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Distribution, genetic diversity, and variable expression of the gene encoding hyaluronate lyase within the Streptococcus suis population.

ABSTRACT: 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.

SUBMITTER: King SJ

PROVIDER: S-EPMC438560 | biostudies-literature | 2004 Jul

REPOSITORIES: biostudies-literature

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Distribution, genetic diversity, and variable expression of the gene encoding hyaluronate lyase within the Streptococcus suis population.

King Samantha J SJ Allen Andrew G AG Maskell Duncan J DJ Dowson Christopher G CG Whatmore Adrian M AM

Journal of bacteriology 20040701 14

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 i ...[more]

PMID: 15231806

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Project description:Streptococcus dysgalactiae subsp. equisimilis (SDSE) causes cellulitis, bacteremia, and invasive diseases, such as streptococcal toxic shock syndrome. Although SDSE infection is more prevalent among elderly individuals and those with diabetes mellitus than infections with Streptococcus pyogenes (Group A streptococci; GAS) and Streptococcus agalactiae (Group B streptococci; GBS), the mechanisms underlying the pathogenicity of SDSE remain unknown. SDSE possesses a gene hylD encoding a hyaluronate lyase (HylD), whose homologue (HylB) is involved in pathogenicity of GBS, while the role of HylD has not been characterized. In this study, we focused on the enzyme HylD produced by SDSE; HylD cleaves hyaluronate (HA) and generates unsaturated disaccharides via a ?-elimination reaction. Hyaluronate-agar plate assays revealed that SDSE promoted dramatic HA degradation. SDSE expresses both HylD and an unsaturated glucuronyl hydrolase (UGL) that catalyzes the degradation of HA-derived oligosaccharides; as such, SDSE was more effective at HA degradation than other ?-hemolytic streptococci, including GAS and GBS. Although HylD shows some homology to HylB, a similar enzyme produced by GBS, HylD exhibited significantly higher enzymatic activity than HylB at pH 6.0, conditions that are detected in the skin of both elderly individuals and those with diabetes mellitus. We also detected upregulation of transcripts from hylD and ugl genes from SDSE wild-type collected from the mouse peritoneal cavity; upregulated expression of ugl was not observed in ?hylD SDSE mutants. These results suggested that disaccharides produced by the actions of HylD are capable of triggering downstream pathways that catalyze their destruction. Furthermore, we determined that infection with SDSE?hylD was significantly less lethal than infection with the parent strain. When mouse skin wounds were infected for 2 days, intensive infiltration of neutrophils was observed around the wound areas infected with SDSE wild-type but not SDSE?hylD. Our investigation suggested that HylD and UGL play important roles in nutrient acquisition from hosts, followed by the bacterial pathogenicity damaging host tissues.

Project description:BackgroundStreptococcus suis is an important infectious agent for pigs and occasionally for humans. The host innate immune system plays a key role in preventing and eliminating S. suis infections. One important constituent of the innate immune system is the protein lysozyme, which is present in a variety of body fluids and immune cells. Lysozyme acts as a peptidoglycan degrading enzyme causing bacterial lysis. Several pathogens have developed mechanisms to evade lysozyme-mediated killing. In the present study we compared the lysozyme sensitivity of various S. suis isolates and investigated the molecular basis of lysozyme resistance for this pathogen.ResultsThe lysozyme minimal inhibitory concentrations of a wide panel of S. suis isolates varied between 0.3 to 10 mg/ml. By inactivating the oatA gene in a serotype 2 and a serotype 9 strain, we showed that OatA-mediated peptidoglycan modification partly contributes to lysozyme resistance. Furthermore, inactivation of the murMN operon provided evidence that additional peptidoglycan crosslinking is not involved in lysozyme resistance in S. suis. Besides a targeted approach, we also used an unbiased approach for identifying factors involved in lysozyme resistance. Based on whole genome comparisons of a lysozyme sensitive strain and selected lysozyme resistant derivatives, we detected several single nucleotide polymorphisms (SNPs) that were correlated with the lysozyme resistance trait. Two SNPs caused defects in protein expression of an autolysin and a capsule sugar transferase. Analysis of specific isogenic mutants, confirmed the involvement of autolysin activity and capsule structures in lysozyme resistance of S. suis.ConclusionsThis study shows that lysozyme resistance levels are highly variable among S. suis isolates and serotypes. Furthermore, the results show that lysozyme resistance in S. suis can involve different mechanisms including OatA-mediated peptidolycan modification, autolysin activity and capsule production.

Project description:The short generation time of many bacterial pathogens allows the accumulation of de novo mutations during routine culture procedures used for the preparation and propagation of bacterial stocks. Taking the major human pathogen Streptococcus pneumoniae as an example, we sought to determine the influence of standard laboratory handling of microbes on within-strain genetic diversity and explore how these changes influence virulence characteristics and experimental outcomes. A single culture of S. pneumoniae D39 grown overnight resulted in the enrichment of previously rare genotypes present in bacterial freezer stocks and the introduction of new variation to the bacterial population through the acquisition of mutations. A comparison of D39 stocks from different laboratories demonstrated how changes in bacterial population structure taking place during individual culture events can cumulatively lead to fixed, divergent change that profoundly alters virulence characteristics. The passage of D39 through mouse models of infection, a process used to standardize virulence, resulted in the enrichment of high-fitness genotypes that were originally rare (<2% frequency) in D39 culture collection stocks and the loss of previously dominant genotypes. In the most striking example, the selection of a <2%-frequency genotype carrying a mutation in sdhB, a gene thought to be essential for the establishment of lung infection, was associated with enhanced systemic virulence. Three separately passaged D39 cultures originating from the same frozen stocks showed considerable genetic divergence despite comparable virulence. IMPORTANCE Laboratory bacteriology involves the use of high-density cultures that we often assume to be clonal but that in reality are populations consisting of multiple genotypes at various abundances. We have demonstrated that the genetic structure of a single population of a widely used Streptococcus pneumoniae strain can be substantially altered by even short-term laboratory handling and culture and that, over time, this can lead to changes in virulence characteristics. Our findings suggest that caution should be applied when comparing data generated in different laboratories using the same strain but also when comparing data within laboratories over time. Given the dramatic reductions in the cost of next-generation sequencing technology in recent years, we advocate for the frequent sampling and sequencing of bacterial isolate collections.

Dataset Information

Distribution, genetic diversity, and variable expression of the gene encoding hyaluronate lyase within the Streptococcus suis population.

Publications

Distribution, genetic diversity, and variable expression of the gene encoding hyaluronate lyase within the Streptococcus suis population.

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