Patent Abstract:
the invention relates to the field of the diagnosis of and vaccination against streptococcal infections , and to the detection of virulence markers of streptococci . the invention discloses a method for modulating virulence of a streptococcus comprising modifying a genomic fragment of the streptococcus , wherein the genomic fragment comprises at least a functional part of a fragment identifiable by hybridization in streptococcus suis to a nucleic acid or fragment thereof .

Detailed Description:
streptococcus suis is an important cause of meningitis , septicemia , arthritis and sudden death in young pigs ( clifton - haclley , 1983 ; vecht et al ., 1985 ). it can also cause meningitis in man ( arends and zanen , 1988 ). attempts to control the disease are still hampered by the lack of sufficient knowledge about the pathogenesis of the disease , the lack of effective vaccines and sensitive diagnostic methods . to meet these shortages , it is necessary to identify the genes that are involved in the pathogenic process . so far , only a limited number of s . suis genes are known ( smith et al ., 1992 ; smith et al ., 1993 ; serhir et al ., 1997 ; segers et al ., 1998 ; smith et al ., 1999 ; and accession nos . af106927 , z95920 and a57222 ) and of these , only a few are putatively involved in virulence ( smith et al ., 1992 ; smith et al ., 1993 ; jacobs et al ., 1994 ; gottschalk et al ., 1995 ; segers et al ., 1998 ; smith et al ., 1999 ). previously , putative virulence factors have been identified after growth of the bacteria in standard laboratory media . however , it is known that many important virulence factors are environmentally regulated and are induced at specific stages of the infection process ( mahan et al ., 1993 ). recently , several approaches have been reported that allow the identification of genes that are specifically expressed in the host . examples are signature - tagged mutagenesis ( stm ) and in vivo expression technology ( ivet ; mahan et al ., 1993 ; camilli and mekalanos , 1995 ; hensel et al ., 1995 ; mahan et al ., 1995 ; mei et al ., 1997 ; young and miller , 1997 ; chiang and mekalanos , 1998 ; coulter et al ., 1998 ; lowe et al ., 1998 ; polissi et al ., 1998 ; camacho et al ., 1999 ; darwin et al ., 1999 ; edeistein et al ., 1999 ; fuller et al ., 1999 ; zhao et al ., 1999 ). in addition , important virulence proteins could also be identified by the selection of genes specifically expressed under conditions mimicking in vivo conditions , for example by growth in iron - restricted conditions ( litwin and calderwood , 1993 ; martinez et al ., 1990 ). the present invention identifies virulence genes of s . suis by selecting environmentally regulated genes by experimental infections of piglets and by the use of iron - restricted conditions in vitro . for this purpose , chromosomal dna fragments of s . suis were cloned in a plasmid in front of a promoterless erythromycin - resistance gene . subsequently , the library was used for the selection of bacteria in which erythromycin resistance was induced under iron - restricted conditions . in addition , erythromycin - resistant bacteria were selected after infection of piglets with the library and treatment of the piglets with erythromycin . pigs were used instead of mice for these experiments since it was recently shown that virulence of s . suis is different in these two animal species ( vecht et al ., 1997 ). using this approach , 18 unique iron - restriction - induced ( iri ) genes , as well as 22 unique in vivo selected ( ivs ) genes , were identified , several of which are putatively involved in virulence ( smith et al ., 1993 ; smith et al ., 1996 ). bacterial strains and growth conditions . the bacterial strains and plasmids used in this study are listed in table 1 . s . suis strains were grown in todd - hewitt broth ( oxoid ), and plated on columbia agar ( oxoid ) containing 6 % ( v / v ) horse blood . for the selection of genes induced in iron - limited conditions , s . suis cells were plated on agar plates containing todd - hewitt medium , 5 % ( w / v ) yeast extract and 75 μm deferoxamine mesylate ( sigma ). control plates were supplemented with 38 μm feso 4 . 7h 2 o ( sigma ). if required , antibiotics were added at the following concentrations : 100 μg spectinomycin ml − 1 and 1 μg erythromycin ml − 1 . e . coli strains were grown in luria broth ( miller , 1972 ) and plated on luria broth containing 1 . 5 % ( w / v ) agar . if required , 50 μg ampicillin ml − 1 or 50 μg spectinomycin ml − 1 was added . construction of pivs - e . the ivs selection vector used in this study comprises a spectinomycin - resistance gene , a promoterless erythromycin - resistance gene and the origin of replication of the plasmid pwvo1 ( van der vossen et al ., 1987 ). to construct this pivs - e , the spectinomycin - resistance gene was amplified from pkun19 - spc ( konings et al ., 1987 ; smith et al ., 1995 ). in a pcr reaction , the primers 5 ′- tgcatgcatggatccatcga ttttcgttcg - 3 ′ ( seq id no : 2 ) and 5 ′- cgagctcggtacctgattaccaattagaat - 3 ′ ( seq id no : 3 ), which contained nsii and saci restriction sites at their respective 5 ′- ends were used . the obtained pcr product was digested with nsii and saci and ligated into pgkv210 ( van der vossen et al ., 1987 ) that had been digested with saci ( partially ) and nsii . the resulting plasmid was designated pgkv210 - spc . pe194 ( horinouchi and weisblum , 1982 ) was used as a template for the amplification of a promoterless erythromycin - resistance gene . to do this , the primers 5 ′- gggtcgaccctataaccaaattaaagaggg - 3 ′ ( seq id no : 4 ) and 5 ′- cccaagcttgggcagttta tgcatcccttaac - 3 ′ ( seq id no : 5 ) were used in a pcr reaction . these primers contained sali and hindiii restriction sites at their respective 5 ′- ends . the amplified fragment was digested with sali and hindiii and the fragment was ligated into pgkv210 - spc that had been digested with sali and hindiii . the resulting plasmid was designated pivs - e . to construct pivs - pe , the promoter region of the mrp gene was inserted into pivs - e 5 ′ to the promoterless erythromycin - resistance gene . the promoter region of the mrp gene was amplified by pcr from pmrp11 ( smith et al ., 1992 ) using the primers 5 ′- cccaagcttgggaattcataatgtttttttgagg - 3 ′ ( seq id no : 6 ) and 5 ′- gcgtcgaca tctacgcataaaaaatccccc - 3 ′ ( seq id no : 7 ). these primers contained ecori and sali sites at their respective 5 ′- ends . amplified dna was digested with ecori and sali and the resulting fragment was ligated into ecori and sali - digested pivs - e . construction of genomic s . suis libraries in pivs - e . alu i partial digests of s . suis serotype 2 strain 10 dna were size fractionated ( 500 - 1000 bp ) on a 0 . 8 % ( w / v ) agarose gel . the purified fragments were ligated to smai and calf intestinal phosphatase digested pivs - e and the ligation mixtures were transformed to e . coli xl2 - blue cells . spectinomycin - resistant colonies were selected . analysis of the transformants by pcr showed that more then 80 % contained an insert . from 15 pools of about 2000 - 3000 independent e . coli transformants , plasmid dna was isolated . this plasmid dna was subsequently used for the electrotransformation of s . suis strain 10 ( smith et al ., 1995 ). this resulted in approximately 30 , 000 independent s . suis transformants . the transformants were pooled and stored at − 80 ° c . dna techniques . routine dna manipulations and pcr reactions were performed as described by sambrook et al . ( 1989 ). dna sequences were determined on a 373a dna sequencing system ( applied biosystems ). samples were prepared by using the abi / prism dye terminator cycle sequencing ready reaction kit ( applied biosystems ). custom - made sequencing primers were purchased from life technologies . sequencing data were assembled and analyzed using the mcmollytetra software package . the blast program was used to search for protein sequences similar to the deduced amino acid sequences . pcr reaction mixtures ( 50 μl ) contained 10 mm tris - hcl , ph 8 . 3 , 1 . 5 mm mgcl 2 , 50 mm kcl , 0 . 2 mm of each of the four deoxynucleotide triphosphates , 1 μm of each of the primers and 1 u of amplitaq gold dna polymerase ( perkin elmer applied biosystems ). dna amplification was carried out in a perkin elmer 9600 thermal cycler and the program included an incubation for ten minutes at 95 ° c . and 30 cycles of one minute at 95 ° c ., two minutes at 56 ° c . and two minutes at 72 ° c . assessment of erythromycin levels in treated piglets . one - week - old specific pathogen - free ( spf ) piglets were treated orally with erythromycin stearate ( abbott , 20 or 40 mg body weight kg − 1 ) or intramuscularly with erythromycin ( erythrocin 200 ; sanofi santé , 20 or 40 mg body weight kg − 1 ). blood samples were collected 3 hours , 6 hours or 24 hours after the administration of the antibiotics to determine erythromycin levels . experimental infections . gnotobiotic great yorkshire and dutch landrace crossed piglets were obtained from sows by cesarian section . the surgery was performed in sterile flexible film isolators . the piglets were allotted to groups , each having 4 piglets , and were housed in sterile stainless steel incubators . housing conditions and feeding regimens were as described ( vecht et al ., 1989 ; vecht et al ., 1992 ). one - week - old piglets were inoculated intravenously with s . suis strain 10 ( pivs - e ), 10 ( pivs - pe ) or 10 ( pivs - re ) as described ( vecht et al ., 1989 ; vecht et al ., 1992 , table 3 ). two hours after infection , the pigs were injected intramuscularly with erythromycin for the first time and thereafter received erythromycin twice a day : once intramuscularly ( erythrocin , 40 mg body weight kg − 1 ) and once orally ( erythromycin stearate , 40 mg body weight kg − 1 ). piglets were monitored twice a day for clinical signs of disease , such as fever , nervous signs and lameness . blood samples were collected three times a week from each pig . leukocyte concentrations were determined using a conducting counter ( contraves a . g ., swizerland ). to monitor infection with s . suis and to check for absence of contaminants , swabs of the nasopharynx and of feces were collected daily . the swabs were directly plated onto columbia agar containing 6 % ( v / v ) horse blood . after the piglets were killed , they were examined for gross pathological changes . tissue specimens were collected from the central nervous system , serosae , joints , lungs , heart and tonsils . the tissues were homogenized in the presence of todd - hewitt medium using an ultra - turrax tissuemizer ( omni international ) and frozen at − 80 ° c . in the presence of 15 % ( v / v ) glycerol . the plasmid pivs - e was constructed to allow introduction of s . suis dna fragments into a number of unique restriction sites in front of a promoterless erythromycin - resistance gene . the plasmid carries the origin of replication of pwvo1 , which functions in e . coli and in s . suis ( smith et al ., 1995 ). s . suis strain 10 cells containing pivs - e were sensitive to 1 μg erythromycin ml − 1 on agar plates . in pivs - pe the promoter of the mrp gene of s . suis ( smith et al ., 1992 ), which is highly expressed in vivo as well as in vitro , drives expression of the erythromycin - resistance gene . s . suis strain 10 cells containing pivs - pe were resistant to high concentrations of erythromycin (& gt ; 256 μg erythromycin ml − 1 ) on agar plates . a s . suis dna library in pivs - e ( pivs - re ) was constructed and 30 , 000 individual s . suis clones or mutants were obtained . as determined by analysis of 24 randomly selected transformants , more than 80 % of these clones or mutants contained an insert ( results not shown ). moreover , 2 % of the clones were resistant to 1 μg erythromycin ml − 1 on agar plates indicating the presence of some promoter sequences that were functional in vitro . gene sequences that were specifically induced on agar plates under iron - restricted conditions were selected . for this purpose , about 96 , 000 c . f . u . were plated under iron - limiting conditions on agar plates containing deferoxamine mesylate and erythromycin . the 1500 colonies that grew on these plates were inoculated onto plates containing erythromycin , deferoxamine mesylate and feso 4 . twenty - four clones showed reduced growth in the presence of feso 4 . the inserts of the 24 selected iri clones were amplified by pcr using primers complementary to the 5 ′ ends of the erythromycin - and spectinomycin - resistance genes and the nucleotide sequences of these fragments were determined . the sequence data showed that the 24 clones contained 18 unique sequences . the 18 sequences were analyzed for similarity to known genes by comparison with the sequences in the genbank / embl and swissprot databases . one sequence , iri31 , was identical to cps2a , a previously identified s . suis gene putatively involved in the regulation of capsule expression ( smith et al ., 1999 ). fourteen iri sequences were similar to sequences of known , non - s . suis , genes . three of these sequences ( iri2 ( seq id no : 15 ), iri1 , 6 and 22 ( seq id no : 8 ), and iri34 ( seq id no : 21 ) were similar to sequences of environmentally regulated genes previously selected by applying the ivet to v . cholerae ( camilli and mekalanos , 1995 ), s . aureus ( lowe et al ., 1998 ) and p . aeruginosa ( wang et al ., 1996 ), respectively . one , contained in iri1 , 6 , and 22 ( seq id no : 8 ), was similar to the agra gene of staphylococcus aureus , a key locus involved in the regulation of numerous virulence proteins . three iri sequences had no significant similarity to any sequences in the databases ( table 2 ). to determine the antibiotic treatment regime required for a successful selection of in vivo - expressed promoter sequences , piglets were treated with different concentrations of erythromycin once a day . the erythromycin was administered either orally or intramuscularly . levels of erythromycin in sera were determined 3 , 6 or 24 hours after treatment over one week . high erythromycin levels were detected three hours and six hours after both treatments ( results not shown ). however , 24 hours after the treatments , the levels decreased dramatically . based on these data , we hypothesized that for efficient promoter selection , it was necessary to treat the animals twice a day with erythromycin ( 40 mg kg − 1 ), once intramuscularly ( at 9 a . m .) and once orally ( at 4 p . m .). to test this hypothesis , pigs were inoculated either with s . suis strain 10 ( pivs - pe ) or with strain 10 ( pivs - e ). in pivs - pe , the promoter of the mrp gene of s . suis ( smith et al ., 1992 ), which is highly expressed in vivo as well as in vitro , drives expression of the erythromycin - resistance gene . the control plasmid , pivs - e , does not contain a promoter in front of the erythromycin - resistance gene . the strains were inoculated intravenously or intranasally . all pigs infected with strain 10 ( pivs - pe ) showed specific s . suis symptoms ( table 3 ) and , except for one , all pigs died in the course of the experiment . moreover , high numbers of bacteria were isolated from the central nervous system , the serosae and the joints . in contrast , none of the pigs inoculated with strain 10 ( pivs - e ) showed specific clinical signs of disease and all survived the infection until the end of the experiment . moreover , bacteria were not isolated from the central nervous system , the serosae or the joints of these animals . these data demonstrated that in vivo - expressed sequences could be selected from pigs using the applied antibiotic treatment regimen . piglets were inoculated intravenously with different doses ( 5 × 10 5 to 5 × 10 8 c . f . u .) of the s . suis library ( table 3 ) and treated with erythromycin as described herein . specific signs of disease developed in all animals three to eight days after infection ( table 3 ). high numbers of bacteria were recovered from tissues ( central nervous system , joints , serosae , lung , liver , spleen , heart and kidney ) of the individual piglets . analysis of the recovered bacteria showed that a limited number of different clones were present in each of the bacterial samples isolated from the diseased pigs . for example , 30 randomly selected clones from the joints of one pig all possessed identical dna inserts as assessed by pcr and dna sequence analysis ( results not shown ). in addition , at 80 % of the 62 sample sites analyzed , four randomly selected clones were identical . however , from different tissues of a single animal , different clones or mutants could be isolated . on the other hand , identical clones could be isolated from different , as well as from corresponding , tissues of different animals . these findings indicated that a limited number of clones had been selected in vivo and were greatly enriched in the affected tissues . the observed selection was not tissue specific . further , none of the selected clones failed to grow on agar plates that contained 1 μg erythromycin ml − 1 . two - hundred forty - five clones were analyzed by pcr and partial sequence analysis . among these , 22 unique ivs clones were found . the 22 sequences were analyzed for similarity to sequences of known genes by comparison with the genbank / embl and swissprot databases ( table 4 ). the sequences of two genes showed similarity to genes encoding putative virulence factors : ivs21 , 26 and 30 which was identical to the epf gene , a previously identified s . suis gene , putatively involved in virulence ( smith et al ., 1993 ; smith et al ., 1996 ); and ivs31 ( seq id no : 37 ), which was similar to the fibronectin - binding protein of s . gordonii . moreover , the sequences of two ivs genes ( ivs25 ( seq id no : 24 ) and ivs6 , 7 , 13 and 14 ( seq id no : 43 )) were homologous to two environmentally regulated ivi genes , previously identified using ivet selection in other bacterial species ( camilli and mekalanos , 1995 ; lowe et al ., 1998 ). four ivs sequences ( ivs25 ( seq id no : 34 ); ivs23 and 24 ( seq id no : 33 ), ivs2 , 4 and 28 ( seq id no : 31 ); and ivs6 , 7 , 13 and 14 ( seq id no : 43 )) were also found when the library was selected using iron - restricted conditions . the remainder of the sequences showed similarity to sequences of known , non - s . suis genes , including two genes showing similarity to mobile elements and five genes showing similarity to genes of unknown function . the identification of environmentally regulated genes of s . suis serotype 2 by the use of iron - restricted conditions and by experimental infection of piglets is described . eighteen unique iri genes and 22 unique ivs genes were found . none of the ivs genes was exclusively expressed in vivo . four iri genes were identical to four clones selected in vivo . the selected gene sequences encode for potential virulence factors , expand our knowledge about the pathogenesis of s . suis infections in pigs and are of value in control of the disease either by the development of effective vaccines or by the development of new diagnostic methods . a promoter trap was used to identify environmentally regulated s . suis genes expressed under specific conditions , i . e ., during iron - restriction or during experimental infection . this system differs from the antibiotic - based ivet system described for s . typhimurium ( mahan et al ., 1995 ) in two ways . one is that the lacz reporter gene fusion is omitted in our vector constructions because inclusion of the lacz gene resulted in structural instability of the vector . the other difference is that a plasmid system was used rather than a chromosomal integration system . a plasmid system was used because the low transformation efficiency of s . suis ( smith et al ., 1995 ) might prevent the generation of a complete gene library using a chromosomal integration system . from the data , it is evident that a number of inducible and environmentally regulated sequences were selected . four iri genes were identical to four ivs genes . because most bacteria require iron for their growth and because there is a limited amount of free iron available within the host ( payne , 1993 ), it might be expected that the expression of some ivs genes is regulated by iron . with the in vivo selection system , tissue - specific colonization was not observed : clones isolated from one piglet were also isolated from other piglets from corresponding as well as from different tissues . this might be due to the mechanisms involved in the molecular pathogenesis of s . suis infections in pigs . furthermore , it was striking and different from the observations made with ivet systems that only a limited number of clones could be selected . in addition , we were not able to demonstrate that we selected for gene sequences that are exclusively expressed in vivo . this could be explained either by the absence of promoter sequences exclusively expressed in vivo among the 22 identified ivs genes , and / or by the inability of this plasmid - based system to identify such sequences due to gene dose effects . a number of interesting genes were selected . two ivs genes showed similarity to genes encoding putative virulence factors . ivs21 , 26 and 30 were shown to be identical to the epf gene of s . suis ( smith et al ., 1993 ), which is found in virulent strains of s . suis serotypes 1 and 2 ( stockhofe - zurwieden et al ., 1996 ; vecht et al ., 1991 ; vecht et al ., 1992 ). ivs31 ( seq id no : 37 ) showed similarity to the fibronectin / fibrinogen - binding protein of s . gordonii ( accession no . x65164 ) and group a streptococci ( courtney et al ., 1994 ). in streptococci , fibronectin / fibrinogen - binding proteins play an important role in adhesion to host cells and are considered to be important virulence factors . the selection of these two ivs genes demonstrated the selectivity of the system and might be indicative for the relevance of the other ivs genes in the pathogenesis of s . suis infections in pigs . the performance of the system was further demonstrated by the observation that two ivs genes , ivs25 ( seq id no : 34 ) and ivs6 , 7 , 13 and 14 ( seq id no : 43 ) showed similarity to environmentally regulated genes previously identified using an ivet selection system in other bacterial species . ivs25 ( seq id no : 34 ) showed significant similarity to the sapr gene of s . mutans ( accession no . p72485 ) and lactobacillus sake lb706 ( axelsson and holck , 1995 ) as well as to the agra gene of s . aureus ( projan and novick , 1997 ), both of which encode response regulator proteins of bacterial two - component signal - transduction systems , thus mediating the response to an environmental signal ( projan and novick , 1997 ). use of an ivet selection system for s . aureus in mice selected the region preceding the agra gene , suggesting induction of agra expression under in vivo conditions ( lowe et al ., 1998 ). moreover , in s . aureus , the agr locus was shown to play an important role in altering the expression of a considerable number of virulence factors in response to cell density ( projan and novick , 1997 ). clones ivs6 , 7 , 13 and 14 ( seq id no : 43 ) showed similarity to a gene , ivi vi , previously identified by ivet selection in v . cholerae ( camilli and mekalanos , 1995 ). the function of ivivi is unknown . however , the genes showed similarity to members of the atp - binding cassette family of transporters . the sequenced portion of ivs6 , 7 , 13 and 14 ( seq id no : 43 ) included an n - terminal atp - binding walker a box motif , which is highly conserved in this transporter family . four ivs genes were identical to four iri genes . the first gene , ivs23 and 24 ( seq id no : 33 ), which is identical to iri24 ( seq id no : 17 ), showed similarity to cpsy of s . agalactiae ( koskiniemi et al ., 1998 ) and to oxyr of various organisms ( demple , 1999 ). cpsy of s . agalactiae is involved in the regulation of capsule expression and environmental induction of expression of the cpsy gene has been suggested by koskiniemi et al . ( 1998 ). in s . suis , ivs23 and 24 ( seq id no : 33 ) and iri24 ( seq id no : 17 ) are not linked to the capsular locus ( smith et al ., 1999 ). the oxyr gene is the central regulator of oxidative stress response in e . coli ( demple , 1999 ) and approximately ten genes are under the control of the oxyr protein . the second gene , ivs2 , 4 and 28 ( seq id no : 31 ), which is identical to iri10 and 20 ( seq id no : 9 ), showed similarity to the yoae gene of e . coli ( accession no . p76262 ), a putative abc transporter protein . the third and the fourth genes , ivs25 ( seq id no : 34 ) and ivs6 , 7 , 13 and 14 ( seq id no : 43 ) were identical to iri1 , 6 and 22 ( seq id no : 8 ) and iri2 ( seq id no : 15 ), respectively . these genes also showed similarity to ivi genes selected using ivet in other bacterial species . based on data presented by niven et al . ( 1999 ), selection of iri genes of s . suis is not expected . the authors described that s . suis does not require iron for growth . however , in their studies the authors used media reduced from iron by using ethylenediamine di - o - hydroxyphenylacetic acid ( edda ). therefore , the different conditions used in vitro may explain the different results obtained . two of the s . suis ivs genes , ivs1 ( seq id no : 25 ) and ivs8 ( seq id no : 44 ), showed similarity to transposon sequences . moreover , one s . suis ivs gene , ivs2 , 4 and 28 ( seq id no : 31 ), had a gc % that was considerably higher than the composition of the rest of the selected genes . it is striking that in s . typhimurium , several of the ivi clones that are required for full virulence have been found to be associated with mobile elements . their atypical base composition and codon usage has led to the suggestion that they have been acquired from other bacterial species by horizontal transfer ( conner et al ., 1998 ). our screen also identified five ivs genes that showed similarity to sequences encoding proteins of unknown function . these genes are not standard housekeeping or metabolic genes . besides the four ivs / iri genes , a considerable number of other iri genes have been selected in this study by plating the library under iron - restricted conditions . interestingly , one of the selected iri genes , iri31 , is identical to the cps2a gene of s . suis . this gene was previously isolated as a part of the capsular locus of s . suis serotype 2 ( smith et al ., 1999 ) and was implicated in the regulation of capsular polysaccharide biosynthesis ( kolkman et al ., 1997 ; smith et al ., 1999 ). moreover , because the capsule of s . suis is expressed in larger size after in vivo growth when compared to growth in vitro ( quessy et al ., 1994 ), regulated expression of cps2a might be expected . another iri gene , iri7 ( seq id no : 23 ), showed similarity to the rpgg gene of s . mutans . this gene was shown to be required for the biosynthesis of rhamnose - glucose polysaccharide ( yamashita et al ., 1999 ). because rhanmose is part of the polysaccharide capsule in s . suis serotype 2 ( elliott and tai , 1978 ), a role of the iri7 ( seq id no : 23 ) gene in capsule biosynthesis can be proposed . iri34 ( seq id no : 21 ) showed similarity to the np 16 gene , previously identified using ivet selection in p . aeruginosa and suspected to encode threonine dehydratase activity ( wang et al ., 1996 ). together with the observation that 4 iri genes could be selected by the in vivo approach , these data show that the iri genes encode important virulence factors for s . suis . contribution of fibronectin - binding protein to pathogenesis of streptococcus suis serotype 2 . streptococcus suis causes severe infections , such as meningitis , septicemia , and arthritis , in piglets . the animals often do not survive the infection ( 6 , 28 ). occasionally , s . suis causes septicemia and meningitis in humans ( 3 ). the pathogenesis of an s . suis infection is rarely understood . sows are symptomless carriers of s . suis on their tonsils and pass the bacteria on to their piglets . the piglets cannot cope with the bacteria and subsequently develop the specific symptoms of an s . suis infection . until now , 35 capsular serotypes of s . suis have been described ( 26 ), but serotype 2 strains are most often isolated from diseased piglets . the capsule is an important virulence factor since piglets infected with an acapsular mutant of s . suis serotype 2 strains do not develop any clinical symptoms ( 22 ). bacterial proteins have been suggested to play a role in the pathogenesis as well ( 2 , 26 ). the expression of murimidase - released protein ( mrp ), extracellular factor ( ef ) and suilysin was shown to be strongly associated with pathogenic strains of s . suis serotype 2 ( 1 , 29 , 30 ). since isogenic mutants lacking mrp and ef and isogenic mutants lacking suilysin were still pathogenic in young piglets , these proteins are not absolutely required for virulence ( 2 , 23 ). recently , a new virulence factor was identified ( 21 ) by using a complementation approach . the function of this virulence factor in the pathogenesis has to be further investigated . many important virulence factors are environmentally regulated and are induced at specific stages of the infection process ( 15 ). to identify these genes in s . suis , promoters and their downstream sequences that are “ on ” during experimental s . suis infection of piglets ( 20 ) were cloned . twenty - two in vivo selected ( ivs ) genes were found . two of the ivs genes were directly linked to virulence since homology was found to genes in the database that encode for known virulence factors . one of these ivs genes ( ivs - 21 ) was identical to the epf gene of virulent s . suis serotype 2 strains ( 30 ). the other ( ivs - 31 ) ( seq id no : 37 ) showed homology to genes encoding fibronectin -/ fibrinogen - binding proteins of streptococcus gordonii ( genbank accession no . x65164 ) and streptococcus pyogenes fbp54 ( 8 ). a considerable number of fibronectin - binding proteins of various bacterial species have been shown to be important virulence factors ( 12 ). in s . pyogenes , fbp54 was shown to be expressed in the human host and to preferentially mediate adherence to human buccal epithelial cells ( 7 ). it was shown that the hfbp54 protein induces protective immunity against s . pyogenes challenge in mice ( 13 ). a fibronectin -/ fibrinogen - binding protein of s . suis ( fbps ) is described herein and the sequence of fbps was determined . binding studies showed that purified fbps bound fibronectin and fibrinogen . a contribution of fbps to the pathogenesis of s . suis serotype 2 was found . bacterial strains and growth conditions . the bacterial strains and plasmids used in this study are listed in table 5 . s . suis strains were grown in todd - hewitt broth ( code cm 189 ; oxoid , ltd .) and plated on columbia blood base agar plates ( code cm331 ; oxoid , ltd ., london , united kingdom ), containing 6 % ( vol / vol ) horse blood . e . coli strains were grown in luria broth ( 17 ) and plated on luria broth containing 1 . 5 % ( wt / vol ) agar . if required , antibiotics were added at the following concentrations : 50 μg / ml of spectinomycin ( sigma , st . louis , mo .) for e . coli and 100 μg / ml for s . suis , 100 μg / ml of ampicillin ( boehringer , mannheim , germany ) for e . coli and 25 μg / ml of kanamycin ( boehringer ) for e . coli . dna techniques and sequence analysis . routine dna manipulations were performed as described by sambrook et al . ( 19 ). dna sequences were determined on a 373a dna sequencing system ( applied biosystems , warrington , great britain ). samples were prepared by use of an abi prism dye terminator cycle sequencing ready reaction kit ( applied biosystems ). sequencing data were assembled and analyzed using the lasergene program ( dnastar ). the blast software package was used to search for protein sequences homologous to the deduced amino acid sequences in the genbank / embl databases . southern blotting and hybridization . chromosomal dna was isolated as described by sambrook et al . ( 19 ). dna fragments were separated on 0 . 8 % agarose gels and transferred to genescreen plus hybridization transfer membrane ( nen ™ life science products , boston , usa ) as described by sambrook et al . ( 19 ). dna probes of the fbps and spc genes were labeled with ( α - 32 p ) dctp ( 3 , 000 ci / mmol ; amersham life science , buckinghamshire , great britain ) by use of a random primed dna labeling kit ( boehringer ). the dna on the blots was pre - hybridized for at least 30 minutes at 65 ° c . and subsequently hybridized for 16 hours at 65 ° c . with the appropriate dna probes in a buffer containing 0 . 5 m sodium phosphate ( ph 7 . 2 ), 1 mm edta and 7 % sodium dodecyl sulphate . after hybridization , the membranes were washed twice with a buffer containing 40 mm sodium phosphate ( ph 7 . 2 ), 1 mm edta and 5 % sodium dodecyl sulphate for 30 minutes at 65 ° c . and twice with a buffer containing 40 mm sodium phosphate ( ph 7 . 2 ), 1 mm edta and 1 % sodium dodecyl sulphate for 30 minutes at 65 ° c . the signal was detected on a phosphor - imager ( storm ; molecular dynamics , sunnyvale , calif .). construction of a fbps knock - out mutant . to construct the mutant strain 10δfbps , the pathogenic strain 10 ( 27 , 29 ) of s . suis serotype 2 was electrotransformed ( 24 ) with the plasmid pfbps7 - 47 . in this plasmid , the fbps gene was inactivated by the insertion of a spectinomycin - resistance gene . to create pfbps7 - 47 ( fig1 ), the 382 bp sali - sali fragment of pfbps7 - 46 was replaced by the 1 . 2 kb ecorv - smai fragment of pic - spc , containing the spectinomycin resistance gene , after the sali sites of the vector were made blunt ( fig1 ). after electrotransformation of strain 10 with pfbps7 - 47 , spectinomycin - resistant colonies were selected on columbia agar plates containing 100 μg / ml of spectinomycin . southern blotting and hybridization experiments were used to select for double cross - over integration events ( data not shown ). fbps expression construct . to construct an fbps expression plasmid , the qiaexpress kit ( qiagen gmbh , hilden , germany ) was used . the primers corresponded to positions 250 to 272 and from 1911 to 1892 of the fbps gene . the sequences of these primers were 5 ′( gcggatccgatgacgatgacaaatcttttgacggattttttttac ) 3 ′ ( seq id no : 46 ) and 5 ′( cccaagcttgggcatgaactagattttcatgg ) 3 ′ ( seq id no : 47 ). the primers contained restriction sites for bamhi and hindiii , respectively , to amplify the fbps gene from pfbps7 - 47 . the amplified pcr product was digested with bamhi and hindiii and the 1 . 8 kb fbps gene was cloned into pqe - 30 digested with bamhi and hindiii , yielding pqe - 30 - fbps . pqe - 30 - fbps was transformed to m15 ( prep4 ). purification of fbps . m15 ( prep4 ) ( pqe - 30 - fbps ) was used to express and purify the fbps using the qiaexpressionist ™ ( qiagen ). in short , m15 ( prep4 ) ( pqe - 30 - fbps ) cells were grown exponentially ; 1 mm iptg was added and the cells were allowed to grow another four hours at 37 ° c . subsequently , cells were harvested and lysed . the cleared supernatants were loaded onto ni 2 + - nta agarose columns . fbps containing a 6 × his tag was bound to the ni 2 + - column . the columns were washed and the protein was eluted . different buffers were used for native and for denaturing purification . fbps purified under denaturing conditions was renaturated on a ni 2 + - nta column by using a linear 6 m - 1 m urea gradient in 500 mm nacl , 20 % glycerol and 20 mm tris - hcl ( ph7 . 4 ), containing protease inhibitors ( 25 μg / ml of pefabloc , 0 . 7 μg / ml of pepstatin , 1 μg / ml of aprotinin , 0 . 5 μg / ml of leupeptin ). all procedures were performed according to the manufacturer &# 39 ; s recommendations . the 6 × his tag was removed from the protein by incubating purified fbps in 20 mm tris - hcl ( ph 7 . 4 ), 50 mm nacl , 2 mm cacl 2 and 0 . 5 u of light chain enterokinase ( new england biolabs , beverly , mass .) for 16 hours at rt . immunization of rabbits with fbps . purified and renaturated fbps was used to immunize two rabbits . to remove urea , the protein was dialyzed against phosphate buffered saline ( 136 mm nacl ; 2 . 68 mm kcl ; 8 . 1 mm na 2 hpo 4 ; 2 . 79 mm kh 2 po 4 ( ph 7 . 2 )) over night at 4 ° c . seven days before immunization , blood was collected from the rabbits to determine the natural titers against fbps . at day one , those rabbits with negative anti - fbps titers were immunized intramuscularly with two times 0 . 5 ml of 100 μg / ml of fbps in a water - in - oil emulsion ( specol ; id - lelystad ). at day 28 , rabbits were immunized for the second time using the same amount of protein and the same route of immunization . three weeks after the second immunization , the rabbits were sacrificed and blood was collected . the blood was coagulated and serum was collected and used for immuno detection of fbps . immunodetection of fbps . proteins were separated by sodium dodecyl sulfate ( sds )- polyacrylamide gel electrophoresis ( page ) by standard procedures ( 19 ). proteins in the gel were visualized using sypro - orange ( molecular probes , sunnyvale , calif .) staining according to the manufacturer &# 39 ; s recommendations . signals were detected on a phosphor imager ( storm ; molecular dynamics ). a known bovine serum albumin concentration range was used as a standard to calculate the amounts of protein present in the gel . the molecular dynamics program was used for the calculations . proteins were transferred to a nitrocellulose membrane by standard procedures ( 19 ). the membranes were blocked in blotto : tris - buffered saline ( tbs ) ( 50 mm tris - hcl ( ph 7 . 5 ), 150 mm nacl ) containing 4 % skimmed milk and 0 . 05 % tween 20 at room temperature ( rt ) for one hour . to detect recombinant purified fbps , membranes were incubated with a monoclonal antibody against the 6 × his tag ( clontech , palo alto , calif .) in a 1 : 10 , 000 dilution in blotto - tbs ( 1 : 1 ) at rt for one hour , followed by an incubation with alkaline phosphatase - conjugated anti - mouse antibody in a 1 : 1 , 000 dilution in blotto - tbs ( 1 : 1 ) at rt for one hour . reactivity of purified fbps was tested by using a convalescent serum of a pig that had survived an s . suis infection . nitrocellulose membranes were incubated with the polyclonal pig serum in a 1 : 200 dilution in blotto - tbs ( 1 : 1 ) at rt for one hour , followed by incubation at rt for one hour with alkaline phosphatase - conjugated anti - swine antibody in a 1 : 2 , 000 dilution in blotto - tbs ( 1 : 1 ). as a substrate , nitro blue tetrazolium ( merck , darmstadt , germany ) bromochloroindolyl phosphate ( sigma ) was used . all washing steps were performed in blotto - tbs ( 1 : 1 ). fibronectin and fibrinogen binding . binding studies were performed by indirect western blotting . proteins were separated by sds - page and transferred to a nitrocellulose membrane as described herein . the membranes were blocked in mpbs : pbs containing 4 % skimmed milk and 0 . 05 % tween 20 . subsequently , the membrane was incubated with 5 μg / ml of human fibronectin ( fn ) ( sigma ) or 5 μg / ml of human fibrinogen ( fgn ) ( sigma ) in pbs containing 5 % fetal calf serum , 2 % nacl , and 0 . 05 % tween 80 at rt for one hour . to detect bound fibronectin and fibrinogen , the membranes were incubated with horse - radish peroxidase - conjugated anti - fibronectin ( dako ) or anti - fibrinogen ( dako ) antibodies in a 1 : 1 , 000 dilution in pbs containing 5 % fetal calf serum , 2 % nacl , and 0 . 05 % tween 80 at rt for one hour . the signal was visualized by using ecl + ( amersham pharmacia biotech , n . j .) according to the manufacturer &# 39 ; s recommendations . signals were detected on a phosphor imager ( storm ; molecular dynamics ). all washing steps were performed in mpbs - pbs ( 1 : 1 ). experimental infections . germ - free piglets , cross - breeds of great yorkshire and dutch landrace , were obtained from sows by cesarean section . the surgery was performed in sterile flexible film isolators . piglets were allotted to groups of four and were housed in sterile stainless steel incubators . housing conditions and feeding regimens were as described ( 27 , 29 ). six - day - old piglets were inoculated intranasally with about 10 7 cfu of bordetella bronchiseptica 92932 to predispose the piglets to infection with s . suis . two days later , the piglets were inoculated intranasally with 10 6 cfu of s . suis strain 10 plus 106 cfu of s . suis strain 10δfbps . to determine differences in virulence between wild - type and mutant strains , ld 50 values should be determined . to do this , large numbers of piglets are required . for ethical reasons , this is not acceptable . to circumvent this problem , co - colonization studies were performed . to monitor for the presence of s . suis and b . bronchiseptica and to check for absence of contaminants , swabs taken from the nasopharynx and the feces were cultured three times a week . the swabs were plated directly onto columbia agar containing 6 % horse blood or grown for 48 hours in todd - hewitt broth and subsequently plated onto columbia agar containing 6 % horse blood . pigs were monitored twice a day for clinical signs and symptoms , such as fever , nervous signs , and lameness . blood samples from each pig were collected three times a week . leukocytes were counted with a cell counter . the piglets were killed when specific signs of an s . suis infection were observed , such as arthritis or meningitis , or when the pigs became mortally ill . the other piglets were killed two weeks after inoculation with s . suis and examined the same way as the piglets that were killed based on their clinical symptoms . all piglets were examined for pathological changes . tissue specimens from heart , lung , liver , kidney , spleen , and tonsil , and from the organs specifically involved in an s . suis infection ( central nervous system ( cns ), serosae , and joints ) were sliced with a scalpel or a tissuenizer . tissue slices from each organ or site were resuspended in 2 - 25 ml of todd - hewitt containing 15 % glycerol depending on the size of the tissue slice . the suspension was centrifuged at 3 , 000 rpm for five minutes . the supernatant was collected and serial dilutions were plated on columbia agar containing 6 % horse blood , as well as on columbia agar plates containing 6 % horse blood and 100 μg / ml of spectinomycin to quantitate the number of wild - type and mutant bacteria present . the number of mutant strain 10δfbps cells was determined by counting the number of cfu on the appropriate serial dilution on the selective plates ; the number of wild - type strain 10 cells was determined by counting the number of cfu on the appropriate serial dilution on the columbia agar blood plates of which the number of cfu counted on the selective plates was subtracted . when wild - type and mutant bacteria were found in tissues , the ratio of wild - type and mutant strain was determined again by toothpicking about 100 individual colonies onto both columbia agar plates and onto columbia agar plates containing 100 μg / ml spectinomycin . all animal experiments were approved by the ethical committee of the institute for animal science and health in accordance with the dutch law on animal experiments . nucleotide sequence accession number . the nucleotide sequence data of fbps have been submitted to genbank , in which the sequence is listed under accession no . af438158 . results . cloning of the s . suis fbps gene . one of the in vivo selected genes ( ivs - 31 ) ( seq id no : 37 ) ( 20 ) showed homology to the 5 ′ part of genes encoding for flpa and fbp54 , fibronectin - binding proteins ( fnbp ) of streptococcus gordonii ( genbank accession no . x65164 ) and streptococcus pyogenes ( 8 ), respectively . to clone the entire fbps gene of s . suis , ivs31 ( seq id no : 37 ) was used as a probe to identify a chromosomal dna fragment of s . suis serotype 2 containing flanking fbps sequences . a 5 kb ecori fragment was identified and cloned in pgem7zf (+) yielding pfbps7 - 46 ( fig1 ). sequence analysis revealed that this fragment contained the entire fbps gene of s . suis serotype 2 . an open reading frame of 1659 bp coding for a polypeptide of 553 amino acids was found . the putative atg start codon is preceded by a sequence similar to ribosome binding sites of gram - positive bacteria . further upstream , two putative promoter sequences could be identified . upstream of these promoter sequences of fbps , a direct repeat was found that could serve as a transcription terminator of the gene located 5 ′ of fbps . downstream of fbps , a gene that showed homology to an alpha - acetolactate decarboxylase was found . this gene is transcribed in the opposite direction of fbps . the deduced amino acid sequence was aligned with that of several previously identified fnbps from other bacteria . as expected , fbps was substantially homologous to flpa of s . gordonii ( 76 %) and also showed homology to fnbp &# 39 ; s of other organisms , like streptococcus pneumoniae ( 73 %), s . pyogenes ( 69 %), lactococcus lactis ( 59 %), and bacillus subtilis ( 41 %). compared to the sequence of fbp54 , fbps has a longer n - terminus with 76 additional amino acids . this longer n - terminus was also seen in other organisms like s . gordonii , s . pneumoniae and b . subtilis . in fbp54 , the primary fibronectin -/ fibrinogen - binding domain was localized to its n - terminal part , to the first 89 amino acids ( 8 ). over this region , the homology of fbps to fbp54 is very high ( 80 %), suggesting that fbps can bind both fibronectin and fibrinogen . binding of fbps to fibronectin and fibrinogen . to confirm the binding of fbps of s . suis to fibronectin ( fn ) and fibrinogen ( fgn ), fbps was purified under native conditions . a protein expression construct , which expresses fbps with a 6 × his tag fused to the n - terminus , was used for purification . four hundred μg of fbps was purified from 50 ml of exponential - phase e . coli cells after induction with iptg . the purity of this fpbs was determined with sds - page and western blotting ( fig2 ). the induced e . coli lysate contained a broad range of proteins , among which the 64 kda protein fbps was present ( panel a , lane 1 ). after purification , highly purified fbps with 6 × his tag was obtained ( panel a , lane 2 ). when both samples were incubated with a monoclonal antibody against the 6 × his tag , fbps was the only protein that was detected ( panel b ). to determine whether fbps binds fn and fgn , a western blot containing purified fbps was incubated with soluble human fn and human fgn ( fig3 , panels a and b ). specific binding of fn and fgn to fbps was detected . no binding of fn and fgn to bsa , a negative control protein , was observed . to exclude possible background signals due to immunoglobulin - binding of fbps , the same experiment was performed without addition of fibronectin or fibrinogen . no binding was found ( fig3 , panels c and d ) indicating that the binding was specific for fibronectin and fibrinogen . to control whether the binding of fn and fgn to fbps was not mediated by the 6 × his tag , the tag was removed by an enterokinase treatment . fig3 , panels e and f , show that fbps without the 6 × his tag still efficiently bound to fn and fgn . therefore , it appears that fbps can specifically bind to fn and fgn . immunogenicity of fbps . since it was shown that fbp54 induced a protective immune response in mice against a lethal dose of s . pyogenes ( 13 ), it was determined whether purified fbps was recognized by convalescent serum of a pig that survived an s . suis infection . as shown in fig2 panel c , the fbps reacted with this anti - serum . when the same experiment was performed with non - immune serum of an spf piglet , no band of the size of fbps was detected ( data not shown ). these findings indicate that fbps is expressed in vivo and that the protein is indeed immunogenic in young pigs . distribution of the fbps gene among the 35 s . suis serotypes . since we were interested in a cross - protective vaccine candidate , the presence of the fbps gene among the various s . suis serotypes was analyzed . ivs - 31 ( seq id no : 37 ), the clone containing the promoter and the 5 ′- part of the fbps gene , was radiolabeled and chromosomal dna of the reference strains of the 35 different s . suis serotypes was hybridized with this probe . the three different phenotypes of s . suis serotype 2 , a pathogenic , a non - pathogenic and a weak pathogenic strain , were included in this study . the fbps gene was present in all s . suis serotypes and phenotypes except for serotypes 32 and 34 ( fig4 ). role of fbps in pathogenesis . to test the role of fbps in the pathogenesis of s . suis , an isogenic knock - out mutant of fbps was constructed in strain 10 , strain 10δfbps . since upstream of fbps a direct repeat was found that could serve as a transcription terminator and downstream of fbps a gene showing homology to an alpha - acetolactate decarboxylase was found that is transcribed in the opposite direction , polar effects to genes upstream or downstream of fbps are not expected . to verify that the mutant strain 10δfbps did not produce fbps , protoplasts of strain 10 and strain 10δfbps were subjected to sds - page and western blotting . fbps was detected using a polyclonal antiserum raised against purified fbps . it was shown that strain 10δfbps expressed no fbps , while strain 10 did express fbps ( data not shown ). subsequently , the virulence of this mutant strain was tested in an experimental infection in piglets . the mutant strain 10δfbps was used in a competition challenge experiment with the wild - type strain to determine the relative attenuation of the mutant strain . using in vitro conditions , the growth rates of the wild - type and mutant strain in todd - hewitt medium were found to be essentially identical ( data not shown ). wild - type and mutant strain were inoculated at an actual ratio of 0 . 65 ( 1 . 63 × 10 6 cfu of wild - type bacteria ml − 1 and 3 . 09 × 10 6 cfu of mutant bacteria ml − 1 ). during the experiment , piglets that developed specific s . suis symptoms ( meningitis , arthritis , or mortal illness ) were killed . piglets that did not develop these symptoms were killed at the end of the experiment . from all piglets , the ratio of wild - type and mutant strain in various organs was determined . as shown in fig5 , panel a , similar numbers of wild - type and mutant bacteria were re - isolated from tonsils . the ratio was similar to the input ratio ( ratio varied from 0 . 33 - 0 . 85 , average 0 . 61 ). this indicates that the efficiency of colonization of wild - type and mutant strain on tonsils was essentially identical . apparently , fbps is not strictly required for colonization of the tonsils of the piglets . three out of four piglets developed clinical signs specific for an s . suis infection . two piglets ( 4664 and 4666 ) showed clinical signs of arthritis and one piglet ( 4668 ) showed clear central nervous signs . the fourth piglet did not develop any clinical signs . these observations coincided with pathomorphological abnormalities of the specific organs of an s . suis infection in post - mortem sections . as shown in fig5 , panel b , exclusively wild - type bacteria were re - isolated from the joints of piglet 4664 and from the cns of piglet 4668 . the numbers of cfu of wild - type bacteria that were re - isolated from these specific organs were very high , while no mutant bacteria were found . from the joints of pig 4666 , low numbers of both wild - type and mutant bacteria were re - isolated in a ratio of 0 . 84 ( 1 . 0 × 10 2 cfu of wild - type bacteria and 5 . 2 × 10 2 cfu of mutant bacteria ), a ratio essentially identical to the input ratio ( fig5 , panel b ). southern blot experiments using the fbps and the spc genes as probes , confirmed that the mutant bacteria isolated from the joint of pig 4666 were indeed identical to the input mutant bacteria . taken together , these data indicate , that the fbps mutant is capable of reaching and colonizing the specific s . suis organs ( at least the joints ), but that the mutant is far less efficiently recovered from organs than the wild type . kolkman , m . a . b ., w . wakarchuk , p . j . m . nuijten and b . a . m . van der zeijst . 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