PATENT DOCUMENT

Abstract:
A vaccine for the treatment or prophylaxis of  C. difficile  associated disease comprises a  C. difficile  gene or a  C. difficile  peptide/polypeptide or a derivative or fragment or mutant or variant thereof which is immunogenic in humans. The gene encodes a  C. difficile  surface layer protein, SlpA or variant or homologue thereof. The peptide/polypeptide is a  C. difficile  surface layer protein, SlpA or variant or homologue thereof. The vaccine may comprise a chimeric nucleic acid sequence.

Full Description:
[0001]     This is a Continuation of application Ser. No. 10/068,870, filed Feb. 11, 2002. 
     
    
     INTRODUCTION  
       [0002]     The invention relates to vaccines to provide immunological protection against  C. difficile  infection.  
       BACKGROUND  
       [0003]      Clostridium difficile  is a common nosocomial pathogen and a major cause of morbidity and mortality among hospitalised patients throughout the world [Kelly et al., 1994]. Outbreaks of  C. difficile  have necessitated ward and partial hospital closure. With the increasing elderly population and the changing demographics of the population,  C. difficile  is set to become a major problem in the 21st century. The spectrum of  C. difficile  diseases range from asymptomatic carriage to mild diarrhoea to fulminant pseudomembranous colitis. Host factors rather than bacterial factors appear to determine the response to  C. difficile  [Cheng et al., 1997; McFarland et al., 1991; Shim et al., 1998].  
         [0004]     Reports indicate that hypogammaglobulinaemia in children appears to predispose to the development of disease due to  C. difficile  and that therapy with intravenously administered gamma globulin can be associated with the clinical resolution of chronic relapsing colitis due to  C. difficile  disease [Leung et al., 1991; Pelmutter et al., 1985]. A study by Mulligan et al. [1993] found elevated levels of immunoglobulins reactive with  C. difficile  in asymptomatic carriers as opposed to symptomatic patients. Recently it has been shown that patients who became colonised with  C. difficile  who had relatively low levels of serum IgG antibody against toxin A had a much greater risk of developing  C. difficile  diarrhoea [Kyne et al., 2000].  
         [0005]     It is clear that any advance in the understanding of  C. difficile  disease and methods of preventing or treating  C. difficile  diarrhoea (CDD) and other related diseases will be of major therapeutic potential.  
       STATEMENTS OF INVENTION  
       [0006]     According to the invention there is provided a vaccine for the treatment or prophylaxis of  C. difficile  associated disease, the vaccine comprising a  C. difficile  gene or a  C. difficile  peptide/polypeptide or a derivative or fragment or mutant or variant thereof which is immunogenic in humans.  
         [0007]     The invention also provides a vaccine for the treatment or prophylaxis of  C. difficile  associated disease, the vaccine comprising a  C. difficile  gene or  C. difficile  peptide/polypeptide or a derivative or fragment or mutant or variant thereof to which immunoreactivity is detected in individuals who have recovered from  C. difficile  infection.  
         [0008]     Preferably the gene encodes a  C. difficile  surface layer protein, SlpA or variant or homologue thereof.  
         [0009]     Preferably the peptide/polypeptide is a  C. difficile  surface layer protein, SlpA or variant or homologue thereof.  
         [0010]     Most preferably the vaccine comprises a chimeric nucleic acid sequence. Preferably the chimeric nucleic acid sequence is derived from the 5′ end of the gene, encoding the mature N-terminal moiety of SlpA from  C. difficile.    
         [0011]     In one embodiment of the invention the vaccine comprises a chimeric peptide/polypeptide. Preferably the amino acid sequence of the chimeric peptide/polypeptide is derived from the mature N-terminal moiety of SlpA from  C. difficile.    
         [0012]     Preferably the vaccine of the invention contains an amino acid sequence SEQ ID No. 1 or a derivative or fragment or mutant or variant thereof.  
         [0013]     Preferably the vaccine contains an amino acid sequence SEQ ID No. 2 or a derivative or fragment or mutant or variant thereof.  
         [0014]     In one embodiment of the invention the vaccine contains a nucleotide sequence SEQ ID No. 3 or a derivative or fragment or mutant or variant thereof; a nucleotide sequence SEQ ID No. 4 or a derivative or fragment or mutant or variant thereof; a nucleotide sequence SEQ ID No. 5 or a derivative or fragment or mutant or variant thereof; a nucleotide sequence SEQ ID No. 6 or a derivative or fragment or mutant or variant thereof; a nucleotide sequence SEQ ID No. 7 or a derivative or fragment or mutant or variant thereof; a nucleotide sequence SEQ ID No. 8 or a derivative or fragment or mutant or variant thereof; a nucleotide sequence SEQ ID No. 9 or a derivative or fragment or mutant or variant thereof or a nucleotide sequence SEQ ID No. 10 or a derivative or fragment or mutant or variant thereof.  
         [0015]     Preferably the vaccine of the invention is in combination with at least one other  C. difficile  sub-unit.  
         [0016]     The invention provides a vaccine for the treatment or prophylaxis of  C. difficile  associated disease, the vaccine comprising the mature N-terminal moiety of a surface layer protein, SlpA of  C. difficile  or variant or homologue thereof which is immunogenic in humans.  
         [0017]     Most preferably the N-terminal moiety of SlpA contains an amino acid sequence SEQ ID No. 1.  
         [0018]     In one embodiment of the invention the N-terminal moiety of SlpA contains an amino acid sequence SEQ ID No. 2.  
         [0019]     The invention also provides a vaccine for the treatment or prophylaxis of  C. difficile  associated disease, the vaccine comprising an immunodominant epitope derived from a  C. difficile  gene or a  C. difficile  peptide/polypeptide or a derivative or fragment or mutant or variant thereof which is immunogenic in humans.  
         [0020]     Preferably the vaccine of the invention comprises a pharmaceutically acceptable carrier. Most preferably the vaccine is in combination with a pharmacologically suitable adjuvant. Ideally the adjuvant is interleukin 12. Alternatively the adjuvant may be a heat shock protein.  
         [0021]     In one embodiment of the invention the vaccine comprises at least one other pharmaceutical product.  
         [0022]     The pharmaceutical product may be an antibiotic, selected from one or more metronidazole, amoxycillin, tetracycline or erythromycin, clarithromycin or tinidazole.  
         [0023]     In one embodiment of the invention the pharmaceutical product comprises an acid-suppressing agent such as omeprazole or bismuth salts.  
         [0024]     The vaccine of the invention may be in a form for oral administration, intranasal administration, intravenous administration or intramuscular administration.  
         [0025]     In one embodiment of the invention the vaccine includes a peptide delivery system.  
         [0026]     The invention also provides an immunodominant epitope derived from a  C. difficile  gene or a  C. difficile  peptide/polypeptide or a derivative or fragment or mutant or variant thereof. Preferably the  C. difficile  peptide/polypeptide contains an amino acid sequence SEQ ID No. 1 or SEQ ID No. 2 or a derivative or fragment or mutant or variant thereof.  
         [0027]     In one embodiment of the invention the  C. difficile  peptide/polypeptide contains an amino acid sequence SEQ ID No. 3 or SEQ ID No. 4 or SEQ ID No. 5 or SEQ ID No. 6 or SEQ ID No. 7 or SEQ ID No. 8 or SEQ ID No. 9 or SEQ ID No. 10 or a derivative or fragment or mutant or variant thereof.  
         [0028]     The invention further provides a chimeric nucleic acid sequence derived from the 5′ end of the slpA gene encoding the mature N-terminal moiety of SlpA from  C. difficile  which is immunogenic in humans.  
         [0029]     The invention also provides a chimeric peptide/polypeptide wherein the amino acid sequence of the chimeric peptide/polypeptide is derived from the mature N-terminal moiety of SlpA from  C. difficile.    
         [0030]     The invention provides a  C. difficile  peptide comprising SEQ ID No. 1 or SEQ ID No. 2 or SEQ ID No. 3 or SEQ ID No. 4 or SEQ ID No. 5 or SEQ ID No. 6 or SEQ ID No. 7 or SEQ ID No. 8 or SEQ ID No. 9 or SEQ ID No. 10.  
         [0031]     One aspect of the invention provides for the use of a  C. difficile  gene or a  C. difficile  peptide/polypeptide or a derivative or fragment or mutant or variant thereof which is immunogenic in humans in the preparation of a medicament for use in a method for the treatment or prophylaxis of  C. difficile  infection or  C. difficile  associated disease in a host.  
         [0032]     Preferably the medicament which is prepared is a vaccine of the invention.  
         [0033]     The invention also provides a method for preparing a vaccine for prophylaxis or treatment of  C. difficile  associated disease, the method comprising; 
        obtaining a  C. difficile  gene or a  C. difficile  peptide/polypeptide or a derivative or fragment or mutant or variant thereof which is immunogenic in humans; and     forming a vaccine preparation comprised of said gene or peptide/polypeptide or derivative or fragment or mutant or variant, which is suitable for administration to a host and which when administered raises an immune response.        
 
         [0036]     Preferably the  C. difficile  peptide/polypeptide contains an amino acid sequence SEQ ID No. 1 or SEQ ID No. 2 or a derivative or fragment or mutant or variant thereof.  
         [0037]     Most preferably the  C. difficile  gene contains an amino acid sequence SEQ ID No. 3 or SEQ ID No. 4 or SEQ ID No. 5 or SEQ ID No. 6 or SEQ ID No. 7 or SEQ ID No. 8 or SEQ ID No. 9 or SEQ ID No. 10 or a derivative or fragment or mutant or variant thereof.  
         [0038]     The invention further provides a method for prophylaxis or treatment of  C. difficile  associated disease, the method comprising; 
        obtaining a  C. difficile  gene or a  C. difficile  peptide/polypeptide or a derivative or fragment or mutant or variant thereof which is immunogenic in humans;     forming a vaccine preparation comprised of said gene or peptide/polypeptide or derivative or fragment or mutant or variant, and     administering the vaccine preparation to a host to raise an immune response.        
 
         [0042]     One aspect of the invention provides monoclonal or polyclonal antibodies or fragments thereof, to a  C. difficile  peptide/polypeptide or a derivative or fragment or mutant or variant thereof which is immunogenic in humans.  
         [0043]     Another aspect of the invention provides monoclonal or polyclonal antibodies or fragments thereof, to  C. difficile  peptide/polypeptide or a derivative or fragment or mutant or variant thereof to which immunoreactivity is detected in individuals who have recovered from  C. difficile  infection.  
         [0044]     The invention also provides purified antibodies or serum obtained by immunisation of an animal with a vaccine of the invention.  
         [0045]     The invention provides the use of the antibodies or fragments of the invention in the preparation of a medicament for treatment or prophylaxis of  C. difficile  infection or  C. difficile  associated disease.  
         [0046]     Preferably the antibodies or serum are used in the preparation of a medicament for treatment or prophylaxis of  C. difficile  infection or  C. difficile  associated disease.  
         [0047]     Most preferably the antibodies or fragments or serum of the invention are used in passive immunotherapy for established  C. difficile  infection.  
         [0048]     In one embodiment of the invention the antibodies or fragment or serum of the invention are used for the eradication of  C. difficile  associated disease.  
         [0049]     The invention also provides use of interleukin 12 as an adjuvant in  C. difficile  vaccine.  
         [0050]     The invention further provides use of humanised antibodies or serum for passive vaccination of an individual with  C. difficile  infection. 
     
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
       [0051]     The invention will be more clearly understood from the following description thereof given by way of example only with reference to the accompanying figures, in which:— 
         [0052]      FIG. 1A  is a Western blot showing recognition of antigens from a crude extract of  C. difficile  171500 (PCR type 1) by serum antibodies from a patient infected with this strain. Lane 1: Pre-infection; Lane 2: Early acute; Lane 3: Late acute; Lane 4: Convalescent;  
         [0053]      FIG. 1B  is a Western blot showing recognition of antigens from a crude extract of  C. difficile  170324 (PCR type 12) by serum antibodies from a patient infected with this strain. Lane 1: Pre-infection; Lanes 2-5: Acute; Lanes 6-7: Convalescent;  
         [0054]      FIG. 2 . is a Western blot showing recognition of antigens from two  C. difficile  strains of different type by serum from convalescent patients. 
        Lane 1: Strain 170324 (PCR type 12), crude antigen preparation     Lane 2: Strain 170324, surface layer protein preparation     Lane 3: Strain 171500 (PCR type 1), crude antigen preparation     Lane 4: Strain 171500, surface layer protein preparation.     Molecular mass markers (kDa) are shown on the left; and          
         [0060]      FIG. 3  is an SDS-PAGE gel showing crude SLP preparations from selected strains of  C. difficile . The gel contains 12% acrylamide, and has been stained for protein with Coomassie Blue. Each lane contains 5 μg of protein. Molecular weight markers are shown on the left. 
        Lane 1: 171500 (PCR type 1)     Lane 2: 172450 (PCR type 5)     Lane 3: 170324 (PCR type 12)     Lane 4: 171448 (PCR type 12)     Lane 5: 171862 (PCR type 17)     Lane 6: 173644 (PCR type 31)     Lane 7: 170444 (PCR type 46)     Lane 8: 170426 (PCR type 92)         
     
    
     DETAILED DESCRIPTION OF THE INVENTION  
       [0069]     Two antigenic peptides containing SEQ ID No. 1 and SEQ ID No. 2, associated with two common infecting types of  C. difficile , were found to be immunogenic in humans. The antigenic peptides were found to induce a strong immune response in individuals who recover from  C. difficile  infection. Individuals who have recovered from  C. difficile  infection are those individuals who have been exposed to  C. difficile  or something strongly related and have recovered. This includes individuals where a carrier state exists in that the  C. difficile  infection has not and will not necessarily become clinically significant.  
         [0070]     These antigenic peptides were found to be products of the slpA gene from  C. difficile  which is the structural gene for the surface layer protein, SlpA. The gene or its products are therefore ideal candidates for the preparation of vaccines against  C. difficile.    
         [0071]     Surface layer proteins (SLPs), also known as S-layers or crystalline surface layers, are associated with a wide range of bacterial species. They form a 2-dimensional array, which covers the surface of the cell completely, and grows with the cell [Sleytr et al., 1993]. The molecular weight can range from 40 000 to 200 000 Da. The proteins are typically acidic, contain a large proportion of hydrophobic amino acid residues, and have few or no sulphur-containing amino acid residues. Glycosylated S-layer proteins occur in some species. The precise function of S-layers is not always known, but since they comprise approximately 15% of the cell protein, it seems likely that they are important for in vivo functioning of the organism. In Gram positive organisms, the SLP has been shown to delay or prevent the excretion of degradative enzymes from the cell to the outside milieu, and may thereby create a space analagous to the periplasmic space of Gram negative bacteria. Many pathogenic species possess SLPs, which have been ascribed functions such as antiphagocytosis ( Campylobacter fetus ), and inhibition of complement-mediated killing ( Aeromonas salmonicida ).  
         [0072]     Kawata et, al. [1984] described the SLPs of  Clostridium difficile . They showed the S-layer to be composed of 2 polypeptides, and demonstrated size heterogeneity for the polypeptides from different strains. Delmée et al. [1986] showed that crude extracts from  C. difficile  strains of different serotype showed different polypeptide profiles in SDS-PAGE. Poxton et al. [1999] made similar observations using purified SLP preparations. Slide agglutination [Delmée et al., 1990] has identified 21 different serotypes, apparently distinguished by the heterogeneity of the SLP.  
         [0073]     Pantosti et al. [1989] isolated  C. difficile  from a number of patients with antibiotic-associated diarrhoea, and prepared SLPs from them. Cerquetti et al. [2000] published N-terminal sequences of SLPs from several strains, indicating wide differences between strains. In 2000 the complete DNA sequence of the  C. difficile  genome was published (available at web address http://www.sanger.ac.uk/Projects/C_difficile/).  
         [0074]     The peptides of the invention were found to be encoded by a single open reading frame (ORF) named slpA from  C. difficile . The peptides identified in our clinical study correspond to a lower molecular weight moiety of the slpA gene product. Since an immune response is also mounted against a higher molecular weight slpA gene product ( FIG. 2 ), this entity may also be included in a vaccine.  
         [0075]     The slpA gene has been sequenced from a number of strains corresponding to different PCR types. The sequences of strains 171500 (PCR type 1)(NCIMB 41081; PHLS R13537), 172450 (PCR type 5)(PHLS R12884), 170324 (PCR type 12) (NCIMB 41080; PHLS R12882), 171448 (PCR type 12) (PHLS R13550), 171862 (PCR type 17) (PHLS R13702), 173644 (PCR type 31) (PHLS R13711), 170444 (PCR type 46) (PHLS R12883) and 170426 (PCR type 92) (PHLS R12871) with translations thereof are given in Appendices 1 to 8. Substantial variation in nucleotide and predicted amino acid sequence was found between strains of PCR types 1, 5, 12, 17 and 31. The genes from strains of PCR types 46 and 92 are almost identical in sequence to those of PCR type 12. When the DNA sequences of genes of different strains within a PCR type are compared, the sequences are almost if not quite identical, indicating that the potential for variation is not infinite. These findings are in agreement with serotyping studies [Delmée et al., 1986, 1990], and indicate that the production of an effective vaccine based on the slpA product is feasible. In this respect, the present invention includes all variant slpA genes and their products, individually and combined, fragments of them, and their mutants and derivatives.  
         [0076]     One aspect of the invention provides the combination of immunodominant eptopes from the slpA gene products from various serotypes into a single vaccine. In this way a single vaccine may be used to immunise against several different  C. difficile  strains.  
         [0077]     The most common PCR types isolated from infections in the clinical study carried out at St. James&#39;s Hospital, Dublin, Ireland were PCR types 1 and 12. However, a vaccine which elicits an intense antibody response against many infecting types would be therapeutically very valuable. Recombinant DNA chimera, or several chimeras, encoding contiguous immunodominant epitopes may be made for use in the vaccine. The recombinant DNA may serve as the active component in a vaccine, or may be inserted into an appropriate expression system for the generation of a chimeric peptide vaccine in a suitable host.  
         [0078]     Chimeras can be generated by PCR amplification of the DNA encoding peptide regions of interest, incorporating cleavage sites for restriction endonucleases into the primers. The amplified fragments can thus be cleaved to generate compatible ends, and spliced together to create chimeras.  
         [0079]     The dominant epitopes may be identified by cleavage of the slpA products into fragments by agents which cleave at known sites, and by immunoblotting with homologous patient serum. Immunodominant peptides may be tested for their capacity to stimulate T-cell proliferative responses in vitro, using mouse splenic T-cells.  
         [0080]     DNA vaccination involves immunisation with recombinant DNA encoding the antigen or epitope of interest, cloned in a vector which promotes high level expression in mammalian cells. Typically, the vector is a plasmid vector which which also replicates in a procaryotic vector such as  Escherichia coli , so that the DNA can be produced in quantity. Following immunisation, the plasmid enters a host cell, where it remains in the nucleus, and directs synthesis of the recombinant polypeptide. The polypeptide stimulates the production of neutralising antibodies, as well as activating cytotoxic T-cells.  
         [0081]     Using a DNA vaccine, it may be necessary to modify the DNA sequence to take account of codon usage in humans. The G+C content of mammalian DNA is much higher than that of  C. difficile . The generation of such synthetic DNA molecules, essentially containing numerous silent mutations, is within the scope of the invention.  
         [0082]     A peptide vaccine will ideally be made using recombinant peptides. Similar considerations apply as in the generation of a DNA vaccine with regard to expression in a different host, such as  Escherichia coli , which has a different codon usage pattern to  C. difficile . Problems of expression may be overcome by the use of a special host strain which carries additional copies of rare tRNAs (e.g.  E. coli  BL21-CodonPlus™-RIL from Stratagene), or by using de novo synthesis of a DNA segment carrying silent mutations which will enable normal expression in  E. coli . There are many expression systems which are likely to allow high-level expression of slpA genes in  E. coli . An example is the pBAD/Thio TOPO vector of Invitrogen, in which expressed genes are under control of the arabinose promoter, which is subject to positive and negative control, enabling very tight control of expression. In this vector, the recombinant protein is typically fused to a modified thioredoxin carrying several histidine residues which enable purification by nickel chromatography. The recombinant protein can be cleaved from the thioredoxin moiety by enterokinase enzyme.  
         [0083]     Affinity chromatography may also be used with fixed antibodies or some other agent which strongly binds the peptide of interest to purify the protein from the native organism.  
         [0084]     Purified immunogenic peptides may be used in combination with other  C. difficile  sub-units as a combined vaccine against  C. difficile . Potential candidates are the products of the other sip genes, which share limited homology with the slpA gene product and with the N-acetylmuramoyl L-alanine amidase, (CwlB), from  Bacillus subtilis , and which may be involved in remodelling of the peptidoglycan.  
         [0085]     Other purified proteins of  C. difficile  to which constitutive antibodies are detected in individuals recovering from  C. difficile  infection are also within the scope of the present invention  
         [0086]     A deposit of  Clostridium difficile  strain 171500, PCR type 1, was made at the NCIMB on Jan. 29, 2001, and accorded the accession number NCIMB 41081.  
         [0087]     A deposit of  Clostridium difficile  strain 170324, PCR type 12, was made at the NCIMB on Jan. 29, 2001, and accorded the accession number NCIMB 41080.  
         [0088]     Two peptides of the invention were found to contain the following sequences:  
                                           33kDa peptide                   SEQ ID No. 1:   DKTKVETADQGYTVVQSKYK                       31kDa peptide           SEQ ID No. 2   ATTGTQGYTVVKNDGKKAVK          
 
         [0089]     The invention will be more clearly understood from the following examples.  
       EXAMPLE 1  
     Clinical Study  
       [0090]     Examination of sequential antibody responses to  C. difficile  among elderly patients who developed the disease was carried out. The study was based on the hypothesis that the host immune response influenced the development of  Clostridium difficile  disease. In particular we determined that a particular pattern of immune response to  C. difficile  antigens correlated with the outcome of CDD.  
         [0000]     Materials and Methods  
         [0000]     Patients  
         [0091]     Serum was collected from over 300 patients and of these 30 patients developed CDD. The infecting strain (homologous strain) was grown from each patient. Strains of  C. difficile  were typed at the Anaerobe Reference Laboratory, Wales [O&#39;Neill et al., 1996]. The most common strains isolated were PCR type 1 (n=15) which is the most common type causing epidemics and PCR type 12 (n=5) which is also a common hospital strain. Pre-infection serum samples were obtained from patients. Acute phase sera were then collected from patients who developed  C. difficile  disease. Convalescent sera were collected from patients who recovered. Protein extracts of patients&#39; infecting  C. difficile  strain were probed with the patients sera using Western blotting. IgG responses to the antigens were examined.  
         [0000]     Western Blotting  
         [0092]     Proteins from SDS-PAGE gels were electroblotted (0.8 mA/cm2 for 1 h) to PVDF membrane using a semi-dry blotting apparatus (Atto). Primary antibodies (human serum: 1/50-1/10,000 dilution) were detected using a 1/5000 dilution of anti-human IgG (horse radish peroxidase-conjugated) in combination with enhanced chemiluminesence (ECL). Blots were washed in phosphate buffered saline (pH 7.5) containing Tween 20 (0.1% v/v), and incubated in the same solution comprising dried skim milk (5% w/v) and antibodies at the appropriate concentration. Blots were exposed to Kodak X-OMAT film for various periods of time and developed.  
         [0000]     Results  
         [0093]     Overall 5 patients made a full recovery and new antibody responses to previously unrecognised antigens were evident in 4 of these patients. Three of these patients had  C. difficile  belonging to PCR type I and one patient had  C. difficile  PCR type 12. These patients developed an acute phase antibody response to previously unrecognised  C. difficile  antigens which persisted during convalescence ( FIGS. 1A and 1B ). These antigens were recognised by antibodies from patients who recovered and represent potential candidate vaccine antigens.  FIG. 1A  shows a strong reaction of convalescent antibodies was observed with the 33 kDa antigen (Lane 4, arrow).  FIG. 1B  shows a strong reaction of convalescent antibodies was observed with the 31 kDa antigen (Lanes 6 and 7, arrow).  
         [0094]     These antibody responses have also been found in some controls in the same ward who were also on antibiotics but who did not develop CDD.  
       EXAMPLE 2  
     Further Characterisation of Protective Antigens  
       [0000]     Materials and Methods  
         [0095]     Partial purification and N-terminal sequencing of the 33 kDa and the 31 kDa proteins The antigens were partially purified from  C. difficile  based on their molecular weight using preparative continuous-elution SDS-PAGE on a model 491 Prep-Cell (Bio-Rad). The appropriate antigens were subsequently identified on Western blots probed with serum obtained from individuals who recovered from  C. difficile  infection.  
         [0000]     Preparation of Surface Layer Proteins (SLPs)  
         [0096]     SLPs were purified from  C. difficile  by extracting washed cells with 8 M urea, in 50 mM Tris HCl, pH 8.3 in the presence of a cocktail of protease inhibitors (Complete®, Boehringer Mannheim), for 1 h at 37° C., followed by centrifugation for 19 000×g for 30 min. The SLPs were recovered in the supernatant and dialysed to remove the urea [Cerquetti et al., 2000].  
         [0000]     Results  
         [0097]     The immunodominant protein which was associated with a positive outcome from  C. difficile  strain 171500 (PCR type 1) was identified and purified using preparative SDS-PAGE. The N-terminal region of the protein was sequenced using an Applied Biosystems Procise Sequencer, viz DKTKVETADQGYTVVQSKYK (SEQ ID No. 1)  
         [0098]     The antigen which was associated with a protective antibody response from the  C. difficile  strain 170324 (PCR type 12) was identified and the N-terminal sequence obtained, viz ATTGTQGYTVVKNDGKKAVK (SEQ ID No. 2).  
         [0099]     These sequences were used to interrogate the  C. diffcile  genome sequence using the TBLASTN programme, which compared our query sequences with those of the genome project (available at web address http://www.sanger.ac.uk/Projects/C_difficile/), translated in all 6 possible reading frames. A nearly identical stretch of sequence was identified when the sequence from strain 1710324 (type 12) was used for interrogation. The same stretch of sequence was picked up with the sequence from strain 171500 (type 1) was used, although the identity was much less strong. Since the homologous sequence belonged to an open reading frame encoding a 719-residue peptide, this result was somewhat surprising. However, when the N-terminal sequences from the higher molecular weight SLP component were later published by Cerquetti et al [2000], it became apparent that they were encoded downstream along the same gene, subsequently identified as slpA, and the reason for the discrepancy in size between the gene and its products became readily apparent.  
         [0100]     The purified SLPs from strains 171500 (PCR type 1) and 170324 (PCR type 12) showed strong reactivity with homologous convalescent serum, and co-migrated with the dominant antigens detected in crude cell extracts as shown in  FIG. 2 . Lanes 1 and 3 contain crude antigen preparations from PCR types 1 and 12 respectively, and Lanes 2 and 4 contain SLP preparations from PCR types 1 and 12, respectively. Panel A was probed with serum from a patient recovering from infection with PCR type 1, and Panel B was probed with serum from a patient recovering from infection with PCR type 12. Each serum detected 2 major antigens in the infecting strain (Panel A, Lane 3); (Panel B, Lane 1), which co-migrated with the 2 SLPs (Panel A, Lane 4; Panel B, Lane 2), with which the sera also reacted strongly. Note that serum from the patient infected with the PCR type 1 strain recognised the higher molecular weight SLP from the PCR type 12 strain (Panel A, Lanes 1 and 2), whereas the converse did not occur (Panel B, Lanes 3 and 4). There is no apparent antigenic cross-reactivity with regard to the lower molecular weight SLPs.  
         [0101]     SLPs were prepared from selected strains by urea extraction, and subjected to SDS-PAGE and staining with Coomassie Blue ( FIG. 3 ). Most strains showed a characteristic profile, with two major bands located in the 29 000 to 36 000 and 45 000 to 50 000 molecular weight range. An exception was strain 172450 ( FIG. 3 , Lane 2), which showed a single, high molecular weight band, approximately 43 000 in size.  
         [0000]     Cloning, Sequencing and Analysis of slpA Genes  
         [0102]     The nucleotide sequences of the slpA genes from the two sample strains of  C. difficile  (PCR types 1 and 12, deposited at the NCIMB) and of several others (PCR types 5, 12, 17, 31, 46 and 92, available from the Anaerobe Reference Unit at the Department of Medical Microbiology and Public Health Laboratory, Cardiff, Wales were obtained. The slpA gene and flanking sequence was amplified by polymerase chain reaction from genomic DNA prepared from  C. difficile  using a commercial kit (Puregene® DNA isolation kit for yeast and Gram positive bacteria, Gentra systems Minneapolis, Minn.). The forward primer (5′ ATGGATTATTATAGAGATGTGAG 3′), was based on sequence from the genome sequencing project, starting 112 nucleotides upstream from the start of the slpA open reading frame. Two reverse primers were used, depending on the PCR type. A downstream primer (5′ CTATTTAAAGTTTTATTAAAACTTATATTAC 3′) was used to amplify slpA from PCR types 12, 17, 31, 46 and 92. A reverse primer based on the 3′ end of the slpA open reading frame from strain 630 and the subsequent nonsense codon (5′ TTACATATCTAATAAATCTTTCATTTTGTTTATAACTG 3′) was used to amplify slpA from PCR types 1 and 5. The choice of primer for the latter two PCR types may have resulted in a small number of systematic errors in the nucleotide sequence obtained. PCR was carried out using HotStar™ Taq polymerase (Qiagen Ltd., Crawley, West Sussex, UK) according to the manufacturer&#39;s instructions. A single fragment of approximately 2 kb was obtained for each strain, which was then cloned into the pBAD/Thio TOPO vector (Invitrogen, Groningen, Netherlands). Inserts were sequenced from both ends by standard procedures in commercial facilities at MWG (Wolverton Mill South, Milton Keynes, UK) and Cambridge University. New primers were designed on the basis of initial sequencing results, enabling sequencing of both strands to be completed (a process known as chromosome walking).  
         [0103]     The results are shown in Appendices 1-8.  
         [0104]     The nucleotide sequences were translated to enable prediction of the amino acid sequence(s) of the product(s) (Appendices 1-8). The N-terminal sequences obtained experimentally for the low molecular weight protective antigens from strains 171500 (PCR type 1) and 170324 (PCR type 12) were almost identical to those predicted from the nucleotide sequences of their respective slpA genes (18/20 identical residues for strain 171500, and 19/20 identical residues for strain 170324).  
         [0105]     Appendix 1 shows the open reading frame with translation for slpA from strain 171500 (PCR type 1), SEQ ID No 3. Since the reverse primer was based on the 35 nucleotides from the 3′ end of the s/pa gene, the sequence is not necessarily 100% accurate in this region. However, this part of the gene does not seem to vary greatly from strain to strain.  
         [0106]     Appendix 2 shows the open reading frame with translation for slpA from strain 172450 (PCR type 5), SEQ ID No 4. Again, the sequence obtained for the 3′ 35 nucleotides is not fully reliable. This gene is considerably smaller than the other slpA genes sequenced, and shows strong sequence divergence from the other PCR types examined.  
         [0107]     Appendix 3 shows the open reading frame with translation for slpA from strain 170324 (PCR type 12), SEQ ID No 5. This gene showed a single base difference when compared with the strain used for the genome sequencing project, strain 630, of the same PCR type. The deduced amino acid sequence is identical.  
         [0108]     Appendix 4 shows the open reading frame with translation for slpA from strain 171448 (PCR type 12), SEQ ID No 6. This gene was almost identical in sequence to that from strain 170324.  
         [0109]     Appendix 5 shows the open reading frame with translation for slpA from strain 171862 (PCR type 17), SEQ ID No 7.  
         [0110]     Appendix 6 shows the open reading frame with translation for slpA from strain 173644 (PCR type 31), SEQ ID No 8. Like the slpA from strain 172450, this sequence is very dissimilar to those of slpA genes from other PCR types encountered.  
         [0111]     Appendix 7 shows the open reading frame with translation for slpA from strain 170444 (PCR type 46), SEQ ID No 9. This sequence is virtually identical to that obtained for slpA from PCR type 12 and 92 strains.  
         [0112]     Appendix 8 shows the open reading frame with translation for slpA from strain 170426 (PCR type 92), SEQ ID No 10. This sequence is virtually identical to that obtained for slpA from PCR type 12 and 46.  
         [0113]     The cleavage site of the putative signal sequences from both genes was determined from experimental evidence (the N-terminal sequence of the mature proteins as determined by Edman degradation), and by the prediction tool of the Centre for Biological Sequence Analysis at the Technical University of Denmark [Nielsen et al., 1997]. The site for cleavage of the slpA gene product to form the mature SLPs was predicted from experimental [Cerquetti et al., 2000, Karjalainen et al., 2001 and Calabi et al., 2001]. The cleavage site is typically preceded by the motif TKS. However, the relevant motif is likely to be TKG in strain 173644 (PCR type 31). No obvious motif appeared for strain 172450 (PCR type 5). However, the protein produced by type 5 strains does appear to be cleaved; hence we predicted the site to occur at a point where the SLP sequence aligns with the cleavage sites of other PCR types.  
         [0114]     The molecular weight and isoelectric point was calculated for each of the predicted mature proteins by the ExPASy server of the Swiss Institute for Bioinformatics (Table 1). In general, the calculated molecular weights were in fair agreement with apparent molecular masses determined from migration in gels ( FIG. 3 ). No lower molecular weight band was apparent for Strain 172450 (PCR type 5; Lane 2). However, a higher molecular weight band is present, which is similar in size to the predicted weight for the C-terminal moiety. We observed a similar profile for another type 5 strain. It is possible that the lower molecular weight species is subject to degradation in this strain. Another possibility is that it is heavily glycosylated, which can affect staining. All peptides had a predicted isoelectric point below 7, typical of acidic proteins, and characteristic of SLPs in general [Sleyter et al, 1993].  
                               TABLE 1                                       MW         C. difficile  strain   pI   pI   MW   (C-       (PCR type)   (N-terminal)   (C-terminal)   (N-terminal)   terminal)                   171500 (Type 1)   4.83   4.66   33365.41   44220.37       172450 (Type 5)   4.86   4.65   19364.46   42757.63       170324 (Type 12)   4.92   4.58   34228.25   39522.24       171448 (Type 12)   4.98   4.58   34156.18   39492.21       171862 (Type 17)   5.09   4.53   33783.73   39407.11       173644 (Type 31)   5.05   4.56   33626.48   41821.69       170444 (Type 46)   5.06   4.58   34230.31   39522.24       170426 (Type 92)   4.99   4.58   34242.32   39522.24                  
 
         [0115]     The translated nucleotide sequences were compared with published SlpA sequences (EMBL Accession numbers AJ300676, and AJ300677 for examples from PCR types 1, and 17 respectively; strain 630 available from the Sanger Institute for PCR type 12; EMBL Accession number AY004256 for a variant from an unnamed PCR type). The Clustal W alignment programme, which is freely available, was used. Where SlpA sequences from our isolates were compared with those of other strains of the same PCR types, they were found to be nearly or quite identical. This observation indicates, together with existing knowledge from serotyping, that the number of variants of slpA is not infinite, and that natural evolution of the gene is not rapid. Table 2 shows a compilation of homologies, based on amino acid residue identity, for the different translated sequences measured against published sequences. Homologies are compiled for the predicted mature peptides, either combined (Table 2A) or as N-terminal (low molecular weight, less conserved moiety) (Table 2B) and C-terminal (high molecular weight, more conserved) (Table 2C) mature peptides according to predicted cleavage sites. It is clear that the SlpA sequences from strains 172450 (PCR type 5) and 173644 (PCR type 31) are quite distinct particularly with respect to N-terminal region.  
                                                     TABLE 2A                           630   AJ300676   AJ300677   AY004256       Strain.type   (type 12)   (type 1)   (type 17)   (type unknown)                                171500.type1   55.2   99.7   55.4   56.42       172450.type5   49.8   54.0   49.9   47.77       170324.type12   100.0   57.8   81.7   59.77       171448.type12   99.7       171862.type17   82.3   58.7   100   57.54       173644.type31   57.9   59.2   60.1   56.88       170444.type46   99.6       170426.type92   99.9                  
 
         [0116]    
       
         
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 2B 
               
               
                   
               
               
                   
               
               
                   
                 630 
                 AJ300676 
                 AJ300677 
                 AY004256 
               
               
                 Strain.type 
                 (type 12) 
                 (type 1) 
                 (type 17) 
                 (type unknown) 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 171500.type1 
                 35.4 
                 100 
                 34.5 
                 33.54 
               
               
                 172450.type5 
                 31.6 
                 32.2 
                 31.0 
                 24.58 
               
               
                 170324.type12 
                 100 
                 34.9 
                 64.6 
                 36.14 
               
               
                 171448.type12 
                 99.7 
               
               
                 171862.type17 
                 64.3 
                 34.4 
                 100 
                 31.55 
               
               
                 173644.type31 
                 37.5 
                 34.1 
                 41.3 
                 31.86 
               
               
                 170444.type46 
                 99.1 
               
               
                 170426.type92 
                 99.7 
               
               
                   
               
             
          
         
       
     
         [0117]    
       
         
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 2C 
               
               
                   
               
               
                   
               
               
                   
                 630 
                 AJ300676 
                 AJ300677 
                 AY004256 
               
               
                 Strain.type 
                 (type 12) 
                 (type 1) 
                 (type 17) 
                 (type unknown) 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 171500.type1 
                 70.2 
                 99.5 
                 71.2 
                 73.80 
               
               
                 172450.type5 
                 58.4 
                 60.4 
                 63.0 
                 57.60 
               
               
                 170324.type12 
                 100 
                 77.3 
                 97.1 
                 80.00 
               
               
                 171448.type12 
                 99.7 
               
               
                 171862.type17 
                 97.3 
                 78.8 
                 100 
                 79.62 
               
               
                 173644.type31 
                 74.1 
                 78.9 
                 75.1 
                 75.38 
               
               
                 170444.type46 
                 100 
               
               
                 170426.type92 
                 100 
               
               
                   
               
             
          
         
       
     
         [0118]     The term antibody used throughout the specification includes but is not limited to polyclonal, monoclonal, chimeric, single chain, Fab fragments and fragments produced by a Fab expression library.  
         [0119]     The antibodies and fragments thereof may be humanised antibodies. Neutralising antibodies such as those which inhibit biological activity of the substance amino acid sequence are especially preferred for diagnostics and therapeutics.  
         [0120]     Antibodies both polyclonal and monoclonal which are directed against epitopes obtainable from a polypeptide or peptide of the present invention are particularly useful in diagnosis and those which are neutralising are useful in passive immunotherapy.  
         [0121]     Antibodies may be produced by any of the standard techniques well known in the art.  
         [0122]     A therapeutically effective amount of the polypeptide, polynucleotide, peptide or antibody of the invention in the form of pharmaceutical composition may be administered. The composition may optionally comprise a pharmaceutically acceptable carrier, diluent or excipients and including combinations thereof. The pharmaceutical composition may be used in conjugation with one or more additional pharmaceutically active compounds and/or adjuvants.  
         [0123]     Different adjuvants depending on the host may be used to increase immunological response. The adjuvant may be selected from the group comprising Freunds, mineral gels such as aluminium hydroxide and surface active substances.  
         [0124]     The vaccine of the invention may be in the form of an immune modulating composition or pharmaceutical composition and may be administered by a number of different routes such as by injection (which includes parenteral, subcutaneous and intramuscular injection) intranasal, intramuscular, mucosal, oral, intra-vaginal, urethral or ocular administration. There may be different formulation/composition requirements dependent on the different delivery systems.  
         [0125]     The invention is not limited to the embodiments hereinbefore described which may be varied in detail.  
       REFERENCES  
       [0000]    
       
          Calabi E., Ward S., Wren B., Paxton T., Panico M., Morris H., Dell A., Dougan G., Fairweather N. (2001). Molecular characterization of the surface layer proteins from  Clostridium difficile . Mol. Microbiol. 40:1187-1199.  
          Cerquetti M., Molinari A., Sebastianelli A., Diociaiuti M., Petruzzelli R., Cap C., Mastrantonio P. (2000). Characterization of surface layer proteins from different  Clostridium difficile  clinical isolates. Microbial Pathogenesis, 28:363-372.  
          Cheng S. H, Lu J. J, Young T. G, Perng C. L, Chi W. M. (1997)  Clostridium difficile -associated diseases: comparison of symptomatic infection versus carriage on the basis of risk factors, toxin production, and genotyping results.  Clin Infect Dis;  25: 157-8.  
          Delmée M., Laroche Y., Avesani V., Cornelis G. (1986). Comparison of serogrouping and polyacrylamide gel electrophoresis for typing  Clostridium difficile . J. Clin. Microbiol. 24:991-994.  
          Delmée M., Avesani V., Delferrière N., Burtonboy G. (1990). Characterization of flagella of  Clostridium difficile  and their role in serogrouping reactions.  
          Karjalainen T., Waligora-Dupriet A.-J., Cerquetti M., Spigaglia P., Maggioni A., Mauri P., Mastrantonio P., (2001). Molecular and genomic analysis of genes encoding surface-anchored proteins from  Clostridium difficile . Infect. Immun. 69:3442-3446.  
          Kawata T., Takeoka A., Takumi K., Masuda K. (1984). Demonstration and preliminary characterization of a regular array in the cell wall of  Clostridium difficile . FEMS Microbiol. Lett 24:323-328.  
          Kelly, C. P., Pothoulakis C and LaMont J. T.  Clostridium difficile  colitis.  New England Journal of Medicine.  1994 330: 257-262.  
          Kyne L, Warny M, Qamar A, Kelly C. Asymptomtic carriage of  Clostridium difficile  and serum levels of IgG antibody against Toxin A.  New England Journal of Medicine  2000; 390-7.  
          Leung Y. M, Kelly C. P, Boguniewicz M, Pothoulakis C, LaMont J. T, Flores A. Treatment with intravenous gamma globulin of chronic relapsing colitis by  Clostridium difficile ; toxin:  J. Pediatr  1991; 118: 633-7.  
          McFarland L. V, Elmer G. W, Stamm W. E, Mulligan M. E. Correlation of immunoblot type, enterotoxin production, and cytokine production with clinical manifestation of  Clostridium difficile  infection in a cohort of hospitalised patients.  Infect Immnun.  1991; 59: 2456-62.  
          Mulligan M. E, Miller S. D, McFarland L. V, Fung H. C, Kwok R. Y. Elevated levels of serum immunoglobulins in asymptomatic carriers of  Clostridium difficile. Clin Infect Dis  1993: 16(Suppl 4); S239-44.  
          Nielsen H., Engelbrecht J., Brunak S., von Heijne G. (1997). Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Protein Eng. 10:1-6.  
          O&#39;Neill G. L., Ogunsota F. T, Brazier J. S, Duerdon B. I, Modification of a PCR ribotyping method for application as a routine typing scheme for  Clostridium difficile. Anaerobe  (1996) 2, 205-209.  
          Pantosti A, Cerquetti M, Viti F, Ortisi G, Mastratonio P. Immunoblot Of Serum Immunoglobulin G Response to Surface Proteins of  Clostridium difficile  in Patients With Antibiotic Associated Diarrhoea.  J. Clin Microbiol  1989: 27; 2594-7.  
          Pelmutter D. H, Leichtnr A. M, Goldman H, Winter H. S. Chronic diarrahoea associated with hypogammaglobulinaemia and enteropathy in infants and children:  Dig Dis Sci  1985; 30; 1149-55.  
          Poxton I. R., Higgins P. G., Currie C. G., McCoubrey J. (1999). Variation in the cell surface proteins of  Clostridium difficile . Anaerobe 5:213-215.  
          Shim J. Johnson S, Samone M, Bliss D Z, Gerding D. N. Primary symptomless colonisation by  Clostridium difficile  and decreased risk of subsequent diarrhoea.  The Lancet  Vol 351 1998: 633-5.  
       
     
         [0144]     Sleytr U. B., Messner P., Pum D., Sára M. (1993). Crystalline bacterial cell surface layers. Mol. Microbiol. 10:911-916.  
                                     APPENDIX 1                       SEQ ID No. 3. Nucleotide sequence of slpA from  Clostridium               difficile  strain 171500, PCR type 1, with translation. The       putative secretory signal cleavage site (□) and site of       cleavage to form the two mature SLPs (♦) are indicated.                                1   ATGAATAAGAAAAATATAGCAATAGCTATGTCAGGTTTAACAGTTTTAGCTTCGGCTGCA   60               ---------+---------+---------+---------+---------+---------       1   M  N  K  K  N  I  A  I  A  M  S  G  L  T  V  L  A  S  A  A   20               61   CCTGTATTTGCAGATGATACAAAAGTTGAAACTGGTGATCAAGGATATACAGTGGTACAA   120           ---------+---------+---------+---------+---------+---------       21   P  V  F  A  D  D  T  K  V  E  T  G  D  Q  G  Y  T  V  V  Q   40                                       □       121   AGCAAGTATAAGAAAGCTGTTGAACAATTACAAAAAGGAATATTAGATGGAAGTATAACA   180           ---------+---------+---------+---------+---------+---------       41   S  K  Y  K  K  A  V  E  Q  L  Q  K  G  I  L  D  G  S  I  T   60               181   GAAATTAAAGTTTTCTTTGAGGGAACTTTAGCATCTACTATAAAAGTAGGTTCTGAGCTT   240           ---------+---------+---------+---------+---------+---------       61   E  I  K  V  F  F  E  G  T  L  A  S  T  I  K  V  G  S  E  L   80               241   AATGCAGCAGATGCAAGTAAATTATTGTTTACACAAGTAGATAATAAACTAGATAATTTA   300           ---------+---------+---------+---------+---------+---------       81   N  A  A  D  A  S  K  L  L  F  T  Q  V  D  N  K  L  D  N  L   100               301   GGTGATGGAGATTATGTAGATTTCTTAATAACTTCTCCAGGTCAAGGGGATAAAATAACT   360           ---------+---------+---------+---------+---------+---------       101   G  D  G  D  Y  V  D  F  L  I  T  S  P  G  Q  G  D  K  I  T   120               361   ACAAGTAAACTTGTTGCATTGAAAGATTTAACAGGTGCTTCAGCAGATGCTATAATTGCT   420           ---------+---------+---------+---------+---------+---------       121   T  S  K  L  V  A  L  K  D  L  T  G  A  S  A  D  A  I  I  A   140               421   GGAACATCTTCAGCAGATGGTGTTGTTACAAATACTGGAGCTGCTAGTGGTTCTACTGAG   480           ---------+---------+---------+---------+---------+---------       141   G  T  S  S  A  D  G  V  V  T  N  T  G  A  A  S  G  S  T  E   160               481   ACAAATTCAGCAGGAACAAAACTTGCAATGTCAGCTATTTTTGACACAGCATATACAGAT   540           ---------+---------+---------+---------+---------+---------       161   T  N  S  A  G  T  K  L  A  M  S  A  I  F  D  T  A  Y  T  D   180               541   TCATCTGAAACTGCGGTTAAGATTACTATAAAAGCAGATATGAATGATACTAAATTTGGT   600           ---------+---------+---------+---------+---------+---------       181   S  S  E  T  A  V  K  I  T  I  K  A  D  M  N  D  T  K  F  G   200               601   AAAGCAGGTGAGACAACTTATTCAACTGGGCTTACATTTGAAGATGGGTCTACAGAAAAA   660           ---------+---------+---------+---------+---------+---------       201   K  A  G  E  T  T  Y  S  T  G  L  T  F  E  D  G  S  T  E  K   220               661   ATTGTTAAATTAGGGGACAGTGATATTATAGATATAACTAAAGCTCTTAAACTTACTGTT   720           ---------+---------+---------+---------+---------+---------       221   I  V  K  L  G  D  S  D  I  I  D  I  T  K  A  L  K  L  T  V   240               721   GTTCCTGGAAGTAAAGCAACTGTTAAGTTTGCTGAAAAAACACCAAGTGCCAGTGTTCAA   780           ---------+---------+---------+---------+---------+---------       241   V  P  G  S  K  A  T  V  K  F  A  E  K  T  P  S  A  S  V  Q   260               781   CCAGTAATAACAAAGCTTAGAATAATAAATGCTAAAGAAGAAACAATAGATATTGACGCT   840           ---------+---------+---------+---------+---------+---------       261   P  V  I  T  K  L  R  I  I  N  A  K  E  E  T  I  D  I  D  A   280               841   AGTTCTAGTAAAACAGCACAAGATTTAGCTAAAAAATATGTATTTAATAAAACTGATTTA   900           ---------+---------+---------+---------+---------+---------       281   S  S  S  K  T  A  Q  D  L  A  K  K  Y  V  F  N  K  T  D  L   300               901   AATACTCTTTATAAAGTATTAAATGGAGATGAAGCAGATACTAATGGATTAATAGAAGAA   960           ---------+---------+---------+---------+---------+---------       301   N  T  L  Y  K  V  L  N  G  D  E  A  D  T  N  G  L  I  E  E   320               961   GTTAGTGGAAAATATCAAGTAGTTCTTTATCCAGAAGGAAAAAGAGTTACAACTAAGAGT   1020           ---------+---------+---------+---------+---------+---------       321   V  S  G  K  Y  Q  V  V  L  Y  P  E  G  K  R  V  T  T  K  S   340               1021   GCTGCAAAGGCTTCAATTGCTGATGAAAATTCACCAGTTAAATTAACTCTTAAGTCAGAT   1080           ---------+---------+---------+---------+---------+---------       341   A  A  K  A  S  I  A  D  E  N  S  P  V  K  L  T  L  K  S  D   360                          ♦       1081   AAGAAGAAAGACTTAAAAGATTATGTGGATGATTTAAGAACATATAATAATGGATATTCA   1140           ---------+---------+---------+---------+---------+---------       361   K  K  K  D  L  K  D  Y  V  D  D  L  R  T  Y  N  N  G  Y  S   380               1141   AATGCTATAGAAGTAGCAGGAGAAGATAGAATAGAAACTGCAATAGCATTAAGTCAAAAA   1200           ---------+---------+---------+---------+---------+---------       381   N  A  I  E  V  A  G  E  D  R  I  E  T  A  I  A  L  S  Q  K   400               1201   TATTATAACTCTGATGATGAAAATGCTATATTTAGAGATTCAGTTGATAATGTAGTATTG   1260           ---------+---------+---------+---------+---------+---------       401   Y  Y  N  S  D  D  E  N  A  I  F  R  D  S  V  D  N  V  V  L   420               1261   GTTGGAGGAAATGCAATAGTTGATGGACTTGTAGCTTCTCCTTTAGCTTCTGAAAAGAAA   1320           ---------+---------+---------+---------+---------+---------       421   V  G  G  N  A  I  V  D  G  L  V  A  S  P  L  A  S  E  K  K   440               1321   GCTCCTTTATTATTAACTTCAAAAGATAAATTAGATTCAAGCGTAAAAGCTGAAATAAAG   1380           ---------+---------+---------+---------+---------+---------       441   A  P  L  L  L  T  S  K  D  K  L  D  S  S  V  K  A  E  I  K   460               1381   AGAGTTATGAATATAAAGAGTACAACAGGTATAAATACTTCAAAGAAAGTTTATTTAGCT   1440           ---------+---------+---------+---------+---------+---------       461   R  V  M  N  I  K  S  T  T  G  I  N  T  S  K  K  V  Y  L  A   480               1441   GGTGGAGTTAATTCTATATCTAAAGAAGTAGAAAATGAATTAAAAGATATGGGACTTAAA   1500           ---------+---------+---------+---------+---------+---------       481   G  G  V  N  S  I  S  K  E  V  E  N  E  L  K  D  M  G  L  K   500               1501   GTTACAAGATTAGCAGGAGATGATAGATATGAAACTTCTCTAAAAATAGCTGATGAAGTA   1560           ---------+---------+---------+---------+---------+---------       501   V  T  R  L  A  G  D  D  R  Y  E  T  S  L  K  I  A  D  E  V   520               1561   GGTCTTGATAATGATAAAGCATTTGTAGTTGGAGGAACAGGATTAGCAGATGCCATGAGT   1620           ---------+---------+---------+---------+---------+---------       521   G  L  D  N  D  K  A  F  V  V  G  G  T  G  L  A  D  A  M  S   540               1621   ATAGCTCCAGTTGCATCTCAATTAAGAAATGCTAATGGTAAAATGGATTTAGCTGATGGT   1680           ---------+---------+---------+---------+---------+---------       541   I  A  P  V  A  S  Q  L  R  N  A  N  G  K  N  D  L  A  D  G   560               1681   GATGCTACACCAATAGTAGTTGTAGATGGAAAAGCTAAAACTATAAATGATGATGTAAAA   1740           ---------+---------+---------+---------+---------+---------       561   D  A  T  P  I  V  V  V  D  G  K  A  K  T  I  N  D  D  V  K   580               1741   GATTTCTTAGATGATTCACAAGTTGATATAATAGGTGGAGAAAACAGTGTATCTAAAGAT   1800           ---------+---------+---------+---------+---------+---------       581   D  F  L  D  D  S  Q  V  D  I  I  G  G  E  N  S  V  S  K  D   600               1801   GTTGAAAATGCAATAGATGATGCTACAGGTAAATCTCCAGATAGATATAGTGGAGATGAT   1860           ---------+---------+---------+---------+---------+---------       601   V  E  N  A  I  D  D  A  T  G  K  S  P  D  R  Y  S  G  D  D   620               1861   AGACAAGCAACTAATGCAAAAGTTATAAAAGAATCTTCTTATTATCAAGATAACTTAAAT   1920           ---------+---------+---------+---------+---------+---------       621   R  Q  A  T  N  A  K  V  I  K  E  S  S  Y  Y  Q  D  N  L  N   640               1921   AATGATAAAAAAGTAGTTAATTTCTTTGTAGCTAAAGATGGTTCTACTAAAGAAGATCAA   1980           ---------+---------+---------+---------+---------+---------       641   N  D  K  K  V  V  N  F  F  V  A  K  D  G  S  T  K  E  D  Q   660               1981   TTAGTTGATGCTTTAGCAGCAGCTCCAGTTGCAGCAAACTTTGGTGTAACTCTTAATTCT   2040           ---------+---------+---------+---------+---------+---------       661   L  V  D  A  L  A  A  A  P  V  A  A  N  F  G  V  T  L  N  S   680               2041   GATGGTAAGCCAGTAGATAAAGATGGTAAAGtATTAACTGGTTCTGATAATGATAAAAAT   2100           ---------+---------+---------+---------+---------+---------       681   D  G  K  P  V  D  K  D  G  K  V  L  T  G  S  D  N  D  K  N   700               2101   AAATTAGTATCTCCAGCACCTATAGTATTAGCTACTGATTCTTTATCTTCAGATCaAAGT   2160           ---------+---------+---------+---------+---------+---------       701   K  L  V  S  P  A  P  I  V  L  A  T  D  S  L  S  S  D  Q  S   720               2161   GTATCTATAAGTAaAGTTCTTGATAAAGATAATGGAGAAAACTTAGTTCAAGTTGGTAAA   2220           ---------+---------+---------+---------+---------+---------       721   V  S  I  S  K  V  L  D  K  D  N  G  E  N  L  V  Q  V  G  K   740               2221   GGTATAGCTACTTCAGTTATAAACAAAATGAAAGATTTATTAGATATG   2268           ---------+---------+---------+---------+--------       741   G  I  A  T  S  V  I  N  K  M  K  D  L  L  D  M   756                  
 
         [0145]    
       
         
               
               
             
               
               
               
               
             
           
               
                 APPENDIX 2 
               
               
                   
               
               
                   
               
               
                 SEQ ID No. 4. Nucleotide sequence of slpA from  Clostridium   
                   
               
               
                   difficile  strain 172450, PCR type 5, with translation. The 
               
               
                 putative secretory signal cleavage site (□) is indicated, 
               
               
                 and an approximation of the and site of cleavage to form 
               
               
                 the two mature SLPs (♦) is also indicated. 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 1 
                 ATGAAAAAAAGAAATTTAGCAATGGCTATGGCAGCTGTTACTGTAGTAGGTTCTGCTGCT 
                 60 
                   
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 1 
                 M  K  K  R  N  L  A  M  A  M  A  A  V  T  V  V  G  S  A  A 
                 20 
               
               
                   
               
               
                 61 
                 CCAGTTTTTGCAGCAGCTTCAGATGTAATATCACTACAAGATGGTACAAATGATAAGTAT 
                 120 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 21 
                 P  V  F  A  A  A  S  D  V  I  S  L  Q  D  G  T  N  D  K  Y 
                 40 
               
               
                   
               
               
                   
                                           □ 
               
               
                   
               
               
                 121 
                 ACAGTATCAAATACTAAAGCTAGTGACTTAGTAAAGGATATTTTAGCAGCACAAAACTTA 
                 180 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 41 
                 T  V  S  N  T  K  A  S  D  L  V  K  D  I  L  A  A  Q  N  L 
                 60 
               
               
                   
               
               
                 181 
                 ACAACAGGTGCAGTTATTTTGAACAAAGATACAAAAGTTACTTTCTATGATGCAAATGAG 
                 240 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 61 
                 T  T  G  A  V  I  L  N  K  D  T  K  V  T  F  Y  D  A  N  E 
                 80 
               
               
                   
               
               
                 241 
                 AAAGATTCTTCAACTCCAACTGGAGATAAAAAAGTTTATTCAGAACAAACTTTAACTACA 
                 300 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 81 
                 K  D  S  S  T  P  T  G  D  K  K  V  Y  S  E  Q  T  L  T  T 
                 100 
               
               
                   
               
               
                 301 
                 GCTAATGGAAATGAAGATTATGTAAAGACAACTTTAAAAAATTTAGATGCAGGAGAATAT 
                 360 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 101 
                 A  N  G  N  E  D  Y  V  K  T  T  L  K  N  L  D  A  G  E  Y 
                 120 
               
               
                   
               
               
                 361 
                 GCTATTATAGATTTAACTTATAATAATGCTAAAACTGTTGAAATTAAAGTAGTAGCAGCT 
                 420 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 121 
                 A  I  I  D  L  T  Y  N  N  A  K  T  V  E  I  K  V  V  A  A 
                 140 
               
               
                   
               
               
                 421 
                 AGTGAAAAAACAGTAGTTGTATCTAGTGATGCGAAAAATAGTGCAAAAGATATAGCTGAA 
                 480 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 141 
                 S  E  K  T  V  V  V  S  S  D  A  K  N  S  A  K  D  I  A  E 
                 160 
               
               
                   
               
               
                 481 
                 AAATATGTGTTTGAAGACAAAGACTTAGAAAATGCACTAAAAACTATAAATGCCTCAGAT 
                 540 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 161 
                 K  Y  V  F  E  D  K  D  L  E  N  A  L  K  T  I  N  A  S  D 
                 180 
               
               
                   
               
               
                 541 
                 TTCAGTAAAACTGATAGTTACTATCAAGTAGTTCTTTATCCAAAAGGAAAGAGATTACAA 
                 600 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 181 
                 F  S  K  T  D  S  Y  Y  Q  V  V  L  Y  P  K  G  K  R  L  Q 
                 200 
               
               
                   
               
               
                 601 
                 GGTTTCTCAACTTATAGAGCTACAAATTATAATGAAGGAACTGCATATGGTAATACACCA 
                 660 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 201 
                 G  F  S  T  Y  R  A  T  N  Y  N  E  G  T  A  Y  G  N  T  P 
                 220 
               
               
                   
               
               
                   
                                 ♦ 
               
               
                   
               
               
                 661 
                 GTAATATTAACTCTAAAATCTACTAGTAAGAGTAATTTAAAGACTGCAGTAGAAGAGTTA 
                 720 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 221 
                 V  I  L  T  L  K  S  T  S  K  S  N  L  K  T  A  V  E  E  L 
                 240 
               
               
                   
               
               
                 721 
                 CAAAAATTGAATGCTAGTTATTCTAATACTACAACTTTAGCTGGTGATGACAGAATACAA 
                 780 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 241 
                 Q  K  L  N  A  S  Y  S  N  T  T  T  L  A  G  D  D  R  I  Q 
                 260 
               
               
                   
               
               
                 781 
                 ACAGCTATAGAGATAAGTAAAGAATATTACAATAATGATGGCGAGAAATCAGATCATTCA 
                 840 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 261 
                 T  A  I  E  I  S  K  E  Y  Y  N  N  D  G  E  K  S  D  H  S 
                 280 
               
               
                   
               
               
                 841 
                 GCTGATGTTAAAGAGAATGTTAAAAATGTTGTATTAGTAGGTGCAAATGCACTAGTAGAT 
                 900 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 281 
                 A  D  V  K  E  N  V  K  N  V  V  L  V  G  A  N  A  L  V  D 
                 300 
               
               
                   
               
               
                 901 
                 GGATTAGTTGCGGCTCCTTTAGCAGCAGAJAAAGATGCTCCACTATTATTAACTTCAAAA 
                 960 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 301 
                 G  L  V  A  A  P  L  A  A  E  K  D  A  P  L  L  L  T  S  K 
                 320 
               
               
                   
               
               
                 961 
                 GATAAATTAGATTCGTCAGTAAAATCTGALATAAAGAGAGTTTTAGACTTAAAAACTTCA 
                 1020 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 321 
                 D  K  L  D  S  S  V  K  S  E  I  K  R  V  L  D  L  K  T  S 
                 340 
               
               
                   
               
               
                 1021 
                 ACAGAAGTAACAGGAAAAACAGTTTATATAGCTGGTGGAGTTAATAGTGTATCTAAAGAA 
                 1080 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 341 
                 T  E  V  T  G  K  T  V  Y  I  A  G  G  V  N  S  V  S  K  E 
                 360 
               
               
                   
               
               
                 1081 
                 GTTGTAACAGAATTAGAATCAATGGGATTAAAAGTTGAAAGATTCTCAGGTGATGATAGA 
                 1140 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 361 
                 V  V  T  E  L  E  S  M  G  L  K  V  E  R  F  S  G  D  D  R 
                 380 
               
               
                   
               
               
                 1141 
                 TATGAAACTTCTTTAAAAATAGCAGGTGAAATAGGCTTAGATAATGATAAGGCTTATGTA 
                 1200 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 381 
                 Y  E  T  S  L  K  I  A  G  E  I  G  L  D  N  D  K  A  Y  V 
                 400 
               
               
                   
               
               
                 1201 
                 GTTGGTGGAACAGGATTAGCAGATGCCATGAGTATAGCTTCAGTTGCTTCTACTAAATTA 
                 1260 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 401 
                 V  G  G  T  G  L  A  D  A  M  S  I  A  S  V  A  S  T  K  L 
                 420 
               
               
                   
               
               
                 1261 
                 GATGGTAATGGTGTTGTAGATAGAACAAATGGACATGCTACTCCAATAGTTGTTGTAGAT 
                 1320 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 421 
                 D  G  N  G  V  V  D  R  T  N  G  H  A  T  P  I  V  V  V  D 
                 440 
               
               
                   
               
               
                 1321 
                 GGAAAAGCTGATAAAATATCTGATGACTTAGATAGTTTCTTAGGAAGCGCTGATGTAGAT 
                 1380 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 441 
                 G  K  A  D  K  I  S  D  D  L  D  S  F  L  G  S  A  D  V  D 
                 460 
               
               
                   
               
               
                 1381 
                 ATAATAGGTGGATTTGCAAGTGTATCTGAAAAGATGGAAGAAGCTATATCAGATGCTACT 
                 1440 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 461 
                 I  I  G  G  F  A  S  V  S  E  K  M  E  E  A  I  S  D  A  T 
                 480 
               
               
                   
               
               
                 1441 
                 GGTAAAGGCGTTACAAGAGTTAAAGGCGACGATAGACAAGACACTAACTCTGAAGTTATA 
                 1500 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 481 
                 G  K  G  V  T  R  V  K  G  D  D  R  Q  D  T  N  S  E  V  I 
                 500 
               
               
                   
               
               
                 1501 
                 AAAACATATTATGCTAATGATACTGAAATAGCTAAAGCTGCAGTTTTAGATAAAGATTCA 
                 1560 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 501 
                 K  T  Y  Y  A  N  D  T  E  I  A  K  A  A  V  L  D  K  D  S 
                 520 
               
               
                   
               
               
                 1561 
                 GGTGCTTCAAGTAGTGATGCAGGAGTATTTAATTTCTATGTAGCTAAAGATGGATCTACA 
                 1620 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 521 
                 G  A  S  S  S  D  A  G  V  F  N  F  Y  V  A  K  D  G  S  T 
                 540 
               
               
                   
               
               
                 1621 
                 AAAGAAGATCAATTAGTTGATGCATTAGCAGTAGGAGCTGTTGCTGGATATAAACTTGCT 
                 1680 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 541 
                 K  E  D  Q  L  V  D  A  L  A  V  G  A  V  A  G  Y  K  L  A 
                 560 
               
               
                   
               
               
                 1681 
                 CCAGTTGTATTAGCTACTGATTCTTTATCTTCTGATCAATCGGTTGCTATAAGCAAAGTT 
                 1740 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 561 
                 P  V  V  L  A  T  D  S  L  S  S  D  Q  S  V  A  I  S  K  V 
                 580 
               
               
                   
               
               
                 1741 
                 GTAGGAGAAAAATATTCTAAAGATTTAACACAAGTTGGTCAAGGAATAGCTAATTCAGTT 
                 1800 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 581 
                 V  G  E  K  Y  S  K  D  L  T  Q  V  G  Q  G  I  A  N  S  V 
                 600 
               
               
                   
               
               
                 1801 
                 ATAAACAAAATGAAAGATTTATTAGATATG 
                 1830 
               
               
                   
                 ---------+---------+---------+ 
               
               
                 601 
                 I  N  K  M  K  D  L  L  D  M 
                 610 
               
               
                   
               
             
          
         
       
     
         [0146]    
       
         
               
               
             
               
               
               
               
             
           
               
                 APPENDIX 3 
               
               
                   
               
               
                   
               
               
                 SEQ ID No. 5. Nucleotide sequence of slpA from  Clostridium   
                   
               
               
                   difficile  strain 170324, PCR type 12, with translation. The 
               
               
                 putative secretory signal cleavage site (□) and site of 
               
               
                 cleavage to form the two mature SLPs (♦) are indicated. 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 1 
                 ATGAATAAGAAAAATATAGCAATAGCTATGTCAGGTTTAACAGTTTTAGCTTCGGCTGCT 
                 60 
                   
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 1 
                 M  N  K  K  N  I  A  I  A  M  S  G  L  T  V  L  A  S  A  A 
                 20 
               
               
                   
               
               
                 61 
                 CCTGTTTTTGCTGCAACTACTGGAACACAAGGTTATACTGTAGTTAAAAACGACTGGAAA 
                 120 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 21 
                 P  V  F  A  A  T  T  G  T  Q  G  Y  T  V  V  K  N  D  W  K 
                 40 
               
               
                   
               
               
                   
                                     □ 
               
               
                   
               
               
                 121 
                 AAAGCAGTAAAACAATTACAAGATGGACTAAAAGATAATAGTATAGGAAAGATAACTGTA 
                 180 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 41 
                 K  A  V  K  Q  L  Q  D  G  L  K  D  N  S  I  G  K  I  T  V 
                 60 
               
               
                   
               
               
                 181 
                 TCTTTTAATGATGGGGTTGTGGGTGAAGTAGCTCCTAAAAGTGCTAATAAGAAAGCGGAC 
                 240 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 61 
                 S  F  N  D  G  V  V  G  E  V  A  P  K  S  A  N  K  K  A  D 
                 80 
               
               
                   
               
               
                 241 
                 AGAGATGCTGCAGCTGAGAAGTTATATAATCTTGTTAACACTCAATTAGATAAATTAGGT 
                 300 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 81 
                 R  D  A  A  A  E  K  L  Y  N  L  V  N  T  Q  L  D  K  L  G 
                 100 
               
               
                   
               
               
                 301 
                 GATGGAGATTATGTTGATTTTTCTGTAGATTATAATTTAGAAAACAAAATAATAACTAAT 
                 360 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 101 
                 D  G  D  Y  V  D  F  S  V  D  Y  N  L  E  N  K  I  I  T  N 
                 120 
               
               
                   
               
               
                 361 
                 CAAGCAGATGCAGAAGCAATTGTTACAAAGTTAAATTCACTTAATGAGAAAACTCTTATT 
                 420 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 121 
                 Q  A  D  A  E  A  I  V  T  K  L  N  S  L  N  E  K  T  L  I 
                 140 
               
               
                   
               
               
                 421 
                 GATATAGCAACTAAAGATACTTTTGGAATGGTTAGTAAAACACAAGATAGTGAAGGTAAA 
                 480 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 141 
                 D  I  A  T  K  D  T  F  G  M  V  S  K  T  Q  D  S  E  G  K 
                 160 
               
               
                   
               
               
                 481 
                 AATGTTGCTGCAACAAAGGCACTTAAAGTTAAAGATGTTGCTACATTTGGTTTGAAGTCT 
                 540 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 161 
                 N  V  A  A  T  K  A  L  K  V  K  D  V  A  T  F  G  L  K  S 
                 180 
               
               
                   
               
               
                 541 
                 GGTGGAAGCGAAGATACTGGATATGTTGTTGAAATGAAAGCAGGAGCTGTAGAGGATAAG 
                 600 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 181 
                 G  G  S  E  D  T  G  Y  V  V  E  M  K  A  G  A  V  E  D  K 
                 200 
               
               
                   
               
               
                 601 
                 TATGGTAAAGTTGGAGATAGTACGGCAGGTATTGCAATAAATCTTCCTAGTACTGGACTT 
                 660 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 201 
                 Y  G  K  V  G  D  S  T  A  G  I  A  I  N  L  P  S  T  G  L 
                 220 
               
               
                   
               
               
                 661 
                 GAATATGCAGGTAAAGGAACAACAATTGATTTTAATAAAACTTTAAAAGTTGATGTAACA 
                 720 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 221 
                 E  Y  A  G  K  G  T  T  I  D  F  N  K  T  L  K  V  D  V  T 
                 240 
               
               
                   
               
               
                 721 
                 GGTGGTTCAACACCTAGTGCTGTAGCTGTAAGTGGTTTTGTAACTAAAGATGATACTGAT 
                 780 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 241 
                 G  G  S  T  P  S  A  V  A  V  S  G  F  V  T  K  D  D  T  D 
                 260 
               
               
                   
               
               
                 781 
                 TTAGCAAAATCAGGTACTATAAATGTAAGAGTTATAAATGCAAAAGAAGAATCAATTGAT 
                 840 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 261 
                 L  A  K  S  G  T  I  N  V  R  V  I  N  A  K  E  E  S  I  D 
                 280 
               
               
                   
               
               
                 841 
                 ATAGATGCAAGCTCATATACATCAGCTGAAAATTTAGCTAAAAGATATGTATTTGATCCA 
                 900 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 281 
                 I  D  A  S  S  Y  T  S  A  E  N  L  A  K  R  Y  V  F  D  P 
                 300 
               
               
                   
               
               
                 901 
                 GATGAAATTTCTGAAGCATATAAGGCAATAGTAGCATTACAAAATGATGGTATAGAGTCT 
                 960 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 301 
                 D  E  I  S  E  A  Y  K  A  I  V  A  L  Q  N  D  G  I  E  S 
                 320 
               
               
                   
               
               
                 961 
                 AACTTAGTTCAGTTAGTTAATGGAAAATATCAAGTGATTTTTTATCCAGAAGGTAAAAGA 
                 1020 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 321 
                 N  L  V  Q  L  V  N  G  K  Y  Q  V  I  F  Y  P  E  G  K  R 
                 340 
               
               
                   
               
               
                 1021 
                 TTAGAAACTAAATCAGCAAATGATACAATAGCTAGTCAAGATACACCAGCTAAAGTAGTT 
                 1080 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 341 
                 L  E  T  K  S  A  N  D  T  I  A  S  Q  D  T  P  A  K  V  V 
                 360 
               
               
                   
               
               
                   
                                       ♦ 
               
               
                   
               
               
                 1081 
                 ATAAAAGCTAATAAATTAAAAGATTTAAAAGATTATGTAGATGATTTAAAAACATATAAT 
                 1140 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 361 
                 I  K  A  N  K  L  K  D  L  K  D  Y  V  D  D  L  K  T  Y  N 
                 380 
               
               
                   
               
               
                 1141 
                 AATACTTATTCAAATGTTGTAACAGTAGCAGGAGAAGATAGAATAGAAACTGCTATAGAA 
                 1200 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 381 
                 N  T  Y  S  N  V  V  T  V  A  G  E  D  R  I  E  T  A  I  E 
                 400 
               
               
                   
               
               
                 1201 
                 TTAAGTAGTAAATATTATAATTCTGATGATAAAAATGCAATAACTGATAAAGCAGTTAAT 
                 1260 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 401 
                 L  S  S  K  Y  Y  N  S  D  D  K  N  A  I  T  D  K  A  V  N 
                 420 
               
               
                   
               
               
                 1261 
                 GATATAGTATTAGTTGGATCTACATCTATAGTTGATGGTCTTGTTGCATCACCATTAGCT 
                 1320 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 421 
                 D  I  V  L  V  G  S  T  S  I  V  D  G  L  V  A  S  P  L  A 
                 440 
               
               
                   
               
               
                 1321 
                 TCAGAAAAAACAGCTCCATTATTATTAACTTCAAAAGATAAATTAGATTCATCAGTAAAA 
                 1380 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 441 
                 S  E  K  T  A  P  L  L  L  T  S  K  D  K  L  D  S  S  V  K 
                 460 
               
               
                   
               
               
                 1381 
                 TCTGAAATAAAGAGAGTTATGAACTTAAAGAGTGACACTGGTATAAATACTTCTAAAAAA 
                 1440 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 461 
                 S  E  I  K  R  V  M  N  L  K  S  D  T  G  I  N  T  S  K  K 
                 480 
               
               
                   
               
               
                 1441 
                 GTTTATTTAGCTGGTGGAGTTAATTCTATATCTAAAGATGTAGAAAATGAATTGAAAAAC 
                 1500 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 481 
                 V  Y  L  A  G  G  V  N  S  I  S  K  D  V  E  N  E  L  K  N 
                 500 
               
               
                   
               
               
                 1501 
                 ATGGGTCTTAAAGTTACTAGATTATCAGGAGAAGACAGATACGAAACTTCTTTAGCAATA 
                 1560 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 501 
                 M  G  L  K  V  T  R  L  S  G  E  D  R  Y  E  T  S  L  A  I 
                 520 
               
               
                   
               
               
                 1561 
                 GCTGATGAAATAGGTCTTGATAATGATAAAGCATTTGTAGTTGGTGGTACTGGATTAGCA 
                 1620 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 521 
                 A  D  E  I  G  L  D  N  D  K  A  F  V  V  G  G  T  G  L  A 
                 540 
               
               
                   
               
               
                 1621 
                 GATGCTATGAGTATAGCTCCAGTTGCTTCTCAACTTAAAGATGGAGATGCTACTCCAATA 
                 1680 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 541 
                 D  A  M  S  I  A  P  V  A  S  Q  L  K  D  G  D  A  T  P  I 
                 560 
               
               
                   
               
               
                 1681 
                 GTAGTTGTAGATGGAAAAGCAAAAGAAATAAGTGATGATGCTAAGAGTTTCTTAGGAACT 
                 1740 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 561 
                 V  V  V  D  G  K  A  K  E  I  S  D  D  A  K  S  F  L  G  T 
                 580 
               
               
                   
               
               
                 1741 
                 TCTGATGTTGATATAATAGGTGGAAAAAATAGCGTATCTAAAGAGATTGAAGAGTCAATA 
                 1800 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 581 
                 S  D  V  D  I  I  G  G  K  N  S  V  S  K  E  I  E  E  S  I 
                 600 
               
               
                   
               
               
                 1801 
                 GATAGTGCAACTGGAAAAACTCCAGATAGAATAAGTGGAGATGATAGACAAGCAACTAAT 
                 1860 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 601 
                 D  S  A  T  G  K  T  P  D  R  I  S  G  D  D  R  Q  A  T  N 
                 620 
               
               
                   
               
               
                 1861 
                 GCTGAAGTTTTAAAAGAAGATGATTATTTCACAGATGGTGAAGTTGTGAATTACTTTGTT 
                 1920 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 621 
                 A  E  V  L  K  E  D  D  Y  F  T  D  G  E  V  V  N  Y  F  V 
                 640 
               
               
                   
               
               
                 1921 
                 GCAAAAGATGGTTCTACTAAAGAAGATCAATTAGTAGATGCCTTAGCAGCAGCACCAATA 
                 1980 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 641 
                 A  K  D  G  S  T  K  E  D  Q  L  V  D  A  L  A  A  A  P  I 
                 660 
               
               
                   
               
               
                 1981 
                 GCAGGTAGATTTAAGGAGTCTCCAGCTCCAATCATACTAGCTACTGATACTTTATCTTCT 
                 2040 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 661 
                 A  G  R  F  K  E  S  P  A  P  I  I  L  A  T  D  T  L  S  S 
                 680 
               
               
                   
               
               
                 2041 
                 GACCAAAATGTAGCTGTAAGTAAAGCAGTTCCTAAAGATGGTGGAACTAACTTAGTTCAA 
                 2100 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 681 
                 D  Q  N  V  A  V  S  K  A  V  P  K  D  G  G  T  N  L  V  Q 
                 700 
               
               
                   
               
               
                 2101 
                 GTAGGTAAAGGTATAGCTTCTTCAGTTATAAACAAAATGAAAGATTTATTAGATATG 
                 2157 
               
               
                   
                 ---------+---------+---------+---------+---------+------- 
               
               
                 701 
                 V  G  K  G  I  A  S  S  V  I  N  K  M  K  D  L  L  D  M 
                 719 
               
               
                   
               
             
          
         
       
     
         [0147]    
       
         
               
               
             
               
               
               
               
             
           
               
                 APPENDIX 4 
               
               
                   
               
               
                   
               
               
                 SEQ ID No 6. Nucleotide sequence of slpA from  Clostridium   
                   
               
               
                   difficile  strain 171448, PCR type 12, with translation. The 
               
               
                 putative secretory signal cleavage site (□) and site of 
               
               
                 cleavage to form the two mature SLPs (♦) are indicated. 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 1 
                 ATGAATAAGAAAAATATAGCAATAGCTATGTCAGGTTTAACAGTTTTAGCTTCGGCTGCT 
                 60 
                   
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 1 
                 M  N  K  K  N  I  A  I  A  M  S  G  L  T  V  L  A  S  A  A 
                 20 
               
               
                   
               
               
                 61 
                 CCTGTTTTTGCTGCAACTACTGGAACACAAGGTTATACTGTAGTTAAAAACGACTGGAAA 
                 120 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 21 
                 P  V  F  A  A  T  T  G  T  Q  G  Y  T  V  V  K  N  D  W  K 
                 40 
               
               
                   
               
               
                   
                                     □ 
               
               
                   
               
               
                 121 
                 AAAGCAGTAAAACAATTACAAGATGGACTAAAAGATAATAGTATAGGAAAGATAACTGTA 
                 180 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 41 
                 K  A  V  K  Q  L  Q  D  G  L  K  D  N  S  I  G  K  I  T  V 
                 60 
               
               
                   
               
               
                 181 
                 TCTTTTAATGATGGGGTTGTGGGTGAAGTAGCTCCTAAAAGTGCTAATAAGAAAGCGGAC 
                 240 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 61 
                 S  F  N  D  G  V  V  G  E  V  A  P  K  S  A  N  K  K  A  D 
                 80 
               
               
                   
               
               
                 241 
                 AGAGATGCTGCAGCTGAGAAGTTATATAATCTTGTTAACACTCAATTAGATAAATTAGGT 
                 300 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 81 
                 R  D  A  A  A  E  K  L  Y  N  L  V  N  T  Q  L  D  K  L  G 
                 100 
               
               
                   
               
               
                 301 
                 GATGGAGATTATGTTGATTTTTCTGTAGATTATAATTTAGAAAACAAAATAATAACTAAT 
                 360 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 101 
                 D  G  D  Y  V  D  F  S  V  D  Y  N  L  E  N  K  I  I  T  N 
                 120 
               
               
                   
               
               
                 361 
                 CAAGCAGATGCAGAAGCAATTGTTACAAAGTTAAATTCACTTAATGAGAAAACTCTTATT 
                 420 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 121 
                 Q  A  D  A  E  A  I  V  T  K  L  N  S  L  N  E  K  T  L  I 
                 140 
               
               
                   
               
               
                 421 
                 GATATAGCAACTAAAGATACTTTTGGAATGGTTAGTAAAACACAAGATAGTGGAGGTAAA 
                 480 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 141 
                 D  I  A  T  K  D  T  F  G  M  V  S  K  T  Q  D  S  G  G  K 
                 160 
               
               
                   
               
               
                 481 
                 AATGTTGCTGCAACAAAGGCACTTAAAGTTAAAGATGTTGCTACATTTGGTTTGAAGTCT 
                 540 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 161 
                 N  V  A  A  T  K  A  L  K  V  K  D  V  A  T  F  G  L  K  S 
                 180 
               
               
                   
               
               
                 541 
                 GGTGGAAGCGAAGATACTGGATATGTTGTTGAAATGAAAGCAGGAGCTGTAGAGGATAAG 
                 600 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 181 
                 G  G  S  E  D  T  G  Y  V  V  E  M  K  A  G  A  V  E  D  K 
                 200 
               
               
                   
               
               
                 601 
                 TATGGTAAAGTTGGAGATAGTACGGCAGGTATTGCAATAAATCTTCCTAGTACTGGACTT 
                 660 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 201 
                 Y  G  K  V  G  D  S  T  A  G  I  A  I  N  L  P  S  T  G  L 
                 220 
               
               
                   
               
               
                 661 
                 GAATATGCAGGTAAAGGAACAACAATTGATTTTAATAAAACTTTAAAAGTTGATGTAACA 
                 720 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 221 
                 E  Y  A  G  K  G  T  T  I  D  F  N  K  T  L  K  V  D  V  T 
                 240 
               
               
                   
               
               
                 721 
                 GGTGGTTCAACACCTAGTGCTGTAGCTGTAAGTGGTTTTGTAACTAAAGATGATACTGAT 
                 780 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 241 
                 G  G  S  T  P  S  A  V  A  V  S  G  F  V  T  K  D  D  T  D 
                 260 
               
               
                   
               
               
                 781 
                 TTAGCAAAATCAGGTACTATAAATGTAAGAGTTATAAATGCAAAAGAAGAATCAATTGAT 
                 840 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 261 
                 L  A  K  S  G  T  I  N  V  R  V  I  N  A  K  E  E  S  I  D 
                 280 
               
               
                   
               
               
                 841 
                 ATAGATGCAAGCTCATATACATCAGCTGAAAATTTAGCTAAAAGATATGTATTTGATCCA 
                 900 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 281 
                 I  D  A  S  S  Y  T  S  A  E  N  L  A  K  R  Y  V  F  D  P 
                 300 
               
               
                   
               
               
                 901 
                 GATGAAATTTCTGAAGCATATAAGGCAATAGTAGCATTACAAAATGATGGTATAGAGTCT 
                 960 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 301 
                 D  E  I  S  E  A  Y  K  A  I  V  A  L  Q  N  D  G  I  E  S 
                 320 
               
               
                   
               
               
                 961 
                 AATTTAGTTCAGTTAGTTAATGGAAAATATCAAGTGATTTTTTATCCAGAAGGTAAAAGA 
                 1020 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 321 
                 N  L  V  Q  L  V  N  G  K  Y  Q  V  I  F  Y  P  E  G  K  R 
                 340 
               
               
                   
               
               
                 1021 
                 TTAGAAACTAAATCAGCAAATGATACAATAGCTAGTCAAGATACACCAGCTAAAGTAGTT 
                 1080 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 341 
                 L  E  T  K  S  A  N  D  T  I  A  S  Q  D  T  P  A  K  V  V 
                 360 
               
               
                   
               
               
                   
                                       ♦ 
               
               
                   
               
               
                 1081 
                 ATAAAAGCTAATAAATTAAAAGATTTAAAAGATTATGTAGATGATTTAAAAACATATAAT 
                 1140 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 361 
                 I  K  A  N  K  L  K  D  L  K  D  Y  V  D  D  L  K  T  Y  N 
                 380 
               
               
                   
               
               
                 1141 
                 AATACTTATTCAAATGTTGTAACAGTAGCAGGAGAAGATAGAATAGAAACTGCTATAGAA 
                 1200 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 381 
                 N  T  Y  S  N  V  V  T  V  A  G  E  D  R  I  E  T  A  I  E 
                 400 
               
               
                   
               
               
                 1201 
                 TTAAGTAGTAAATATTATAATTCTGATGATAAAAATGCAATAACTGATAAAGCAGTTAAT 
                 1260 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 401 
                 L  S  S  K  Y  Y  N  S  D  D  K  N  A  I  T  D  K  A  V  N 
                 420 
               
               
                   
               
               
                 1261 
                 GATATAGTATTAGTTGGATCTACATCTATAGTTGATGGTCTTGTTGCATCACCATTAGCT 
                 1320 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 421 
                 D  I  V  L  V  G  S  T  S  I  V  D  G  L  V  A  S  P  L  A 
                 440 
               
               
                   
               
               
                 1321 
                 TCAGAAAAAACAGCTCCATTATTATTAGCTTCAAAAGATAAATTAGATTCATCAGTAAAA 
                 1380 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 441 
                 S  E  K  T  A  P  L  L  L  A  S  K  D  K  L  D  S  S  V  K 
                 460 
               
               
                   
               
               
                 1381 
                 TCTGAAATAAAGAGAGTTATGAACTTAAAGAGTGACACTGGTATAAATACTTCTAAAAAA 
                 1440 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 461 
                 S  E  I  K  R  V  M  N  L  K  S  D  T  G  I  N  T  S  K  K 
                 480 
               
               
                   
               
               
                 1441 
                 GTTTATTTAGCTGGTGGAGTTAATTCTATATCTAAAGATGTAGAAAATGAATTGAAAAAC 
                 1500 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 481 
                 V  Y  L  A  G  G  V  N  S  I  S  K  D  V  E  N  E  L  K  N 
                 500 
               
               
                   
               
               
                 1501 
                 ATGGGTCTTAAAGTTACTAGATTATCAGGAGAAGACAGATACGAAACTTCTTTAGCAATA 
                 1560 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 501 
                 M  G  L  K  V  T  R  L  S  G  E  D  R  Y  E  T  S  L  A  I 
                 520 
               
               
                   
               
               
                 1561 
                 GCTGATGAAATAGGTCTTGATAATGATAA.AGCATTTGTAGTTGGTGGTACTGGATTAGCA 
                 1620 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 521 
                 A  D  E  I  G  L  D  N  D  K  A  F  V  V  G  G  T  G  L  A 
                 540 
               
               
                   
               
               
                 1621 
                 GATGCTATGAGTATAGCTCCAGTTGCTTCTCAACTTAAAGATGGAGATGCTACTCCAATA 
                 1680 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 541 
                 D  A  M  S  I  A  P  V  A  S  Q  L  K  D  G  D  A  T  P  I 
                 560 
               
               
                   
               
               
                 1681 
                 GTAGTTGTAGATGGAAAAGCAAAAGAAATAAGTGATGATGCTAAGAGTTTCTTAGGAACT 
                 1740 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 561 
                 V  V  V  D  G  K  A  K  E  I  S  D  D  A  K  S  F  L  G  T 
                 580 
               
               
                   
               
               
                 1741 
                 TCTGATGTTGATATAATAGGTGGAAAAAATAGCGTATCTAAAGAGATTGAAGAGTCAATA 
                 1800 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 581 
                 S  D  V  D  I  I  G  G  K  N  S  V  S  K  E  I  E  E  S  I 
                 600 
               
               
                   
               
               
                 1801 
                 GATAGTGCAACTGGAAAAACTCCAGATAGAATAAGTGGAGATGATAGACAAGCAACTAAT 
                 1860 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 601 
                 D  S  A  T  G  K  T  P  D  R  I  S  G  D  D  R  Q  A  T  N 
                 620 
               
               
                   
               
               
                 1861 
                 GCTGAAGTTTTAAAAGAAGATGATTATTTCACAGATGGTGAAGTTGTGAATTACTTTGTT 
                 1920 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 621 
                 A  E  V  L  K  E  D  D  Y  F  T  D  G  E  V  V  N  Y  F  V 
                 640 
               
               
                   
               
               
                 1921 
                 GCAAAAGATGGTTCTACTAAAGAAGATCAATTAGTAGATGCCTTAGCAGCAGCACCAATA 
                 1980 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 641 
                 A  K  D  G  S  T  K  E  D  Q  L  V  D  A  L  A  A  A  P  I 
                 660 
               
               
                   
               
               
                 1981 
                 GCAGGTAGATTTAAGGAGTCTCCAGCTCCAATCATACTAGCTACTGATACTTTATCTTCT 
                 2040 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 661 
                 A  G  R  F  K  E  S  P  A  P  I  I  L  A  T  D  T  L  S  S 
                 680 
               
               
                   
               
               
                 2041 
                 GACCAAAATGTAGCTGTAAGTAAAGCAGTTCCTAAAGATGGTGGAACTAACTTAGTTCAA 
                 2100 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 681 
                 D  Q  N  V  A  V  S  K  A  V  P  K  D  G  G  T  N  L  V  Q 
               
               
                   
               
               
                 2101 
                 GTAGGTAAAGGTATAGCTTCTTCAGTTATAAACAAAATGAAAGATTTATTAGATATG 
                 2157 
               
               
                   
                 ---------+---------+---------+---------+---------+------- 
               
               
                 701 
                 V  G  K  G  I  A  S  S  V  I  N  K  M  K  D  L  L  D  M 
                 719 
               
               
                   
               
             
          
         
       
     
         [0148]    
       
         
               
               
             
               
               
               
               
             
           
               
                 APPENDIX 5 
               
               
                   
               
               
                   
               
               
                 SEQ ID No. 7. Nucleotide sequence of slpA from  Clostridium   
                   
               
               
                   difficile  strain 171862, PCR type 17, with translation. The 
               
               
                 putative secretory signal cleavage site (□) and site of 
               
               
                 cleavage to form the two mature SLPs (♦) are indicated. 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 1 
                 ATGAATAAGAAAAACTTAGCAATGGCTATGGCAGCAGTTACTGTTGTGGGTTCTGCAGCG 
                 60 
                   
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 1 
                 M  N  K  K  N  L  A  M  A  M  A  A  V  T  V  V  G  S  A  A 
                 20 
               
               
                   
               
               
                 61 
                 CCAATATTTGCAGATAGTACTACGCCAGGTTATACTGTAGTGAAAAATGATTGGAAAAAA 
                 120 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 21 
                 P  I  F  A  D  S  T  T  P  G  Y  T  V  V  K  N  D  W  K  K 
                 40 
               
               
                   
               
               
                   
                                     □ 
               
               
                   
               
               
                 121 
                 GCAGTAAAACAATTACAAGATGGGTTGAAAAATAAAACTATATCAACAATAAAGGTGTCT 
                 180 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 41 
                 A  V  K  Q  L  Q  D  G  L  K  N  K  T  I  S  T  I  K  V  S 
                 60 
               
               
                   
               
               
                 181 
                 TTTAATGGAAACTCTGTTGGAGAAGTTACACCAGCCAGTTCTGGAGCAAAAAAAGCAGAT 
                 240 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 61 
                 F  N  G  N  S  V  G  E  V  T  P  A  S  S  G  A  K  K  A  D 
                 80 
               
               
                   
               
               
                 241 
                 AGAGATGCTGCAGCTGAAAAGTTATATAATTTAGTAAATACACAATTAGATAAACTAGGT 
                 300 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 81 
                 R  D  A  A  A  E  K  L  Y  N  L  V  N  T  Q  L  D  K  L  G 
                 100 
               
               
                   
               
               
                 301 
                 GATGGAGATTACGTTGACTTTGAAGTAACTTATAATTTAGCTACTCAAATAATTACAAAA 
                 360 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 101 
                 D  G  D  Y  V  D  F  E  V  T  Y  N  L  A  T  Q  I  I  T  K 
                 120 
               
               
                   
               
               
                 361 
                 GCAGAAGCAGAGGCAGTTCTTACAAAATTACAACAATATAATGATAAAGTACTTATAAAT 
                 420 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 121 
                 A  E  A  E  A  V  L  T  K  L  Q  Q  Y  N  D  K  V  L  I  N 
                 140 
               
               
                   
               
               
                 421 
                 TCTGCAACAGATACAGTAAAAGGTATGGTATCTGATACACAAGTTGATAGCAAAAATGTT 
                 480 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 141 
                 S  A  T  D  T  V  K  G  M  V  S  D  T  Q  V  D  S  K  N  V 
                 160 
               
               
                   
               
               
                 481 
                 GCAGCTAACCCACTTAAAGTTAGTGATATGTATACAATACCATCTGCTATTACTGGAAGT 
                 540 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 161 
                 A  A  N  P  L  K  V  S  D  M  Y  T  I  P  S  A  I  T  G  S 
                 180 
               
               
                   
               
               
                 541 
                 GATGATTCTGGGTATAGTATTGCTAAACCAACAGAAAAGACTACAaGTTTATTGTATGGT 
                 600 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 181 
                 D  D  S  G  Y  S  I  A  K  P  T  E  K  T  T  S  L  L  Y  G 
                 200 
               
               
                   
               
               
                 601 
                 ACGGTTGGTGATGCAACTGCAGGTAAAGCAATAACAGTAGATACAGCTTCAAATGAAGCT 
                 660 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 201 
                 T  V  G  D  A  T  A  G  K  A  I  T  V  D  T  A  S  N  E  A 
                 220 
               
               
                   
               
               
                 661 
                 TTTGCTGGAAATGGAAAGGTTATTGACTACAATAAATCATTCAAAGCAACTGTACAAGGA 
                 720 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 221 
                 F  A  G  N  G  K  V  I  D  Y  N  K  S  F  K  A  T  V  Q  G 
                 240 
               
               
                   
               
               
                 721 
                 GATGGAACAGTTAAGACAAGCGGGGTTGTACTTAAAGATGCAAGTGATATGGCTGCAACA 
                 780 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 241 
                 D  G  T  V  K  T  S  G  V  V  L  K  D  A  S  D  M  A  A  T 
                 260 
               
               
                   
               
               
                 781 
                 GGTACTATAAAAGTTAGAGTTACAAGTGCAAAAGAAGAATCTATTGATGTGGATTCAAGT 
                 840 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 261 
                 G  T  I  K  V  R  V  T  S  A  K  E  E  S  I  D  V  D  S  S 
                 280 
               
               
                   
               
               
                 841 
                 TCATATATTAGTGCTGAAAATTTAGCTAAAAAATATGTATTTAATCCTAAAGAGGTTTCT 
                 900 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 281 
                 S  Y  I  S  A  E  N  L  A  K  K  Y  V  F  N  P  K  E  V  S 
                 300 
               
               
                   
               
               
                 901 
                 GAAGCTTATAATGCAATAGTTGCATTACAAAATGATGGAATAGAATCTGATTTAGTACAA 
                 960 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 301 
                 E  A  Y  N  A  I  V  A  L  Q  N  D  G  I  E  S  D  L  V  Q 
                 320 
               
               
                   
               
               
                 961 
                 TTAGTTAATGGAAAATATCAAGTTATTTTCTATCCAGAAGGAAAAAGATTAGAAACTAAA 
                 1020 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 321 
                 L  V  N  G  K  Y  Q  V  I  F  Y  P  E  G  K  R  L  E  T  K 
                 340 
               
               
                   
               
               
                 1021 
                 TCTGCAGATATAATAGCTGATGCAGATAGTCCAGCTAAAATAACTATAAAAGCTAATAAA 
                 1081 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 341 
                 S  A  D  I  I  A  D  A  D  S  P  A  K  I  T  I  K  A  N  K 
                 360 
               
               
                   
               
               
                   
                           ♦ 
               
               
                   
               
               
                 1081 
                 TTAAAAGATTTAAAAGATTATGTAGATGATTTAAAAACATACAATAATACTTACTCAAAT 
                 1140 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 361 
                 L  K  D  L  K  D  Y  V  D  D  L  K  T  Y  N  N  T  Y  S  N 
                 380 
               
               
                   
               
               
                 1141 
                 GTTGTAACAGTAGCAGGAGAAGATAGAATAGAAACTGCTATAGAATTAAGTAGTAAATAT 
                 1200 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 381 
                 V  V  T  V  A  G  E  D  R  I  E  T  A  I  E  L  S  S  K  Y 
                 400 
               
               
                   
               
               
                 1201 
                 TATAATTCTGATGATAAAAATGCAATAACTGATGATGCAGTTAATAATATAGTATTAGTT 
                 1260 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 401 
                 Y  N  S  D  D  K  N  A  I  T  D  D  A  V  N  N  I  V  L  V 
                 420 
               
               
                   
               
               
                 1261 
                 GGATCTACATCTATAGTTGATGGTCTTGTTGCATCACCATTAGCTTCAGAAAAAACAGCT 
                 1320 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 421 
                 G  S  T  S  I  V  D  G  L  V  A  S  P  L  A  S  E  K  T  A 
                 440 
               
               
                   
               
               
                 1321 
                 CCATTATTATTAACTTCAAAAGATAAATTAGATTCATCAGTAAAATCTGAGATAAAAAGA 
                 1380 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 441 
                 P  L  L  L  T  S  K  D  K  L  D  S  S  V  K  S  E  I  K  R 
                 460 
               
               
                   
               
               
                 1381 
                 GTTATGAACTTAAAGAGTGATACTGGTATAAATACTTCTAAAAAAGTTTATTTAGCTGGT 
                 1440 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 461 
                 V  M  N  L  K  S  D  T  G  I  N  T  S  K  K  V  Y  L  A  G 
                 480 
               
               
                   
               
               
                 1441 
                 GGAGTTAATTCTATATCTAAAGATGTAGAAGATGAATTGAAAAATATGGGCCTTAAAGTT 
                 1500 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 481 
                 G  V  N  S  I  S  K  D  V  E  D  E  L  K  N  M  G  L  K  V 
                 500 
               
               
                   
               
               
                 1501 
                 ACTAGATTATCAGGAGAAGACAGATACGAAACTTCTTTAGCAATAGCTGATGAAATAGGT 
                 1560 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 501 
                 T  R  L  S  G  E  D  R  Y  E  T  S  L  A  I  A  D  E  I  G 
                 520 
               
               
                   
               
               
                 1561 
                 CTTGATAATGATAAAGCATTTGTAGTTGGTGGTACTGGATTGGCAGATGCTATGAGTATA 
                 1620 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 521 
                 L  D  N  D  K  A  F  V  V  G  G  T  G  L  A  D  A  M  S  I 
                 540 
               
               
                   
               
               
                 1621 
                 GCTCCAGTTGCTTCTCAACTTAAAGATGGAGATGCTACTCCAATAGTAGTTGTAGATGGA 
                 1680 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 541 
                 A  P  V  A  S  Q  L  K  D  G  D  A  T  P  I  V  V  V  D  G 
                 560 
               
               
                   
               
               
                 1681 
                 AAAGCAAAAGAAATAAGTGATGATGCTAAGAGTTTCTTAGGAACTTCTGATGTTGATATA 
                 1740 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 561 
                 K  A  K  E  I  S  D  D  A  K  S  F  L  G  T  S  D  V  D  I 
                 580 
               
               
                   
               
               
                 1741 
                 ATAGGTGGAAAAAATAGCGTATCTAAAGAGATTGAAGAGTCAATAGATAGTGCAACTGGA 
                 1800 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 581 
                 I  G  G  K  N  S  V  S  K  E  I  E  E  S  I  D  S  A  T  G 
                 600 
               
               
                   
               
               
                 1801 
                 AAAACTCCAGATAGAATAAGTGGAGATGACAGACAAGCAACTAATGCTGAAGTTTTAAAA 
                 1860 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 601 
                 K  T  P  D  R  I  S  G  D  D  R  Q  A  T  N  A  E  V  L  K 
                 620 
               
               
                   
               
               
                 1861 
                 GAAGATGATTATTTCAAAGATGGTGAAGTTGTGAATTACTTTGTTGCAAAAGATGGTTCT 
                 1920 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 621 
                 E  D  D  Y  F  K  D  G  E  V  V  N  Y  F  V  A  K  D  G  S 
                 640 
               
               
                   
               
               
                 1921 
                 ACTAAAGAAGATCAATTAGTAGATGCATTAGCAGCAGCACCAATAGCAGGTAGATTTAAG 
                 1980 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 641 
                 T  K  E  D  Q  L  V  D  A  L  A  A  A  P  I  A  G  R  F  K 
                 660 
               
               
                   
               
               
                 1981 
                 GAGTCTCCAGCTCCAATCATACTAGCTACTGATACTTTATCTTCTGACCAAAATGTAGCT 
                 2040 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 661 
                 E  S  P  A  P  I  I  L  A  T  D  T  L  S  S  D  Q  N  V  A 
                 680 
               
               
                   
               
               
                 2041 
                 GTAAGTAAAGCAGTTCCTAAAGATGGTGGAACTAACTTAGTTCAAGTAGGTAAAGGTATA 
                 2100 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 681 
                 V  S  K  A  V  P  K  D  G  G  T  N  L  V  Q  V  G  K  G  I 
                 700 
               
               
                   
               
               
                 2101 
                 GCTTCTTCAGTTATAAACAAAATGAAAGATTTATTAGATATGTAA 
                 2145 
               
               
                   
                 ---------+---------+---------+---------+---------+----- 
               
               
                 701 
                 A  S  S  V  I  N  K  M  K  D  L  L  D  M  * 
                 715 
               
               
                   
               
             
          
         
       
     
         [0149]    
       
         
               
               
             
               
               
               
               
             
           
               
                 APPENDIX 6 
               
               
                   
               
               
                   
               
               
                 SEQ ID No 8. Nucleotide sequence of slpA from  Clostridium   
                   
               
               
                   difficile  strain 173644, PCR type 31, with translation. The 
               
               
                 putative secretory signal cleavage site (□) and site of 
               
               
                 cleavage to form the two mature SLPs (♦) are indicated. 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 1 
                 ATGAATAAGAAGGATATAGCAATAGCTATGTCAGGATTAACAGTATTAGCTTCTGCAGCA 
                 60 
                   
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 1 
                 M  N  K  K  D  I  A  I  A  M  S  G  L  T  V  L  A  S  A  A 
                 20 
               
               
                   
               
               
                 61 
                 CCTGTATTTGCTGCTAGTAGTTTTACAGCAGATTATAATTATACTGTAGTGCAAGGAAAA 
                 120 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 21 
                 P  V  F  A  A  S  S  F  T  A  D  Y  N  Y  T  V  V  Q  G  K 
                 40 
               
               
                   
               
               
                   
                                     □ 
               
               
                   
               
               
                 121 
                 TATCAAAAAGTTATAACTGGATTACAAGATGGTTTAAAAAATGGAAAAATAACAAATATT 
                 180 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 41 
                 Y  Q  K  V  I  T  G  L  Q  D  G  L  K  N  G  K  I  T  N  I 
                 60 
               
               
                   
               
               
                 181 
                 GATGTAATATTTGATGGAAGTTCAATTGGTGAGGTAGTGCCAGGTTCTGATGCTGCAGCT 
                 240 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 61 
                 D  V  I  F  D  G  S  S  I  G  E  V  V  P  G  S  D  A  A  A 
                 80 
               
               
                   
               
               
                 241 
                 GCAGCTACTAAATTAAAAAGTTTAGTTGATGATAAGTTAGATAACTTAGGTGATGGAAAA 
                 300 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 81 
                 A  A  T  K  L  K  S  L  V  D  D  K  L  D  N  L  G  D  G  K 
                 100 
               
               
                   
               
               
                 301 
                 TACGTTCAATTTAATGTTACTTATACTACTAAATCTATAATAACTAAAGCAGAATTAAAA 
                 360 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 101 
                 Y  V  Q  F  N  V  T  Y  T  T  K  S  I  I  T  K  A  E  L  K 
                 120 
               
               
                   
               
               
                 361 
                 AATTATTATAATCAATTAGAAAGTAGTAAAGATAGAATACTTATAGGAAATGAACCTCAA 
                 420 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 121 
                 N  Y  Y  N  Q  L  E  S  S  K  D  R  I  L  I  G  N  E  P  Q 
                 140 
               
               
                   
               
               
                 421 
                 GATACAGGAACTAAAGGTCTTATAAAAGCTGATACTGATGGTACTACTGCTGTTGCAGCA 
                 480 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 141 
                 D  T  G  T  K  G  L  I  K  A  D  T  D  G  T  T  A  V  A  A 
                 160 
               
               
                   
               
               
                 481 
                 GCTGCACCATTGAAATTATCAGATATATTTACGTTTAGTTATGATGAAGTAACAGGTGTA 
                 540 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 161 
                 A  A  P  L  K  L  S  D  I  F  T  F  S  Y  D  E  V  T  G  V 
                 180 
               
               
                   
               
               
                 541 
                 CTTAAAGCAGAACCAACAAGTAAAGTAAGCGCTGGTAAAGTTCAAGGTCTAAAATATGGA 
                 600 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 181 
                 L  K  A  E  P  T  S  K  V  S  A  G  K  V  Q  G  L  K  Y  G 
                 200 
               
               
                   
               
               
                 601 
                 AATACAGGAGCAACTAACTATACTTCTGGAGCTGAAATATCTGTTCCTACTACAGGCTTA 
                 660 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 201 
                 N  T  G  A  T  N  Y  T  S  G  A  E  I  S  V  P  T  T  G  L 
                 220 
               
               
                   
               
               
                 661 
                 ACATTAACTGCTGATACAACTGCAACAACAGATGTAAATATTTCTGATGTTATGAGTGCA 
                 720 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 221 
                 T  L  T  A  D  T  T  A  T  T  D  V  N  I  S  D  V  M  S  A 
                 240 
               
               
                   
               
               
                 721 
                 TTTAAATTTAATGGTACTGATACGATTAGTGGATTCCCAGCTGGTTCATCAGCTTCTACT 
                 780 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 241 
                 F  K  F  N  G  T  D  T  I  S  G  F  P  A  G  S  S  A  S  T 
                 260 
               
               
                   
               
               
                 781 
                 CTTAGAGCAAGTATAAAAGTAATAAATGCAAAAGAAGAATCTATAGATGTTGATTCAAGT 
                 840 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 261 
                 L  R  A  S  I  K  V  I  N  A  K  E  E  S  I  D  V  D  S  S 
                 280 
               
               
                   
               
               
                 841 
                 TCACATAGAACAGCTGAAGATTTAGCTGAAAAATATGTATTTAAACCAGAAGATGTGAAT 
                 900 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 281 
                 S  H  R  T  A  E  D  L  A  E  K  Y  V  F  K  P  E  D  V  N 
                 300 
               
               
                   
               
               
                 901 
                 AAAACTTATGAGGCACTGACTGATTTATATAAAGAAGGTATAACAAGTAATCTTATCACT 
                 960 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 301 
                 K  T  Y  E  A  L  T  D  L  Y  K  E  G  I  T  S  N  L  I  T 
                 320 
               
               
                   
               
               
                 961 
                 CAAGATGGTGGAAAATATCAAGTTGTTTTATTTGCTCAAGGAAAGAGATTAACTACTAAA 
                 1020 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 321 
                 Q  D  G  G  K  Y  Q  V  V  L  F  A  Q  G  K  R  L  T  T  K 
                 340 
               
               
                   
               
               
                 1021 
                 GGAGCAACTGGAACTTTAGCAGATGAAAATTCTCCTCTTAAAGTAACAATAAAAGCAGAT 
                 1080 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 341 
                 G  A  T  G  T  L  A  D  E  N  S  P  L  K  V  T  I  K  A  D 
                 360 
               
               
                   
               
               
                   
                            ♦ 
               
               
                   
               
               
                 1081 
                 AAAGTAAAAGACTTAAAAGATTATGTTGAAGATTTAAAAAATGCTAACAATGGATATTCA 
                 1140 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 361 
                 K  V  K  D  L  K  D  Y  V  E  D  L  K  N  A  N  N  G  Y  S 
                 380 
               
               
                   
               
               
                 1141 
                 AATTCTGTTGTTGTAGCAGGTGAAGATAGAATAGAAACAGCAATAGAGTTAAGTAGCAAA 
                 1200 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 381 
                 N  S  V  V  V  A  G  E  D  R  I  E  T  A  I  E  L  S  S  K 
                 400 
               
               
                   
               
               
                 1201 
                 TACTATAACTCTGATGATGACAATGCAATAACTAAAGATCCAGTTAACAATGTTGTTTTA 
                 1260 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 401 
                 Y  Y  N  S  D  D  D  N  A  I  T  K  D  P  V  N  N  V  V  L 
                 420 
               
               
                   
               
               
                 1261 
                 GTTGGTTCTCAAGCTGTAGTTGATGGGCTTGTAGCTTCACCTTTAGCATCTGAAAAAAGA 
                 1320 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 421 
                 V  G  S  Q  A  V  V  D  G  L  V  A  S  P  L  A  S  E  K  R 
                 440 
               
               
                   
               
               
                 1321 
                 GCTCCTTTACTATTAACTTCAGCAGGAAAATTAGATTCAAGTGTTAAAGCTGAGTTGAAA 
                 1380 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 441 
                 A  P  L  L  L  T  S  A  G  K  L  D  S  S  V  K  A  E  L  K 
                 460 
               
               
                   
               
               
                 1381 
                 AGAGTAATGGATTTAAAATCTACAACAGGTGTAAATACTTCTAAAAAAGTTTACTTAGCT 
                 1440 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 461 
                 R  V  M  D  L  K  S  T  T  G  V  N  T  S  K  K  V  Y  L  A 
                 480 
               
               
                   
               
               
                 1441 
                 GGTGGAGTAAACTCTATATCTAAAGATGTAGAAAATGAATTAAAAGATATGGGACTTAAA 
                 1500 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 481 
                 G  G  V  N  S  I  S  K  D  V  E  N  E  L  K  D  M  G  L  K 
                 500 
               
               
                   
               
               
                 1501 
                 GTTACAAGATTATCAGGAGATGATAGATATGAAACTTCTTTAGCTATAGCTGATGAAATA 
                 1560 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 501 
                 V  T  R  L  S  G  D  D  R  Y  E  T  S  L  A  I  A  D  E  I 
                 520 
               
               
                   
               
               
                 1561 
                 GGTCTTGATAATGATAAAGCTTTTGTAGTTGGAGGAACAGGATTAGCGGATGCTATGAGT 
                 1620 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 521 
                 G  L  D  N  D  K  A  F  V  V  G  G  T  G  L  A  D  A  M  S 
                 540 
               
               
                   
               
               
                 1621 
                 ATAGCTCCAGTTGCTTCTCAATTAAGAAACTCAAATGGAGAACTTGACTTAAAAGGTGAT 
                 1680 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 541 
                 I  A  P  V  A  S  Q  L  R  N  S  N  G  E  L  D  L  K  G  D 
                 560 
               
               
                   
               
               
                 1681 
                 GCAACTCCAATAGTAGTTGTTGATGGAAAAGCTAAAGATATAAATTCTGAAGTAAAAGAT 
                 1740 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 561 
                 A  T  P  I  V  V  V  D  G  K  A  K  D  I  N  S  E  V  K  D 
                 580 
               
               
                   
               
               
                 1741 
                 TTCTTAGATGATTCACAAGTTGATATAATAGGTGGTGTAAATAGTGTTTCTAAAGAAGTA 
                 1800 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 581 
                 F  L  D  D  S  Q  V  D  I  I  G  G  V  N  S  V  S  K  E  V 
                 600 
               
               
                   
               
               
                 1801 
                 ATGGAAGCAATAGATGATGCTACTGGAAAATCACCTGAGAGATATAGTGGAGAAGATAGA 
                 1860 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 601 
                 M  E  A  I  D  D  A  T  G  K  S  P  E  R  Y  S  G  E  D  R 
                 620 
               
               
                   
               
               
                 1861 
                 CAAGCAACAAATGCTAAAGTTATAAAAGAAGATGATTTCTTTAAAAATGGAGAAGTTACA 
                 1920 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 621 
                 Q  A  T  N  A  K  V  I  K  E  D  D  F  F  K  N  G  E  V  T 
                 640 
               
               
                   
               
               
                 1921 
                 AACTTCTTTGTAGCTAAAGATGGTTCAACTAAAGAAGATCAATTAGTAGATGCTTTAGCA 
                 1980 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 641 
                 N  F  F  V  A  K  D  G  S  T  K  E  D  Q  L  V  D  A  L  A 
                 660 
               
               
                   
               
               
                 1981 
                 GGTGCTGCAATTGCTGGTAACTTTGGTGTAACAGTAGATAATGAAGGAAAACCTACAGTT 
                 2040 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 661 
                 G  A  A  I  A  G  N  F  G  V  T  V  D  N  E  G  K  P  T  V 
                 680 
               
               
                   
               
               
                 2041 
                 GCTGATAAAAAAGCTTCTCCAGCACCAATTGTTTTAGCAACAGATTCTTTATCTTCTGAT 
                 2100 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 681 
                 A  D  K  K  A  S  P  A  P  I  V  L  A  T  D  S  L  S  S  D 
                 700 
               
               
                   
               
               
                 2101 
                 CAAAATGTAGCTATAAGTAAAGCTGTAAATGATGACGCTAATACTAAGAATCTAGTTCAA 
                 2160 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 701 
                 Q  N  V  A  I  S  K  A  V  N  D  D  A  N  T  K  N  L  V  Q 
                 720 
               
               
                   
               
               
                 2161 
                 GTTGGTAAAGGTATAGCTACTTCAGTTGTAAGTAAAATAAAAGATTTATTAGATATG 
                 2217 
               
               
                   
                 ---------+---------+---------+---------+---------+------- 
               
               
                 721 
                 V  G  K  G  I  A  T  S  V  V  S  K  I  K  D  L  L  D  M 
                 739 
               
               
                   
               
             
          
         
       
     
         [0150]    
       
         
               
               
             
               
               
               
               
             
           
               
                 APPENDIX 7 
               
               
                   
               
               
                   
               
               
                 SEQ ID No 9. Nucleotide sequence of slpA from  Clostridium   
                   
               
               
                   difficile  strain 170444, PCR type 46, with translation. The 
               
               
                 putative secretory signal cleavage site (□) and site of 
               
               
                 cleavage to form the two mature SLPs (♦) are indicated. 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 1 
                 ATGAATAAGAAAAATATAGCAATAGCTATGTCAGGTTTAACAGTTTTAGCTTCGGCTGCT 
                 60 
                   
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 1 
                 M  N  K  K  N  I  A  I  A  M  S  G  L  T  V  L  A  S  A  A 
                 20 
               
               
                   
               
               
                 61 
                 CCTGTTTTTGCTGCAACTACTGGAACACAAGGTTATACTGTAGTTAAAAACGACTGGAAA 
                 120 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 21 
                 P  V  F  A  A  T  T  G  T  Q  G  Y  T  V  V  K  N  D  W  K 
                 40 
               
               
                   
               
               
                   
                                     □ 
               
               
                   
               
               
                 121 
                 AAAGCAGTAAAACAATTACAAGATGGACTAAAAGATAATAGTATAGGAAAGATAACTGTA 
                 180 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 41 
                 K  A  V  K  Q  L  Q  D  G  L  K  D  N  S  I  G  K  I  T  V 
                 60 
               
               
                   
               
               
                 181 
                 TCTTTTAATGATGGGGTTGTGGGTGAAGTAGCTCCTAAAAGTGCTAATAAGAAAGCGGAC 
                 240 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 61 
                 S  F  N  D  G  V  V  G  E  V  A  P  K  S  A  N  K  K  A  D 
                 80 
               
               
                   
               
               
                 241 
                 AGAGATGCTGCAGCTGAGAAGTTATATAATCTTGTTAACACTCAATTAGATAAATTAGGT 
                 300 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 81 
                 R  D  A  A  A  E  K  L  Y  N  L  V  N  T  Q  L  D  K  L  G 
                 100 
               
               
                   
               
               
                 301 
                 GATGGAGATTATGTTGATTTTTCTGTAGATTATAATTTAGAAAAAAAAATAATAACTAAT 
                 360 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 101 
                 D  G  D  Y  V  D  F  S  V  D  Y  N  L  E  K  K  I  I  T  N 
                 120 
               
               
                   
               
               
                 361 
                 CAAGCAGATGCAGAAGCAATTGTTACAAAGTTAAATTCACTTAATGAGAAAACTCTTATT 
                 420 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 121 
                 Q  A  D  A  E  A  I  V  T  K  L  N  S  L  N  E  K  T  L  I 
                 140 
               
               
                   
               
               
                 421 
                 GATATAGCAACTAAAGATACTTTTGGAATGGTTAGTAAAACACAAGATAGTGAAGGTAAA 
                 480 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 141 
                 D  I  A  T  K  D  T  F  G  M  V  S  K  T  Q  D  S  E  G  K 
                 160 
               
               
                   
               
               
                 481 
                 AATGTTGCTGCAACAAAGGCACTTAAAGTTAAAGATGTTGCTACATTTGGTTTGAAGTCT 
                 540 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 161 
                 N  V  A  A  T  K  A  L  K  V  K  D  V  A  T  F  G  L  K  S 
                 180 
               
               
                   
               
               
                 541 
                 GGTGGAAGCGAAGATACTGGATATGTTATTGAAATGAAAGCAGGAGCTGTAGAGGATAAG 
                 600 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 181 
                 G  G  S  E  D  T  G  Y  V  I  E  M  K  A  G  A  V  E  D  K 
                 200 
               
               
                   
               
               
                 601 
                 TATGGTAAAGTTGGAGATAGTACGGCAGGTATTGCAATAAATCTTCCTAGTACTGGACTT 
                 660 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 201 
                 Y  G  K  V  G  D  S  T  A  G  I  A  I  N  L  P  S  T  G  L 
                 220 
               
               
                   
               
               
                 661 
                 GAATATGCAGGTAAAGGAACAACAATTGATTTTAATAAAACTTTAAAAGTTGATGTAACA 
                 720 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 221 
                 E  Y  A  G  K  G  T  T  I  D  F  N  K  T  L  K  V  D  V  T 
                 240 
               
               
                   
               
               
                 721 
                 GGTGGTTCAACACCTAGTGCTGTAGCTGTAAGTGGTTTTGTAACTAAAGATGATACTGAT 
                 780 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 241 
                 G  G  S  T  P  S  A  V  A  V  S  G  F  V  T  K  D  D  T  D 
                 260 
               
               
                   
               
               
                 781 
                 TTAGCAAAATCAGGTACTATAAATGTAAGAGTTATAAATGCAAAAGAAGAATCAATTGAT 
                 840 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 261 
                 L  A  K  S  G  T  I  N  V  R  V  I  N  A  K  E  E  S  I  D 
                 280 
               
               
                   
               
               
                 841 
                 ATAGATGCAAGCTCATATACATCAGCTGAAAATTTAGCTAAAAGACATGTATTTGATCCA 
                 900 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 281 
                 I  D  A  S  S  Y  T  S  A  E  N  L  A  K  R  H  V  F  D  P 
                 300 
               
               
                   
               
               
                 901 
                 GATGAAATTTCTGAAGCATATAAGGCAATAGTAGCATTACAAAATGATGGTATAGAGTCT 
                 960 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 301 
                 D  E  I  S  E  A  Y  K  A  I  V  A  L  Q  N  D  G  I  E  S 
                 320 
               
               
                   
               
               
                 961 
                 AATTTAGTTCAGTTAGTTAATGGAAAATATCAAGTGATTTTTTATCCAGAAGGTAAAAGA 
                 1020 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 321 
                 N  L  V  Q  L  V  N  G  K  Y  Q  V  I  F  Y  P  E  G  K  R 
                 340 
               
               
                   
               
               
                 1021 
                 TTAGAAACTAAATCAGCAAATGATACAATAGCTAGTCAAGATACACCAGCTAAAGTAGTT 
                 1080 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 341 
                 L E T K S A N D T I A S Q D T P A K V V 
                 360 
               
               
                   
               
               
                   
                                       ♦ 
               
               
                   
               
               
                 1081 
                 ATAAAAGCTAATAAATTAAAAGATTTAAAAGATTATGTAGATGATTTAAAAACATATAAT 
                 1140 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 361 
                 I  K  A  N  K  L  K  D  L  K  D  Y  V  D  D  L  K  T  Y  N 
                 380 
               
               
                   
               
               
                 1141 
                 AATACTTATTCAAATGTTGTAACAGTAGCAGGAGAAGATAGAATAGAAACTGCTATAGAA 
                 1200 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 381 
                 N  T  Y  S  N  V  V  T  V  A  G  E  D  R  I  E  T  A  I  E 
                 400 
               
               
                   
               
               
                 1201 
                 TTAAGTAGTAAATATTATAATTCTGATGATAAAAATGCAATAACTGATAAAGCAGTTAAT 
                 1260 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 401 
                 L  S  S  K  Y  Y  N  S  D  D  K  N  A  I  T  D  K  A  V  N 
                 420 
               
               
                   
               
               
                 1261 
                 GATATAGTATTAGTTGGATCTACATCTATAGTTGATGGTCTTGTTGCATCACCATTAGCT 
                 1320 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 421 
                 D  I  V  L  V  G  S  T  S  I  V  D  G  L  V  A  S  P  L  A 
                 440 
               
               
                   
               
               
                 1321 
                 TCAGAAAAAACAGCTCCATTATTATTAACTTCAAAAGATAAATTAGATTCATCAGTAAAA 
                 1380 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 441 
                 S  E  K  T  A  P  L  L  L  T  S  K  D  K  L  D  S  S  V  K 
                 460 
               
               
                   
               
               
                 1381 
                 TCTGAAATAAAGAGAGTTATGAACTTAAAGAGTGACACTGGTATAAATACTTCTAAAAAA 
                 1440 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 461 
                 S  E  I  K  R  V  M  N  L  K  S  D  T  G  I  N  T  S  K  K 
                 480 
               
               
                   
               
               
                 1441 
                 GTTTATTTAGCTGGTGGAGTTAATTCTATATCTAAAGATGTAGAAAATGAATTGAAAAAC 
                 1500 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 481 
                 V  Y  L  A  G  G  V  N  S  I  S  K  D  V  E  N  E  L  K  N 
                 500 
               
               
                   
               
               
                 1501 
                 ATGGGTCTTAAAGTTACTAGATTATCAGGAGAAGACAGATACGAAACTTCTTTAGCAATA 
                 1560 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 501 
                 M  G  L  K  V  T  R  L  S  G  E  D  R  Y  E  T  S  L  A  I 
                 520 
               
               
                   
               
               
                 1561 
                 GCTGATGAAATAGGTCTTGATAATGATAAAGCATTTGTAGTTGGTGGTACTGGATTAGCA 
                 1620 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 521 
                 A  D  E  I  G  L  D  N  D  K  A  F  V  V  G  G  T  G  L  A 
                 540 
               
               
                   
               
               
                 1621 
                 GATGCTATGAGTATAGCTCCAGTTGCTTCTCAACTTAAAGATGGAGATGCTACTCCAATA 
                 1680 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 541 
                 D  A  M  S  I  A  P  V  A  S  Q  L  K  D  G  D  A  T  P  I 
                 560 
               
               
                   
               
               
                 1681 
                 GTAGTTGTAGATGGAAAAGCAAAAGAAATAAGTGATGATGCTAAGAGTTTCTTAGGAACT 
                 1740 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 561 
                 V  V  V  D  G  K  A  K  E  I  S  D  D  A  K  S  F  L  G  T 
                 580 
               
               
                   
               
               
                 1741 
                 TCTGATGTTGATATAATAGGTGGAAAAAATAGCGTATCTAAAGAGATTGAAGAGTCAATA 
                 1800 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 581 
                 S  D  V  D  I  I  G  G  K  N  S  V  S  K  E  I  E  E  S  I 
                 600 
               
               
                   
               
               
                 1801 
                 GATAGTGCAACTGGAAAAACTCCAGATAGAATAAGTGGAGATGATAGACAAGCAACTAAT 
                 1860 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 601 
                 D  S  A  T  G  K  T  P  D  R  I  S  G  D  D  R  Q  A  T  N 
                 620 
               
               
                   
               
               
                 1861 
                 GCTGAAGTTTTAAAAGAAGATGATTATTTCACAGATGGTGAAGTTGTGAATTACTTTGTT 
                 1920 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 621 
                 A  E  V  L  K  E  D  D  Y  F  T  D  G  E  V  V  N  Y  F  V 
               
               
                   
               
               
                 1921 
                 GCAAAAGATGGTTCTACTAAAGAAGATCAATTAGTAGATGCCTTAGCAGCAGCACCAATA 
                 1980 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 641 
                 A  K  D  G  S  T  K  E  D  Q  L  V  D  A  L  A  A  A  P  I 
                 660 
               
               
                   
               
               
                 1981 
                 GCAGGTAGATTTAAGGAGTCTCCAGCTCCAATCATACTAGCTACTGATACTTTATCTTCT 
                 2040 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 661 
                 A  G  R  F  K  E  S  P  A  P  I  I  L  A  T  D  T  L  S  S 
                 680 
               
               
                   
               
               
                 2041 
                 GACCAAAATGTAGCTGTAAGTAAAGCAGTTCCTAAAGATGGTGGAACTAACTTAGTTCAA 
                 2100 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 681 
                 D  Q  N  V  A  V  S  K  A  V  P  K  D  G  G  T  N  L  V  Q 
                 700 
               
               
                   
               
               
                 2101 
                 GTAGGTAAAGGTATAGCTTCTTCAGTTATAAACAAAATGAAAGATTTATTAGATATG 
                 2157 
               
               
                   
                 ---------+---------+---------+---------+---------+------- 
               
               
                 701 
                 V  G  K  G  I  A  S  S  V  I  N  K  M  K  D  L  L  D  M 
                 719 
               
               
                   
               
             
          
         
       
     
         [0151]    
       
         
               
               
             
               
               
               
               
             
           
               
                 APPENDIX 8 
               
               
                   
               
               
                   
               
               
                 SEQ ID No 10. Nucleotide sequence of slpA from  Clostridium   
                   
               
               
                   difficile  strain 170426, PCR type 92, with translation. The 
               
               
                 putative secretory signal cleavage site (□) and site of 
               
               
                 cleavage to form the two mature SLPs (♦) are indicated. 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 1 
                 ATGAATAAGAAAAATATAGCAATAGCTATGTCAGGTTTAACAGTTTTAGCTTCGGCTGCT 
                 60 
                   
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 1 
                 M  N  K  K  N  I  A  I  A  M  S  G  L  T  V  L  A  S  A  A 
                 20 
               
               
                   
               
               
                 61 
                 CCTGTTTTTGCTGCAACTACTGGAACACAAGGTTATACTGTAGTTAAAAACGACTGGAAA 
                 120 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 21 
                 P  V  F  A  A  T  T  G  T  Q  G  Y  T  V  V  K  N  D  W  K 
                 40 
               
               
                   
               
               
                   
                                    □ 
               
               
                   
               
               
                 121 
                 AAAGCAGTAAAACAATTACAGGATGGACTAAAAGATAATAGTATAGGAAAGATAACTGTA 
                 180 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 41 
                 K  A  V  K  Q  L  Q  D  G  L  K  D  N  S  I  G  K  I  T  V 
                 60 
               
               
                   
               
               
                 181 
                 TCTTTTAATGATGGGGTTGTGGGTGAAGTAGCTCCTAAAAGTGCTAATAAGAAAGCGGAC 
                 240 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 61 
                 S  F  N  D  G  V  V  G  E  V  A  P  K  S  A  N  K  K  A  D 
                 80 
               
               
                   
               
               
                 241 
                 AGAGATGCTGCAGCTGAGAAGTTATATAATCTTGTTAACACTCAATTAGATAAATTAGGT 
                 300 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 81 
                 R  D  A  A  A  E  K  L  Y  N  L  V  N  T  Q  L  D  K  L  G 
                 301 
               
               
                   
               
               
                 301 
                 GATGGAGATTATGTTGATTTTTCTGTAGATTATAATTTAGAAAAAAAAATAATAACTAAT 
                 360 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 101 
                 D  G  D  Y  V  D  F  S  V  D  Y  N  L  E  K  K  I  I  T  N 
                 120 
               
               
                   
               
               
                 361 
                 CAAGCAGATGCAGAAGCAATTGTTACAAAGTTAAATTCACTTAATGAGAAAACTCTTATT 
                 420 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 121 
                 Q  A  D  A  E  A  I  V  T  K  L  N  S  L  N  E  K  T  L  I 
                 140 
               
               
                   
               
               
                 421 
                 GATATAGCAACTAAAGATACTTTTGGAATGGTTAGTAAAACACAAGATAGTGAAGGTAAA 
                 480 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 141 
                 D  I  A  T  K  D  T  F  G  M  V  S  K  T  Q  D  S  E  G  K 
                 160 
               
               
                   
               
               
                 481 
                 AATGTTGCTGCAACAAAGGCACTTAAAGTTAAAGATGTTGCTACATTTGGTTTGAAGTCT 
                 540 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 161 
                 N  V  A  A  T  K  A  L  K  V  K  D  V  A  T  F  G  L  K  S 
                 180 
               
               
                   
               
               
                 541 
                 GGTGGAAGCGAAGATACTGGATATGTTGTTGAAATGAAAGCAGGAGCTGTAGAGGATAAG 
                 600 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 181 
                 G  G  S  E  D  T  G  Y  V  V  E  M  K  A  G  A  V  E  D  K 
                 200 
               
               
                   
               
               
                 601 
                 TATGGTAAAGTTGGAGATAGTACGGCAGGTATTGCAATAAATCTTCCTAGTACTGGACTT 
                 660 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 201 
                 Y  G  K  V  G  D  S  T  A  G  I  A  I  N  L  P  S  T  G  L 
                 220 
               
               
                   
               
               
                 661 
                 GAATATGCAGGTAAAGGAACAACAATTGATTTTAATAAAACTTTAAAAGTTGATGTAACA 
                 720 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 221 
                 E  Y  A  G  K  G  T  T  I  D  F  N  K  T  L  K  V  D  V  T 
                 240 
               
               
                   
               
               
                 721 
                 GGTGGTTCAACACCTAGTGCTGTAGCTGTAAGTGGTTTTGTAACTAAAGATGATACTGAT 
                 780 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 241 
                 G  G  S  T  P  S  A  V  A  V  S  G  F  V  T  K  D  D  T  D 
                 260 
               
               
                   
               
               
                 781 
                 TTAGCAAAATCAGGTACTATAAATGTAAGAGTTATAAATGCAAAAGAAGAATCAATTGAT 
                 840 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 261 
                 L  A  K  S  G  T  I  N  V  R  V  I  N  A  K  E  E  S  I  D 
                 280 
               
               
                   
               
               
                 841 
                 ATAGATGCAAGCTCATATACATCAGCTGAAAATTTAGCTAAAAGATATGTATTTGATCCA 
                 900 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 281 
                 I  D  A  S  S  Y  T  S  A  E  N  L  A  K  R  Y  V  F  D  P 
                 300 
               
               
                   
               
               
                 901 
                 GATGAAATTTCTGAAGCATATAAGGCAATAGTAGCATTACAAAATGATGGTATAGAGTCT 
                 960 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 301 
                 D  E  I  S  E  A  Y  K  A  I  V  A  L  Q  N  D  G  I  E  S 
                 320 
               
               
                   
               
               
                 961 
                 AATTTAGTTCAGTTAGTTAATGGAAAATATCAAGTGATTTTTTATCCAGAAGGTAAAAGA 
                 1020 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 321 
                 N  L  V  Q  L  V  N  G  K  Y  Q  V  I  F  Y  P  E  G  K  R 
                 340 
               
               
                   
               
               
                 1021 
                 TTAGAAACTAAATCAGCAAATGATACAATAGCTAGTCAAGATACACCAGCTAAAGTAGTT 
                 1080 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 341 
                 L  E  T  K  S  A  N  D  T  I  A  S  Q  D  T  P  A  K  V  V 
                 360 
               
               
                   
               
               
                   
                                       ♦ 
               
               
                   
               
               
                 1081 
                 ATAAAAGCTAATAAATTAAAAGATTTAAAAGATTATGTAGATGATTTAAAAACATATAAT 
                 1140 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 361 
                 I  K  A  N  K  L  K  D  L  K  D  Y  V  D  D  L  K  T  Y  N 
                 380 
               
               
                   
               
               
                 1141 
                 AATACTTATTCAAATGTTGTAACAGTAGCAGGAGAAGATAGAATAGAAACTGCTATAGAA 
                 1200 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 381 
                 N  T  Y  S  N  V  V  T  V  A  G  E  D  R  I  E  T  A  I  E 
                 400 
               
               
                   
               
               
                 1201 
                 TTAAGTAGTAAATATTATAATTCTGATGATAAAAATGCAATAACTGATAAAGCAGTTAAT 
                 1260 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 401 
                 L  S  S  K  Y  Y  N  S  D  D  K  N  A  I  T  D  K  A  V  N 
                 420 
               
               
                   
               
               
                 1261 
                 GATATAGTATTAGTTGGATCTACATCTATAGTTGATGGTCTTGTTGCATCACCATTAGCT 
                 1320 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 421 
                 D  I  V  L  V  G  S  T  S  I  V  D  G  L  V  A  S  P  L  A 
                 440 
               
               
                   
               
               
                 1321 
                 TCAGAAAAAACAGCTCCATTATTATTAACTTCAAAAGATAAATTAGATTCATCAGTAAAA 
                 1380 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 441 
                 S  E  K  T  A  P  L  L  L  T  S  K  D  K  L  D  S  S  V  K 
                 460 
               
               
                   
               
               
                 1381 
                 TCTGAAATAAAGAGAGTTATGAACTTAAAGAGTGACACTGGTATAAATACTTCTAAAAAA 
                 1440 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 461 
                 S  E  I  K  R  V  M  N  L  K  S  D  T  G  I  N  T  S  K  K 
                 480 
               
               
                   
               
               
                 1441 
                 GTTTATTTAGCTGGTGGAGTTAATTCTATATCTAAAGATGTAGAAAATGAATTGAAAAAC 
                 1500 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 481 
                 V  Y  L  A  G  G  V  N  S  I  S  K  D  V  E  N  E  L  K  N 
                 500 
               
               
                   
               
               
                 1501 
                 ATGGGTCTTAAAGTTACTAGATTATCAGGAGAAGACAGATACGAAACTTCTTTAGCAATA 
                 1560 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 501 
                 M  G  L  K  V  T  R  L  S  G  E  D  R  Y  E  T  S  L  A  I 
                 520 
               
               
                   
               
               
                 1561 
                 GCTGATGAAATAGGTCTTGATAATGATAAAGCATTTGTAGTTGGTGGTACTGGATTAGCA 
                 1620 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 521 
                 A  D  E  I  G  L  D  N  D  K  A  F  V  V  G  G  T  G  L  A 
                 540 
               
               
                   
               
               
                 1621 
                 GATGCTATGAGTATAGCTCCAGTTGCTTCTCAACTTAAAGATGGAGATGCTACTCCAATA 
                 1680 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 541 
                 D  A  M  S  I  A  P  V  A  S  Q  L  K  D  G  D  A  T  P  I 
                 560 
               
               
                   
               
               
                 1681 
                 GTAGTTGTAGATGGAAAAGCAAAAGAAATAAGTGATGATGCTAAGAGTTTCTTAGGAACT 
                 1740 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 561 
                 V  V  V  D  G  K  A  K  E  I  S  D  D  A  K  S  F  L  G  T 
                 580 
               
               
                   
               
               
                 1741 
                 TCTGATGTTGATATAATAGGTGGAAAAAATAGCGTATCTAAAGAGATTGAAGAGTCAATA 
                 1800 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 581 
                 S  D  V  D  I  I  G  G  K  N  S  V  S  K  E  I  E  E  S  I 
                 600 
               
               
                   
               
               
                 1801 
                 GATAGTGCAACTGGAAAAACTCCAGATAGAATAAGTGGAGATGATAGACAAGCAACTAAT 
                 1860 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 601 
                 D  S  A  T  G  K  T  P  D  R  I  S  G  D  D  R  Q  A  T  N 
                 620 
               
               
                   
               
               
                 1861 
                 GCTGAAGTTTTAAAAGAAGATGATTATTTCACAGATGGTGAAGTTGTGAATTACTTTGTT 
                 1920 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 621 
                 A  E  V  L  K  E  D  D  Y  F  T  D  G  E  V  V  N  Y  F  V 
                 640 
               
               
                   
               
               
                 1921 
                 GCAAAAGATGGTTCTACTAAAGAAGATCAATTAGTAGATGCCTTAGCAGCAGCACCAATA 
                 1980 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 641 
                 A  K  D  G  S  T  K  E  D  Q  L  V  D  A  L  A  A  A  P  I 
                 660 
               
               
                   
               
               
                 1981 
                 GCAGGTAGATTTAAGGAGTCTCCAGCTCCAATCATACTAGCTACTGATACTTTATCTTCT 
                 2040 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 661 
                 A  G  R  F  K  E  S  P  A  P  I  I  L  A  T  D  T  L  S  S 
                 680 
               
               
                   
               
               
                 2041 
                 GACCAAAATGTAGCTGTAAGTAAAGCAGTTCCTAAAGATGGTGGAACTAACTTAGTTCAA 
                 2100 
               
               
                   
                 ---------+---------+---------+---------+---------+--------- 
               
               
                 681 
                 D  Q  N  V  A  V  S  K  A  V  P  K  D  G  G  T  N  L  V  Q 
                 700 
               
               
                   
               
               
                 2101 
                 GTAGGTAAAGGTATAGCTTCTTCAGTTATAAACAAAATGAAAGATTTATTAGATATG 
                 2157 
               
               
                   
                 ---------+---------+---------+---------+---------+------- 
               
               
                 701 
                 V  G  K  G  I  A  S  S  V  I  N  K  M  K  D  L  L  D  M 
                 719 
               
               
                   
               
             
          
         
       
     
         [0152]