Patent Application: US-59292309-A

Abstract:
the invention relates to the field of 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 , the method comprising modifying a genomic fragment of 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 as shown in fig . 5 .

Description:
bacterial strains and growth conditions . the bacterial strains and plasmids used herein are listed in table 1 . s . suis strains were grown in todd - hewitt broth ( code cm189 , oxoid ) and plated on columbia agar blood base ( code cm331 , oxoid ) containing 6 % ( v / v ) horse blood . if required , antibiotics were added at the following concentrations : erythromycin , 1 μg / ml . e . coli strains were grown in luria broth and plated on luria broth containing 1 . 5 % ( w / v ) agar . if required , 200 μg / ml of erythromycin was added . pcom1 . pcom1 ( fig1 ) is based on the replication functions of pwvo1 . further , the vector contained the erythromycin - resistance gene of pe194 preceded by the promoter region of the mrp gene , as well as the saci - psti part of the multiple cloning site of pkun19 . as a result , pcom1 contained a unique bamhi site ( fig1 ). construction of the genomic s . suis library in pcom1 . sau3ai partial digests of the dna of the pathogenic s . suis serotype 2 , strain 10 were size fractionated (& gt ; 3 kb ) by precipitation with 4 . 6 % of peg 6000 ( bdh chemicals , 19 ). the fragments were ligated to bamhi - digested pcom1 and the ligation mixtures were transformed to e . coli xl2 - blue cells . erythromycin - resistant colonies were selected . about 17 , 000 independent e . coli clones were obtained . analysis of 55 of the transformants showed that 64 % contained an insert of greater than 3 kb . from the pool of e . coli transformants , plasmid dna was isolated and subsequently used for the electrotransformation of the weak - pathogenic s . suis strain s735 . this resulted in approximately 30 , 000 independent s . suis transformants . the s . suis library was designated s735 ( pcom - l ). the transformants were pooled and stored at − 80 ° c . dna techniques . routine dna manipulations were performed as described by sambrook et al . dna sequences were determined on a 373a dna sequencing system ( applied biosystems , warrington , gb ). samples were prepared by use of an abi / prism dye terminator cycle sequencing - ready reaction kit ( applied biosystems ). custom - made sequencing primers were purchased from life technologies . sequencing data was assembled and analyzed using the mcmollytetra software package . the blast program was used to search for protein sequences homologous to the deduced amino acid sequences . for pcr reaction mixtures ( 50 μl ), the pcr expand high fidelity system ( boehringer , mannheim , germany ) was used as described by the supplier . dna amplification was carried out in a perkin elmer 9600 thermal cycler and the program included an incubation for two minutes at 95 ° c ., ten cycles of 20 seconds at 95 ° c ., one minute at 60 ° c . and four minutes at 68 ° c ., 30 cycles of 20 seconds at 95 ° c ., one minute at 60 ° c . and four minutes , extended with 20 seconds for each cycle , at 68 ° c . and ten minutes at 72 ° c . southern blotting and hybridization . chromosomal dna was isolated as described by sambrook et al . dna fragments were separated on 0 . 8 % agarose gels and transferred to gene - screen plus membranes ( nen ) as described by sambrook et al . dna probes were labeled with [( α - 32 p ] dctp ( 3000 ci mmol − 1 ; amersham ) by use of a random primed labeling kit ( boehringer ). the dna on the blots was hybridized at 65 ° c . with the appropriate dna probes as recommended by the supplier of the gene - screen plus membranes . after hybridization , the membranes were washed twice with a solution of 40 mm sodium phosphate , ph 7 . 2 , 1 mm edta , 5 % sds for 30 minutes at 65 ° c ., and twice with a solution of 40 mm sodium phosphate , ph 7 . 2 , 1 mm edta , 1 % sds for 30 minutes at 65 ° c . construction of pcom - v10 - orf2 and pcom - v10 - orf3 . to construct pcom - v10 - orf2 , the primers 5 ′- cgagctcggaagaattggttattgcgcgtg - 3 ′ ( seq id no : 1 ) and 5 ′- cgggatcccgggggatgacctgttgcttg - 3 ′ ( seq id no : 2 ) were used in a pcr reaction on chromosomal dna of s . suis strain 10 to amplify the orf2 encoding region . the resulting fragment was purified , digested with saci and bamhi and cloned into saci and bamhi - digested pcom1 . to construct pcom - v10 - orf3 , the primers 5 ′- tcccccgggggacaagcaacgggtcatcccc - 3 ′ ( seq id no : 3 ) and 5 ′- cgggatcccggttgaatgcccggcaaagcg - 3 ′ ( seq id no : 4 ) were used to amplify the orf3 encoding region . the resulting fragment was digested with smai and bamhi and cloned into pkun19 . the resulting plasmid was designated pkun - orf3 . because the orf2 and orf3 encoding regions are most probably co - transcribed , the promoter region of orf2 was subsequently amplified with primers 5 ′- cgagctcggaagaattggttattgcgcgtg - 3 ′ ( seq id no : 1 ) and 5 ′- tcccccgggggagtcgtgtgtattcgacagcgg - 3 ′ ( seq id no : 5 ). the fragments were digested with saci and smai and cloned into saci and smai - digested pkun - orf3 . the resulting plasmid was digested with saci and bamhi , and the insert fragment was purified and cloned into saci and bamhi - digested pcom1 . this resulted in pcom - v10 - orf3 . experimental infections . germfree pigs , crossbreeds of great yorkshire and dutch landrace , were obtained from sows by caesarian sections . the surgery was performed in sterile flexible film isolators . pigs were allotted to groups , each including 4 or 5 pigs , and were housed in sterile stainless - steel incubators . housing conditions and feeding regimes were performed as described by vecht et al . one - week - old pigs were intravenously inoculated with s . suis strains as described by vecht et al . pigs received erythromycin orally twice a day ( erythromycin stearate , abbott b . v ., amstelveen , the netherlands , 40 mg / kg body weight ). two hours after the infection , the pigs were treated with erythromycin for the first time . pigs 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 . white blood cells were counted with a cell counter . to monitor infection with s . suis , swabs of nasopharynx and feces were collected daily . the swabs were directly plated onto columbia agar containing 6 % horse blood . after the pigs were sacrificed , they were examined for pathological changes . further , tissue specimens were collected from the central nervous system , serosa , joints , lungs , liver , kidney , spleen , heart and tonsils . the tissues were homogenized in the presence of todd - hewitt medium by using an ultra - turrax tissuemizer ( omni international , waterbury , usa ), centrifuged for five minutes at 3 , 000 rpm and the supernatants were frozen at − 80 ° c . in the presence of 15 % glycerol . complementation system . a genomic library of the pathogenic s . suis strain 10 was constructed into the weak - pathogenic strain s735 as described in materials and methods . the plasmid pcom1 allowed the insertion of large dna fragments into the unique bamhi site ( fig1 ). the plasmid carries the origin of replication of pwvo1 that functions in e . coli and in s . suis . this allowed the construction of a dna library in e . coli first . plasmid dna , isolated from the pool of e . coli transformants , was subsequently electrotransformed into s . suis strain s735 . 30 , 000 individual s . suis clones were obtained . as determined by analysis of 24 randomly selected transformants , more than 30 % of the s735 ( pcom - l ) transformants contained an insert greater than 3 kb . selection of genomic fragments associated with virulence . to select for genetic determinants of the pathogenic s . suis strain 10 that could increase the virulence of the weak - pathogenic strain s735 , pigs were inoculated with the s . suis library s735 ( pcom - l ). a dose of either 10 7 or 10 8 cfu was used and the pigs were treated with erythromycin as described in materials and methods . all pigs showed specific s . suis symptoms ( table 2 , a ) three to seven days after the infection and , except for one , all pigs died during the course of the experiment . from five of the pigs , bacteria could be re - isolated from the cns , and from two other pigs , bacteria were isolated from the joints ( table 2 , a ). in previously performed experiments in which pigs were inoculated with weak - pathogenic strains , specific s . suis symptoms were observed with a very low frequency . in addition , from those pigs , bacteria could not be re - isolated from the cns or from the joints . therefore , the data indicated that , compared to virulence of strain s735 , bacteria isolated from pigs inoculated with the s . suis library s735 ( pcom - l ) are more virulent due to the presence of a dna fragment of the pathogenic strain 10 . the plasmid content of 90 randomly selected clones isolated from the cns or the joints of the seven diseased pigs was analyzed by pcr and restriction analysis . the results showed that 88 of the 90 clones analyzed ( 19 of which are shown in fig2 ) contained an insert of about 3 kb and had substantially identical restriction patterns . moreover , the inserts of ten randomly selected clones having substantially identical restriction patterns also showed identical dna sequences ( results not shown ). plasmid dna of ten randomly selected clones from the original s735 ( pcom - l ) library showed ten different restriction patterns ( fig2 ). the data suggest that one specific clone , which was designated s735 ( pcom - v10 ), was greatly enriched in seven different pigs . further , this particular clone was isolated from the cns and from the joints of the various pigs , indicating that the observed enrichment was not tissue specific . virulence - associated properties of the selected fragment v10 . to further analyze the virulence properties of strain s735 ( pcom - v10 ), pigs were intravenously inoculated with 10 6 cfu of strain s735 ( pcom1 ) or strain s735 ( pcom - v10 ). the results ( table 2 , b ) show that , compared to the virulence of strain s735 ( pcom1 ), the virulence of strain s735 ( pcom - v10 ) was greatly enhanced . all pigs inoculated with strain s735 ( pcom - v10 ) showed specific s . suis symptoms and died within one day after infection . in contrast , except for one , none of the pigs inoculated with the control strain s735 ( pcom1 ) showed specific clinical symptoms and these pigs survived until the end of the experiment ( 15 days after infection ). the data proved that introduction of fragment v10 of strain 10 into s735 transformed the weak - pathogenic strain s735 into a highly pathogenic strain . this strongly suggests that the protein ( s ) encoded by v10 are important virulence determinants and play an important role in the pathogenesis of s . suis serotype 2 infections in pigs . to find out whether the observed increase of the fragment v10 on virulence was specific for strain s735 , pcom1 and pcom - v10 were introduced into another weak - pathogenic strain , strain 24 . subsequently , the virulence properties of the strains 24 ( pcom1 ) and 24 ( pcom - v10 ) were determined . as shown in table 2 c and d , similar effects of v10 on the virulence of strains s735 and 24 were observed . both strains 24 ( pcom - v10 ) and s735 ( pcom - v10 ) were highly pathogenic for young piglets , whereas strains 24 ( pcom1 ) and s735 ( pcom1 ) were shown to be weakly - pathogenic ( table 2 , c and d ). this strongly indicates that v10 has a more general ability to transform weak - pathogenic serotype 2 strains into highly pathogenic strains . because a plasmid system for the complementation approach was used , gene - dose effects cannot be excluded . plasmid pcom1 is based on the replication functions of pwvo1 . in gram - positive bacteria , the latter plasmid has a copy number of between 3 and 6 . to find out whether copy effects play a role , the genomic region of strain s735 homologous to fragment v10 of strain 10 ( see below ) was cloned into plasmid pcom1 . this plasmid was designated pcom - v735 . the virulence of strains s735 ( pcom - v735 ), and 24 ( pcom - v735 ) was subsequently compared to that of s735 ( pcom - v10 ), s735 ( pcom1 ), 24 ( pcom - v10 ) and 24 ( pcom1 ). the results ( table 2 , c and d ) show that , in contrast to pcom - v10 , the plasmid pcom - v735 did not carry virulence - enhancing activity . pigs infected with strains s735 ( pcom - v10 ) and 24 ( pcom - v10 ) died within one or two days after infection , whereas most of the pigs infected with strains s735 ( pcom - v735 ), 24 ( pcom - v735 ), s735 ( pcom1 ) and 24 ( pcom1 ) survived until the end of the experiment ( 17 days after infection ). compared to pigs infected with strains containing pcom1 , pigs infected with strains containing pcom - v735 developed more general and specific signs of disease , but much less than pigs infected with strains containing pcom - v10 ( table 2 , c and d ). from these data , it was concluded that the differences in virulence observed between the strains containing pcom - v10 and the strains containing pcom - vs735 are caused by differences between the fragments v10 and v735 ( see below ). the differences in virulence observed between the strains containing pcom1 and the strains containing pcom - vs735 may be due to gene - dose effects . sequence analysis of fragments v10 and v735 . by using the fragment v10 as a probe , a 3 . 1 kb psti - hindiii fragment of strain s735 ( v735 ) was identified and cloned into pcom1 ( fig3 ). to analyze the differences between the fragments v10 and v735 , the nucleotide sequences of the fragments v10 and v735 were determined and the sequences were analyzed for homology to known genes by comparison with the genbank / embl and swissprot databases . the sequence of v10 revealed two complete and two incomplete open reading frames ( fig3 ). orf1 ( nucleotides 1 to 461 ) coded for a polypeptide of 153 amino acids . this protein showed homology ( 49 % identity ) to the c - terminal region of acetate kinase of clostridium thermocellum ( accession number af041841 ) and various other bacterial species . orf2 ( nucleotides 625 to 1327 ) coded for a protein of 233 amino acids . no significant similarities were found between the predicted amino acid sequence of this protein and other proteins present in the data libraries . orf3 ( nucleotides 1382 to 2639 ) coded for a protein of 418 amino acids . this protein showed homology ( 36 % identity ) to folc ( folylpolyglutamate synthetase ) of bacillus subtilis . compared to the other orfs , orf4 is transcribed in the opposite direction . orf4 ( nucleotides 2684 to 2972 ) coded for a polypeptide of 96 amino acids . this polypeptide showed homology ( 67 % identity ) to the c - terminal part of pepa ( glutamyl - aminopeptidase ) of lactococcus lactis . both orfs 2 and 3 possessed putative initiation codons and ribosome - binding sites . putative − 35 ( tggaca ) and − 10 ( tacaat ) sequences , which may function as promoter sequences , were found preceding orf2 . orfs 2 and 3 were separated by 55 nucleotides . in this region , no putative promoter sequences could be observed . this could indicate that the orfs 2 and 3 are co - transcribed . downstream of the orfs 1 and 3 , regions of extended dyad symmetry were found which may function as transcription termination signals . the sequence of the fragment v735 was determined and compared to the sequence of the fragment v10 . no major deletions or insertions were found between the sequenced regions . the orfs 1 , 3 and 4 of strains 10 and s735 were highly homologous . the putative protein fragments encoded by the orfs 1 differed in 2 ( 1 . 3 %) amino acids ; the putative proteins encoded by the orfs 3 differed in 19 ( 4 . 5 %) amino acids ( fig4 b ), whereas the putative protein fragments of the orfs 4 were identical . however , major differences were observed between the orfs 2 of strains 10 and s735 . in the pathogenic strain 10 , an orf of 699 bases was found with a protein product of 233 amino acids . in contrast , due to a frame - shift mutation in the weak - pathogenic strain s735 , an orf of 569 bases was found and coded for a polypeptide of 183 amino acids . compared to the putative protein encoded by strain 10 , the putative protein encoded by strain s735 lacked the n - terminal 50 amino acids ( fig4 a ). besides these n - terminal differences , the putative proteins differed at 9 amino acid positions ( 4 . 9 %). in addition , the putative − 35 regions that may be part of the promoter sequences involved in the expression of orfs 2 and 3 differed between the two strains . a tggaca sequence was found in strain 10 , whereas a tggtca sequence was found in strain s735 . the sequence data suggest that the differences in the virulence - enhancing effects of the fragments v10 and v735 may be the result of functional differences between the putative proteins expressed by the orfs 2 and / or 3 , and / or by differences in their levels of expression . to examine whether the observed increase of the fragment v10 on virulence resulted from orf2 or orf3 or both , the plasmids pcom - v10 - orf2 and pcom - v10 orf3 containing the individual orf2 and orf3 encoding regions were constructed . because orf3 is probably co - transcribed with orf2 , in pcom - v10 - orf3 the orf3 encoding region was preceded by the promoter region of orf2 . subsequently , the virulence properties of the strains s735 ( pcom - v10 ), s735 ( pcom - v10 - orf2 ), s735 ( pcom - v10 - orf3 ) and s735 ( pcom1 ) were determined . as shown at e in table 2 , the fragments v10 and orf2 showed similar effects on the virulence of strain s735 while no effect of orf 3 could be observed on the virulence of strain s735 . these data show that orf2 is responsible for the observed effect on virulence and that the orf2 protein is an important virulence factor . distribution of the orf2 and orf3 sequences among all known 35 s . suis serotypes . to examine the homology between the orf2 and orf3 genes and genes of other s . suis serotypes , cross - hybridization experiments were performed . dna fragments of the orf2 and 3 genes were amplified by pcr , labeled by 32 p , and hybridized to chromosomal dnas of the reference strains of the 35 different s . suis serotypes . as a positive control , a probe specific for 16s rrna was used . the 16s rrna probe hybridized with almost equal intensities with all serotypes tested ( results not shown ). probes orf2 and orf3 hybridized with all serotypes , except for serotypes 32 and 34 ( results not shown ). this indicates that the proteins encoded by orf2 and 3 are common among most streptococcus species . herein , the development and the successful application of an in vivo complementation approach for the identification of important molecular determinants that determine the differences in virulence between pathogenic and weak - pathogenic strains of streptococcus is described . using the complementation approach , one unique clone containing a 3 . 0 kb fragment of pathogenic strain ( v10 ) was selected . the selected fragment was greatly enriched in seven different pigs and the observed enrichment was not tissue specific . the selected fragment showed similar enhancing effects on the virulence of two different weak - pathogenic strains . large differences were observed between the effects of the selected fragment v10 of the pathogenic strain 10 and the corresponding fragment v735 isolated from the weak - pathogenic strain s735 on virulence . in contrast to v10 , which had a strong virulence - enhancing effect on weak - pathogenic strains , v735 showed only minor effects . therefore , differences between these two fragments are considered responsible for the observed differences on virulence . sequence data showed that the fragments v10 and v735 were highly homologous . both fragments contained two complete orfs ( orfs 2 and 3 ), both of which can potentially express proteins that may further contribute to the observed effect on virulence . the orfs 3 are highly homologous and differ in only 19 amino acids . the proteins encoded by the orfs 3 showed homology to folc ( folylpolyglutamate synthetase ) of various pro - and eukaryotic organisms . folylpolyglutamate synthetase catalyzes the conversion of folates to polyglutamate derivatives . bacteria require folates for the biosynthesis of glycin , methionine , formylmethionine , thymidine , purines and pantothenate . whether the folc proteins encoded by the fragments v10 and v735 have different enzymatic activities or different substrate specificities is unknown so far . in e . coli , an folc mutant is methionine deficient ; however , so far , a role of folc in virulence has not been described . significant differences were also observed between the orfs 2 of the fragments v10 and v735 . compared to the putative orf2 protein encoded by strain 10 , the putative protein encoded by strain s735 lacked the n - terminal 50 amino acids . in strain s735 , a strong ribosome - binding site precedes the methionine start codon of orf2 . in contrast , however , the sequence in strain 10 did not indicate the presence of a strong ribosome - binding site preceding the methionine start codon of orf2 . therefore , although orf2 of strain 10 is extended compared to orf2 of strain s735 , it is not clear whether the proteins expressed by these two orfs differ in length . in addition to the putative n - terminal differences , the putative orf2 proteins differed at nine amino acid positions ( 4 . 9 %). except for one amino acid , these amino acid substitutions were clustered at two different positions in the putative protein . the function of the orf2 protein is unknown so far . not even distant or partial homologies were found between the orf2 protein sequences and protein sequences present in the data libraries . hydrophobicity profiles showed that the orf2 - encoded protein ( s ) are very hydrophobic , thus suggesting a role of the orf2 protein in the cellular membrane . the putative − 35 region preceding the orfs 2 and 3 differed between strains s735 and 10 . therefore , differences in the expression levels rather than functional differences responsible for the observed effects on virulence are not excluded . in previous experiments , it was found that pigs infected with weak - pathogenic strains showed only mild clinical signs of disease and that bacteria could never be re - isolated from the cns or the joints . surprisingly , in the experiments described herein in which weak - pathogenic strains containing the control plasmid pcom1 were used , bacteria could ( with a low frequency ) be re - isolated from the cns as well as from the joints . several possible explanations for these observed differences exist . one explanation is that the presence of the plasmid somehow affects the ( virulence ) properties of the strains . another possibility is that the treatment of the pigs with erythromycin makes the pigs more sensitive for s . suis infections , and a third possibility is that , compared to the pigs previously used , the pigs used for the current experiments were more sensitive for s . suis infections . anson k . j ., s . movahedi , h . g . griffin , m . j . gasson and f . mulholland . 1995 . a non - essential glutamyl aminopeptidase is required for optimal growth of lactococcus lactis mg1363 in milk . microbiol . 141 : 2873 - 2881 . arends j . p . and h . c . zanen . 1988 . meningitis caused by streptococcus suis in humans . rev . infect . dis . 10 : 131 - 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