Journal of Bacteriology, July 2004, p . 4740-4747, Vol . 186, No . 14

Distribution, Genetic Diversity, and Variable Expression of the Gene Encoding Hyaluronate Lyase within the Streptococcus suis Population

Samantha J . King,^1, Andrew G . Allen,^2, Duncan J . Maskell,² Christopher G . Dowson,¹ and Adrian M . Whatmore^1*

Infectious Disease Research Group, Department of Biological Sciences, University of Warwick, Coventry CV4 7AL,¹ Centre for Veterinary Science, Department of Clinical Veterinary Medicine, University of Cambridge, Cambridge CB3 0ES, United Kingdom²

Received 12 January 2004/ Accepted 6 April 2004

 
Although Streptococcus suis is an economically important pathogen of pigs and an occasional cause of zoonotic infections of humans knowledge of crucial virulence factors, and as a consequence targets for therapeutic or prophylactic intervention, remains limited . Here we describe a detailed study of the distribution, diversity, and in vitro expression of hyaluronate lyase, a protein implicated as a virulence factor of many mucosal pathogens . The gene encoding hyaluronate lyase, hyl, was present in all 309 bona fide S . suis isolates examined representing diverse serotypes, geographic sources, and clinical backgrounds . Examination of the genetic diversity of hyl by RFLP and sequence analysis indicated a pattern of diversity shared by many gram-positive surface proteins with a variable 5' region encoding the most distal cell surface-exposed regions of the protein and a much more conserved 3' region encoding domains more closely associated with the bacterial cell . Variation occurs by several mechanisms, including the accumulation of point mutations and deletion and insertion events, and there is clear evidence that genetic recombination has contributed to molecular variation in this gene . Despite the ubiquitous presence of hyl, the corresponding enzyme activity was detected in fewer than 30% of the 309 isolates . In several cases this lack of activity correlates with the presence of mutations (either sequence duplications or point mutations) within hyl that result in a truncated polypeptide . There is a striking absence of hyaluronate lyase activity in a large majority of isolates from classic S . suis invasive disease, indicating that this protein is probably not a crucial virulence factor, although activity is present in significantly higher numbers of isolates associated with pneumonia .

 
Streptococcus suis is an important pathogen that has substantial welfare and economic consequences for the pig industry . S . suis is frequently carried asymptomatically but is also a major cause of septicemia, meningitis, and pneumonia in swine . Occasionally, serious zoonotic infections of humans, including septicemia, meningitis, and endocarditis are caused by S . suis (4, 36) . The treatment of S . suis infection is increasingly compromised by antibiotic resistance, and its control in pigs by using prophylactic antibiotics is becoming unacceptable (1, 31, 38) . The search for therapeutic and vaccine targets is hampered by the lack of understanding of S . suis pathogenesis and virulence determinants . Some 35 distinct serotypes of S . suis are currently recognized on the basis of capsular antigenic differences (10-12, 16, 29) . The majority of disease is associated with a small number of these serotypes, such as serotypes 1, 2, and 14, but the prevalence of particular serotypes varies geographically and over time (13) . In addition, not all isolates of these serotypes cause disease, suggesting roles for other factors in S . suis pathogenesis .

The hyaluronate lyase or hyaluronidase of S . suis is a secreted protein (A . G . Allen, unpublished data) that progresively degrades hyaluronic acid (HA) into unsaturated disaccharides . HA is a high-molecular-weight polysaccharide consisting of repeating disaccharide units [ß-1,4-D-glucuronic acid-ß-1,3-N-acetyl-ß-D-glucosamine]_n . HA is a major component of the extracellular matrix of body tissues and a major or sole component of the capsular material of certain microorganisms (23) . Human tissues known to contain HA include blood, plasma, brain, articular cartilage, liver, synovial fluid, umbilical tissue, amniotic fluid, and skin . Hyaluronate lyases are produced by a variety of gram-positive organisms and are also associated with some gram-negative organisms (19) . These enzymes are variously proposed to have roles in providing nutrients for the cell and in pathogenesis . Many members of gram-positive genera capable of elaborating hyaluronidase are able to cause infections initiated at mucosal or skin surfaces of humans or animals . It is proposed that the decrease in viscosity due to depolymerization of HA results in increased permeability of the connective tissues, increasing the ability to spread and hence the virulence of these microorganisms . In addition, hyaluronidases may degrade HA cell surface coatings, thereby allowing direct contact between the bacterium and specific receptors on the cell surface .

Members of several streptococcal species (including Streptococcus pneumoniae and Streptococcus agalactiae that, like S . suis, can cause meningitis and septicemia) are known to produce a cell surface-associated hyaluronate lyase (32, 37) and various lines of evidence have indicated potential importance in pathogenesis . Virulent S . agalactiae isolates associated with invasive disease produce higher levels of extracellular hyaluronate lyase than isolates carried asymptomatically (28, 34) . In addition, whereas all strains of group B streptococci examined contained the gene encoding hyaluronate lyase, in some isolates it is interrupted by an IS element abolishing activity (14, 34) . The possession of an intact hyaluronidase gene is a feature of the group B streptococcus III-3 lineage responsible for most neonatal invasive disease (6) . Studies with S . pneumoniae have shown that addition of hyaluronidase increases pathogenicity in a mouse model of meningitis when intranasal inoculation is used (43) . Furthermore, signature-tagged mutagenesis studies have provided evidence that hyaluronidase is important in pneumococcal pneumonia, although it apparently has little role in septicemia (30) . Expression of hyaluronidase in the group of streptococci previously known as "Streptococcus milleri" has also been strongly associated with isolates obtained from internal abscesses rather than those obtained as normal flora from uninfected sites (39) .

The hyaluronate lyase-encoding gene (hyl) was recently identified in and cloned from S . suis . The DNA sequence of this gene was deposited in GenBank under accession number AJ308330 . The gene apparently encodes a classic gram-positive signal peptide and cell wall-associated LPXTG motif, but hyl from this strain (P1/7) had a premature stop codon resulting in a truncated protein of 522 amino acids lacking hyaluronidase activity . In contrast, a serotype 7 isolate, 1307, harbored full-length hyl (3,495 bp, AJ308328) and possessed hyaluronidase activity in vitro . Any role for S . suis hyaluronate lyase or the truncated protein in virulence is unknown . Although hyaluronidase may contribute to virulence and may therefore be a useful vaccine component, there is a clear need to understand more about the relationship of this protein to virulence, its genetic stability, and the extent of genetic diversity . In order to address these issues, the aims of the present study were to examine the presence of hyl and hyaluronidase activity in a large sample of field isolates, to relate these findings to the clinical background of the isolates and phenotypic characteristics such as serotype, and to examine the nature and extent of hyl genetic diversity .

 
Bacterial isolates. A total of 309 S . suis isolates were used in the present study . Reference strains (8) of 34 serotypes (serotypes 1, 2 to 20, and 22 to 34) were supplied by L . A . Devriese (Faculty of Veterinary Medicine, University of Ghent, Ghent, Belgium), M . Gottschalk (Faculté de Médicine Vétérinaire, Université de Montréal, Montreal, Quebec, Canada), and P . Heath (Veterinary Laboratories Agency, Bury St . Edmunds, United Kingdom) . Twenty-two serotype 2 isolates, including many isolates characterized in previous virulence studies, and twelve serotype 14 isolates from meningitis cases were supplied by P . Norton (Institute for Animal Health, Newbury, United Kingdom) . A total of 140 United Kingdom field isolates obtained by the Veterinary Laboratories Agency from various geographical sources were included in the present study . These were selected to represent isolates of diverse serotypes and sites of isolation and clinical background, including "invasive" disease isolates (meningitis, septicemia, and arthritis) and lung isolates from cases of pneumonia . A similar sample of 84 Spanish field isolates obtained by C . Tarradas and I . Luque was included in the present study . The sample consisted of 39 "carried" isolates obtained from the tonsils of healthy pigs and 45 isolates obtained from symptomatic disease again representing diverse clinical backgrounds (25) . Two porcine isolates, one previously described as atypical (22), provided by C . Lammler, Institut fuer Tierarztliche, Giessen, Germany, were included, as were 16 isolates obtained from S . suis disease of humans . These isolates were obtained from Augustine Cheng (Department of Microbiology, Faculty of Medicine, The Chinese University of Hong Kong [six isolates]), M . Gottschalk (four isolates), G . Grise (Centre Hosptialier d'Elbeuf, Saint-Aubin les Elbeuf, Elbeuf, France [three isolates]) (7), C . Lammler (one isolate), P . Heath (one isolate), and B . Francois (Hospital Universitaire Dupuytren, Limoges, France [one isolate]) (9) .

Preparation of chromosomal DNA. Chromosomal DNA was prepared from all isolates as described previously (42) .

PCR analysis. PCR was performed under standard conditions with 30 cycles of 95°C for 1 min, 50°C for 1 min, and 72°C for 1 min per kb of predicted product . Products were visualized by agarose gel electrophoresis on 1.0% agarose in the presence of 1 µg of ethidium bromide ml^–1 . Details of all oligonucleotides used in the present study are given in Table 1 .

 
Analysis of hyaluronidase genetic diversity. Approximately 5 µl of PCR product was digested by restriction enzymes according to manufacturer's instructions in a total volume of 25 µl . Digests were then separated on 4 and 8% vertical polyacrylamide gels and visualized under UV illumination after staining for 15 min in 0.3 µg of ethidium bromide ml^–1 . Alleles were designated by visual comparison of restriction fragment length polymorphism (RFLP) profiles . In the case of the 5' region of hyaluronidase, a fraction of PCR products used in the RFLP analysis were purified by passage through QiaQuick PCR product purification columns (Qiagen) and directly sequenced . After the RFLP analysis of the full-length PCR product, a fraction of the product was cloned into pGEM-T (Promega), and sequencing was performed after plasmid extraction with the Qiaprep spin miniprep kit (Qiagen) by using the Beckman CEQ2000 system according to the manufacturer's instructions . Sequences of the novel hyaluronidase alleles described here have been submitted to GenBank and assigned the accession numbers indicated in Table 2 .

 
Bioinformatics. Primary sequence analysis was performed by using the package DNASTAR . The similarity of sequences identified in the present study to those already present in the databases was examined by basic local alignment search tool (BLAST) analysis (http://www.ncbi.nlm.nih.gov/BLAST) . Analysis of variable sites was performed by using the MEGA package, version 1.01 (21) .

Hyaluronidase assays. Hyaluronidase assays were performed by using the method of Smith and Willett (35) . Plates were prepared by using sterile brain heart infusion broth containing 1% (wt/vol) agar cooled to 46°C . An aqueous 2-mg ml^–1 solution of filter-sterilized (0.22-µm pore size) HA was added to a final concentration of 400 µg ml^–1 . A 5% (wt/vol) filter-sterilized solution of bovine serum albumin fraction V was then added with constant stirring to give a final concentration of 1% (wt/vol) in the medium . A single colony of isolates to be tested was subcultured to a postage stamp-sized square on an assay plate . After overnight incubation at 37°C, the plate was flooded with 2 M acetic acid and allowed to stand for 10 min . Hyaluronidase activity was detected as a zone of clearing around the patch in a cloudy background, resulting from acetic acid precipitation of an albumin-nondegraded HA complex .

 
Investigations of the distribution and diversity of hyl by PCR, RFLP, and sequencing of full-length PCR products. Chromosomal DNA was purified from all 309 isolates of S . suis in the Warwick collection described in Materials and Methods . From these, 171 randomly selected DNA preparations were screened for the presence of hyl by using primers 1 and 2 designed on the basis of the three extant S . suis hyl sequences (A . Allen, unpublished data; AJ308328 to AJ308330) . Primer 2 binds at the 3' of hyl of strains 1307 and P5/11/88 that encode full-length hyaluronidases, but external to the hyl coding region of the serotype 2 strain P1/7 that encodes the truncated, inactive hyaluronidase . For simplicity, the region covering the coding sequence for the active allele is hereafter termed the hyl locus for all isolates . A product of 3.6 kb (as predicted on the basis of sequence AJ308330) was amplified from 156 of 171 isolates . However, PCR products from 60 of these 156 isolates were deemed too weak for RFLP analysis . A second primer pair designed to amplify the entire hyl locus was tested by using a range of annealing temperatures and magnesium chloride concentrations but did not improve the PCR product yield (data not shown) .

A limited RFLP study of the 96 hyl PCR products obtained with primers 1 and 2 and deemed suitable for analysis was performed . Use of the enzymes HinfI, BslI, Hsp92II, HaeIII/DdeI, Tsp509I, and MwoI indicated that there is substantial genetic diversity between isolates and identified nine distinct alleles (data not shown) . In order to examine the nature of this diversity hyl, PCR products representative of six of the RFLP variants were cloned and sequenced in full . Analysis of nucleotide sequences obtained from these six representatives (isolates 3, 6, 21, 34, 100, and 256) and the three previously identified allelic variants (P1/7, 1307, and P5/11/88) demonstrates that the majority of sequence variation occurs within the 5' region of hyl . A summary of the main features is presented in Fig . 1 . Much of the diversity apparent in this region reflects deletions and/or insertions and an increasing frequency of point mutations toward the 5' end of hyl . Although no structural studies of S . suis hyaluronidase have been carried out, a number of residues identified as forming the active site in S . pneumoniae hyaluronidase (24) can be identified in S . suis . The S . suis residues equivalent to the proposed active site residues Asn349, His399, and Tyr408 are all present and completely conserved in all of the S . suis sequences . Despite the occurrence of deletions and insertions, all nine alleles for which sequence is available remained in frame until the residue (523 within the sequence from strain P1/7) previously identified as the termination codon responsible for an inactive, truncated hyaluronidase protein . Of the six full-length sequences examined in the present study, only one (isolate 21) possesses the early termination codon corresponding to 523 within P1/7, resulting from the identical 2-bp duplication some 12 bp upstream . The remaining five sequences are predicted to encode uninterrupted peptide sequences ranging from 1,140 to 1,172 residues . Nucleotide diversity between the alleles varies from 0.03 to 5.09%, and amino acid diversity of the seven alleles encoding full-length hyaluronidase ranges from 0.09% to 5.43% (considering only residues present in all alleles) . In some cases, deletion or insertion events appear to be mediated by short direct repeats . For example, as demonstrated in Fig . 2, isolate 21 contains a deletion of 138 bp, the first 8 bp of which are identical to those immediately distal to the deletion . A 21-bp insertion previously identified within P1/7 was also identified within the hyl sequence of isolate 6, and this insert includes a direct repeat of a 13-bp sequence present immediately proximal to the insertion . However, in other cases, such as a 74-bp deletion in isolate 256 and a 13-bp insertion in isolate 21 hyl, no obvious mechanism could be identified .

 
PCR amplification and RFLP of the 5' variable region. Given the difficulties in obtaining a full-length hyl PCR product and the fact that the preliminary studies described above indicated that most of the diversity is within the 5' part of hyl, further studies focused on this region . Primers 3 and 4 were designed to amplify an 862-bp fragment of the 5' region of hyl . Comparison of the nine full-length sequences described above demonstrates that nucleotide diversity in this 5' region ranges from 0.15 to 15.88% (for nucleotides present in all alleles), whereas the maximum diversity of the remaining 3' region in the corresponding isolates is 2.59% . A PCR product of approximately the predicted size was amplified from 271 of the 309 isolates screened . All 271 PCR products were of suitable quality for RFLP analysis, and allelic diversity was assessed by restriction of each PCR product independently with two frequently cutting restriction enzymes, MwoI and BfaI . Restriction resulted in the identification of 21 distinct RFLP profiles, although the majority of isolates (62%) possessed profile 1 . The frequency of these 21 profiles and their distribution with respect to serotype and country of origin is shown in Table 3 .

 
Genetic diversity of the 5' region of hyl confirmed by sequencing of RFLP alleles. In order to enhance our understanding of the extent and nature of genetic diversity within the 5' region of hyl, a PCR product representative of each of the 21 RFLP profiles was sequenced directly . The sequence diversity is illustrated in Fig. 3, which shows only residues that are polymorphic (not absent) in one or more isolates relative to the 1307 sequence . Nucleotide sequence from these 21 representatives and the three published sequences corresponded to 18 distinct amino acid sequences . All but 3 of the 24 sequences are predicted to encode uninterrupted amino acid sequences through the region examined . The hyl14 sequence contains a unique single-nucleotide alteration, leading to a stop codon early in the predicted amino acid sequence, while the hyl18 sequence also contains an early stop codon generated as a result of an 18-bp deletion . In addition, the hyl9 sequence contains a 4-bp duplication that causes a frameshift, leading to a stop codon some 37 amino acid residues later . Comparison of the sequences reveals further examples of the role of deletion and insertion events in generating hyl diversity in addition to those already described . Thus, as already mentioned, the hyl18 sequence has an 18-bp deletion, whereas a 12-bp duplication is present within the hyl17 and hyl20 sequences . The diversity between the sequenced alleles at residues present in all 24 sequences ranges from 0 to 17.42% at the nucleotide level and from 0 to 19.8% at the amino acid level .

 
Comparison of the sequences strongly indicates that recombinational shuffling also plays a significant role in the evolution of hyl . This is illustrated in Fig . 3, where three distinct sequence types corresponding to hyl1307 (shown in blue), hyl20 (shown in pink), and hyl21 (shown in red) can be differentiated . There are a number examples of other sequences consisting of components of two or more sequence types that clearly reflect recombination either within the species or perhaps with an hyl gene in closely related species . Perhaps the most obvious example is hyl17 with the 5' 340 bp being closely related to the hyl20 sequence but the remaining 3' region being identical to the hyl1307 sequence and clearly divergent from the equivalent region of hyl20 . Similarly, the 5' hyl13 sequence is virtually identical to the hyl21 sequence but dramatically switches at approximately residue 575, after which point the sequence is very divergent from hyl21 and resembles the hyl1307 and hyl20 sequences (the decreasing diversity toward the 3' of the sequences makes it impossible to distinguish with certainty sequences originating from these two types) . Similar mosaic patterns are apparent in at least two other sequences, hyl7 and hyl14, providing further support for the role of recombination in the evolution of S . suis hyl .

Determination of hyaluronate lyase activity. Plate assays were performed to determine the proportion of the 309 isolates that produce active hyaluronate lyase (Table 3) . In the case of the vast majority of the 309 S . suis isolates, the results were unequivocal, with 91 isolates giving a distinct zone of clearing in the assay indicative of active hyaluronidase, whereas 215 isolates displayed no zone of clearing . The remaining three isolates, scored as "+/–," displayed a weak zone of clearing in three independent tests . Of the 34 different serotype reference strains screened, activity was visualized for serotypes 2, 3, 4, 7, 8, 11, 12, 15, 27, and 30 . In addition, a weak (i.e., +/–) response was visualized for the serotype 33 reference strain . In the majority of cases the presence or absence of hyaluronidase activity was consistent within an RFLP type, and isolates shown to contain interrupted hyl sequences, such as hyl14, hyl18, and hyl9, lacked hyaluronidase activity as would be predicted . However, 4 of the 21 RFLP profiles contained different isolates that had either positive or negative enzyme activity (Table 3) . In most cases an overwhelming majority of isolates showed one state of activity, whereas a small proportion of isolates represented the other . This finding supports the idea that some of the RFLP profiles identified contain some isolates that harbor alterations not detected by our limited RFLP study .

Confirmation of the presence of a hyl homologue in isolates negative by hyl PCR and hyaluronate lyase activity. The attempted amplification of hyl from 38 of 309 isolates with primers 3 and 4 failed to generate a PCR product . These isolates included the reference strains for serotypes 6, 9, 10, 13, 16, 17, 18, 19, 20, 22, 23, 24, 25, 26, 28, 29, 31, 32, 33, and 34 . Of these 38 isolates, 37 also failed to produce detectable hyaluronidase activity by the plate assay, and the remaining isolate (type strain 33) gave only a weak zone of clearing . To determine whether these isolates contain at least part of the hyl locus, a dot blot was performed that included 16 field isolates and 20 reference strains that lack hyaluronidase activity and are hyl PCR negative . A probe was constructed by labeling a PCR product of 1,292 bp amplified from strain P1/7 by using primers 5 and 6 . This probe hybridized to chromosomal DNA from P1/7 used as a positive control, and no hybridization was detected with an E . coli negative control . Strong hybridization of the probe was detected for all test isolates, with the exception of the reference strains for serotypes 32 and 34, which are known to be phylogenetically distantly related to most S . suis isolates (data not shown) . A control probe consisting of the 16S rRNA gene of P1/7 hybridized to all of the chromosomal DNA samples, including the serotype 32 and 34 isolates . Thus, hyl or a fragment thereof does appear to be present in all bona fide S . suis isolates, although in many cases it possesses inactivating mutations and/or no activity could be detected under the conditions examined in the present study .

Relationship between hyaluronidase activity and serotype or disease state. Table 4 shows the distribution of hyaluronidase activity detected by plate assay from field isolates from both asymptomatic carriage and different disease states . Hyaluronidase activity was not strongly associated with classic S . suis invasive disease isolates from cases of septicemia, meningitis, and arthritis being present in only between 0 and 29.6% of isolates associated with these conditions . In contrast, although numbers of isolates are much smaller, about 50% of the isolates obtained from pneumonia possessed hyaluronidase activity . The relationship between hyaluronidase activity and serotype was also investigated when sufficient numbers of strains from individual serotypes had been examined . Within the study there were nine serotypes that had eight or more representatives, and the distribution of hyaluronidase positives varied among these groups . Activity was detected in 75% (9 of 12) of serotype 3 isolates and 77% (10 of 13) of serotype 7 isolates, whereas only 12.5% of serotype 9 isolates, 14.3% (2 of 14) of serotype 1 isolates, 21% (8 of 30) of serotype 14 isolates, and none of the 11 isolates cross-reactive to antisera for both serotypes 1 and 14 gave a positive result . Serotype 2 accounted for 136 of the 309 isolates, and only 26.5% possessed detectable hyaluronidase activity . The other two serotypes contained similar numbers of isolates in which hyaluronidase activity was or was not detected; 50% (4 of 8) serotype 1/2 isolates and 66% (6 of 9) serotype 15 isolates yielded positive results on the plate assay . Hyaluronidase activity was not detected in any representatives of serotypes 6, 13, 17, 18, 19, 20, 22, 23, 24, 25, 26, 29, 31, 32, and 34 (one isolate screened from each) .

 
Prior to the present study, little was known about the diversity and distribution of S . suis hyl and its relationship with virulence, reflecting the general lack of understanding of S . suis pathogenesis and the virulence factors required for S . suis disease . From these studies it is clear that hyaluronate lyase shares features with many other streptococcal surface proteins with a variable N-terminal region and a more conserved C-terminal region containing cell wall-associated motifs . As with many of these surface proteins diversity, and potentially antigenic variation, within the N-terminal region most distal to the cell surface appears to be driven by a number of mechanisms, including the accumulation of point mutations, the generation of deletions and insertions, and recombination events importing DNA from unknown sources . This diversity likely reflects selective pressures imposed by host immune surveillance and contrasts strongly with that seen in non-cell-surface-associated S . suis proteins such as suilysin (20) . Although there are few data regarding the diversity of genes encoding hyaluronate lyase, a bacteriophage-encoded hyaluronidase of Streptococcus pyogenes also displays substantial allelic polymorphism and strong evidence that recombinational processes contribute to molecular variation (27) . The diversity identified within the hyl locus in the present study is likely to be an underestimate of that present in the population . This view is supported by the lack of PCR products from some strains shown to possess the locus by hybridization .

In contrast to the apparent widespread distribution of hyl in S . suis, hyaluronate lyase activity was detected in vitro in fewer than 30% of the corresponding isolates . In many cases the lack of activity of particular alleles correlates with mutations present in the gene; thus, for example, a duplication in hyl9 interrupting the reading frame and a point mutation introducing a stop codon in hyl14 are reflected in the lack of hyaluronidase activity in the parent strain . In other cases, the lack of activity was not associated with any obvious mutation . However, since only the most 5' 20% of the hyl sequence was obtained from a large proportion of isolates lack of activity may reflect uncharacterized mutations further downstream or outside the gene, as well as the influence of regulatory elements on expression . In the majority of cases, the presence or absence of activity was consistent with an RFLP type, but four restriction profiles contained small numbers of isolates giving contradictory assay results . This is likely to be a consequence of the relatively low resolution of the 5' RFLP, with the contradictory isolates representing closely related but distinct sequence variants . An alternative possibility, that some isolates contain another gene with hyaluronate lyase activity, cannot be discounted, particularly since some other streptococcal species can possess multiple hyaluronidases (17, 18) .

The lack of detectable hyaluronate lyase activity in the majority of strains (ca . 75%) isolated from invasive human and porcine disease indicates that hyaluronidase is not an essential virulence determinant . Interestingly, hyaluronidase activity is present in a higher proportion of pneumonia-associated isolates . This might suggest a role for hyaluronidase in lung disease, although any extrapolations need to be treated with caution since, although the proportion of isolates possessing hyaluronidase activity is much higher than with meningitis, septicemia, or arthritis isolates, only 50% of pneumonia-associated isolates still possess activity . In addition, there is continued debate about whether S . suis is a primary cause of pneumonia as many isolations of S . suis are made in conjunction with organisms considered to be more significant respiratory pathogens, such as Actinobacillus pleuropneumoniae, Haemophilus parasuis, Pasteurella multocida, and swine influenza virus (15, 26, 33) . However, there are reports of isolation of S . suis in pure culture from swine with acute respiratory distress or pneumonia, suggesting a potential causative role of this organism in respiratory disease (3, 15) . Interestingly, signature-tagged mutagenesis studies have provided evidence that S . pneumoniae hyaluronidase is important in a model of pneumococcal pneumonia but a septicemia model suggested no major role once the organism has entered the bloodstream (30) . In spite of any potential role of S . suis hyaluronidase in lung disease, it is the classic invasive diseases (meningitis and septicemia) and not localized pneumonia that present the major animal welfare and economic problems associated with S . suis, and the evidence presented here overwhelmingly suggests that hyaluronidase is not a major virulence factor of S . suis . Thus, in contrast to the situation with Streptococcus pneumoniae, where the use of hyaluronidase as a vaccine target (5) or as a target for inhibitory compounds (2) has been suggested, S . suis hyaluronidase appears not to be a relevant target for prophylactic or therapeutic approaches .

One caveat in the argument against a role of hyaluronidase in virulence is the observation that many of the attenuating mutations involve the duplication of short tandem repeats . Thus, the hyl9 isolate possesses a 4-bp duplication, whereas the hyl1 representative sequenced possesses a 2-bp duplication and an additional A in a poly(A) tract . Such direct sequence duplications are likely to be intrinsically unstable and readily reversible . Indeed, the generation and excision of similar short sequence duplications in various capsular biosynthetic genes of S . pneumoniae has recently been associated with capsule phase variation (40, 41) . Thus, the possibility that hyaluronidase is regulated by the generation and excision of these repeats cannot be formally ruled out, although at least two observations argue against this . First, the overwhelming majority (>95%) of isolates possessing hyl1 lack hyaluronidase activity; if the gain and loss of sequence repeats was a common occurrence and the expression of hyaluronidase was important for virulence one might expect more isolates to possess hyaluronidase activity . Second, hyl1 isolates have two frameshift mutations and a further point mutation downstream, and all of these would need to revert to restore a fully coding open reading frame . Although the reversion of multiple changes could occur under appropriate selective pressures, this would presumably be much less likely than the reversion of the single duplications seen in pneumococcal capsular genes . A further possibility that could explain the apparent maintenance of the 5' region of the truncated hyl is that this gene encodes a multifunctional protein and that the 5' region encodes other currently unrecognized activities .

In summary, the present study shows that although the hyl locus is present in virtually all S . suis isolates tested, only 29.4% of isolates express an active protein . The gene, in common with many encoding streptococcal surface proteins, has a variable 5' end evolving by the generation of point mutations, insertions or deletions, and recombination events and a more conserved 3' end . Despite these observations suggesting the corresponding protein is subject to host immune surveillance, the lack of activity in many virulent field isolates, often associated with the disruption of the hyaluronidase open reading frame, suggests that hyaluronidase is not an important virulence factor of S . suis . Thus, the search for the crucial virulence factors of this pathogen and suitable targets for prophylactic and therapeutic intervention continues .

 
This study was supported by project grant 88/S11598 from the BBSRC . A.M.W . was supported by a Wellcome Trust Research Fellowship in Biodiversity (053589), and A.G.A . was supported by a Wellcome Trust Research Career Development Fellowship .

We gratefully acknowledge all colleagues listed in Materials and Methods who provided strains from their respective collections, and we thank Peter Heath (Veterinary Laboratories Agency, Bury St . Edmunds, United Kingdom) for helpful discussions .

 
* Corresponding author . Mailing address: Department of Statutory and Exotic Bacterial Diseases, Veterinary Laboratories Agency, Addlestone, KT15 3NB Surrey, United Kingdom . Phone: 01932-357311 . Fax: 01932-357423 . E-mail: a.whatmore@vla.defra.gsi.gov.uk .

Present address: Department of Microbiology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104 .

Present address: Arrow Therapeutics, Ltd., London SE1 1DA, United Kingdom .

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