF1331 TF2123.
13. A use of an antibody specific for a protein, peptide or fragment thereof described in Table 1 or 2 herein for determining whether an individual has a Tannerella infection.
14. A use according to claim 13, wherein the antibody is specific for TF1342, TF0090, TF0091 , TF1331 TF2123.
15. A use of a polynucleotide for detecting a nucleic acid that encodes or controls the expression of a protein, peptide or fragment thereof described in Table 1 or 2 herein for determining whether an individual or periodontal site has a Tannerella infection.
16. A kit for determining whether an individual or periodontal site has a Tannerella infection including:
- an antibody specific for a protein, peptide or fragment thereof described in Table 1 or 2 herein; or
- a polynucleotide capable of detecting a nucleic acid encoding or controlling the expression of a protein, peptide or fragment thereof described in Table 1 or 2 herein.
17. A kit according to claim 16 wherein the antibody is specific for a protein selected from the group consisting of TF1342, TF0090, TF0091 , TF1331 TF2123.
18. A use of an antigen in the form of:
- a protein, peptide or fragment thereof that is expressed in a Tannerella or;
- a protein, peptide or fragment thereof described in Table 1 or 2 herein
in the manufacture of means for determining whether an individual or periodontal site is infected with a Tannerella.
19. A use of an antibody specific for:
- a protein, peptide or fragment thereof that is expressed in a Tannerella or;
- a protein, peptide or fragment thereof described in Table 1 or 2 herein for determining whether an individual or periodontal site is infected with a Tannerella.
20. A use of a polynucleotide for detecting a nucleic acid that encodes or controls the expression of:
- a protein, peptide or fragment thereof that is expressed in a Tannerella or;
- a protein, peptide or fragment thereof described in Table 1 or 2 herein
in the manufacture of means for determining whether an individual or periodontal site is infected with a Tannerella.
21. A kit for determining whether an individual or periodontal site is infected with a Tannerella including:
- an antigen being a protein, peptide or fragment thereof that is expressed in a Tannerella; or
- an antigen being a protein, peptide or fragment thereof described in Table 1 or 2 herein; or
- an anW-Tannerella antibody specific for at least one of the above described antigens; or
- an antibody specific for an idiotype of an above described ar\t\-Tannerella antibody; or
- a polynucleotide capable of detecting a nucleic acid encoding or controlling the expression of at least one of the above described antigens.
22. An immune complex including an antigen being a protein, peptide or fragment thereof described in Table 1 or 2 herein bound to an antibody specific for said protein, peptide or fragment thereof.
PCT/AU2009/001495 2008-11-17 2009-11-17 Detection of tannerella forsythia infection Ceased WO2010054445A1 (en)

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