Patent Application: US-40926106-A

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 .

Description:
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 . 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 . 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 ). 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 . 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 /). 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 ( fig2 ), this entity may also be included in a vaccine . 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 . 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 . 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 . 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 . 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 . 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 . 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 . 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 . 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 . 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 . 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 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 . 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 . 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 the invention will be more clearly understood from the following examples . 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 . 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 . 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 . 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 ( fig1 a and 1b ). these antigens were recognised by antibodies from patients who recovered and represent potential candidate vaccine antigens . fig1 a shows a strong reaction of convalescent antibodies was observed with the 33 kda antigen ( lane 4 , arrow ). fig1 b shows a strong reaction of convalescent antibodies was observed with the 31 kda antigen ( lanes 6 and 7 , arrow ). 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 . 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 . 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 ]. 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 ) 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 ). 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 . 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 fig2 . 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 . slps were prepared from selected strains by urea extraction , and subjected to sds - page and staining with coomassie blue ( fig3 ). 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 ( fig3 , lane 2 ), which showed a single , high molecular weight band , approximately 43 000 in size . 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 ). 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 ). 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 . 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 . 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 . 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 . appendix 5 shows the open reading frame with translation for slpa from strain 171862 ( pcr type 17 ), seq id no 7 . 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 . 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 . 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 . 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 . 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 ( fig3 ). 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 ]. 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 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 . 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 . 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 . antibodies may be produced by any of the standard techniques well known in the art . 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 . 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 . 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 . the invention is not limited to the embodiments hereinbefore described which may be varied in detail . 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 . 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( 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 . 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 seq id no . 4 . nucleotide sequence of slpa from clostridium seq id no . 5 . nucleotide sequence of slpa from clostridium seq id no 6 . nucleotide sequence of slpa from clostridium seq id no . 7 . nucleotide sequence of slpa from clostridium seq id no 8 . nucleotide sequence of slpa from clostridium seq id no 9 . nucleotide sequence of slpa from clostridium l e t k s a n d t i a s q d t p a k v v seq id no 10 . nucleotide sequence of slpa from clostridium