Epithelial invasion and cell lysis by virulent strains of Streptococcus suis is enhanced by the presence of suilysin

Norton, P

doi:10.1016/s0928-8244(99)00116-9

Cited by 32 publications

(46 citation statements)

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“…Binding between bacterial adhesins and host proteins is an essential step in the colonization of mucosal surfaces, and has also been implicated in bacterial invasion of host cells (Tamura et al, 1994). Indeed, S. suis is able to bind to, and in some cases invade, endothelial and epithelial cells of human and porcine origin (Benga et al, 2005;Lalonde et al, 2000;Norton et al, 1999;Vanier et al, 2004). However, the specific mechanisms involved in these interactions are unknown.…”

Section: Introductionmentioning

confidence: 99%

Isolation and characterization of α-enolase, a novel fibronectin-binding protein from Streptococcus suis

et al. 2008

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Streptococcus suis is an important swine pathogen that causes meningitis, endocarditis, arthritis and septicaemia. As a zoonotic agent, S. suis also causes similar diseases in humans. Binding of pathogenic bacteria to extracellular matrix components enhances their adhesion to and invasion of host cells. In the present study we isolated and identified a novel fibronectin-binding protein from S. suis. The native protein (designated SsEno) possessed not only high homology with other bacterial enolases but also enolase activity. We cloned, expressed and purified SsEno and showed that it is ubiquitously expressed by all S. suis serotypes and we identified its surface localization using immunoelectron microscopy. ELISA demonstrated that SsEno binds specifically to fibronectin and plasminogen in a lysine-dependent manner. Additional surface plasmon resonance assays demonstrated that SsEno binds to fibronectin or plasminogen with low nanomolar affinity. Inhibition experiments with anti-SsEno antibodies also showed that bacterial SsEno is important for the adhesion to and invasion of brain microvascular endothelial cells by S. suis. Overall, the present work is the first study, to our knowledge, to demonstrate a fibronectinbinding activity of a bacterial enolase, and shows that, similar to other bacterial fibronectin-binding proteins, SsEno may contribute to the virulence of S. suis. INTRODUCTIONStreptococcus suis is a major swine pathogen that causes septicaemia, meningitis, endocarditis and arthritis (Higgins & Gottschalk, 2005). Of the 35 known serotypes, serotype 2 is the most frequently isolated and associated with disease (Higgins & Gottschalk, 2005). It has been proposed that two serotypes (serotypes 32 and 34) be excluded from S. suis and redesignated Streptococcus orisratti (Hill et al., 2005). S. suis, especially serotype 2, has also been described as an important zoonotic agent that affects people in close contact with infected pigs or pork-derived products (Lun et al., 2007). Indeed, an important number of cases of human disease with a high rate of mortality in China were linked directly to a concurrent outbreak of S. suis infection in pigs (Ye et al., 2006).Little is known about S. suis virulence factors. The capsule polysaccharide (CPS) is a critical virulence factor, given that unencapsulated isogenic mutants are completely avirulent and rapidly cleared from the circulation in pig and mouse infection models (Charland et al., 2000;Smith et al., 1999). However, non-virulent strains are also encapsulated, indicating that the virulence of this pathogen is a multifactorial process . Other potential virulence factors have also been described in S. suis, including a haemolysin (suilysin), a 136 kDa muramidase-released protein (MRP), a 110 kDa extracellular factor (EF) protein, a hyaluronidase, a superoxide dismutase, various proteases, a serum opacity factor and different adhesins (Baums et al., 2006;.The pathogenesis of S. suis infection is not fully understood and likely involves many steps . Binding between b...

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Section: Introductionmentioning

confidence: 99%

Isolation and characterization of α-enolase, a novel fibronectin-binding protein from Streptococcus suis

et al. 2008

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“…MLST findings in relation to isolates previously characterized in virulence studies. Fourteen reference isolates included in this study had previously been included in studies of S. suis virulence using porcine infection models (Table 4) (33). The virulence of these strains had been determined by one of three different methodologies, either intravenously (i.v.)…”

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“…The virulence of these strains had been determined by one of three different methodologies, either intravenously (i.v.) (33,36) or by using the method of Vecht et al (50) or the method of Galina et al (15). The methods of both Vecht et al (50) and Galina et al (15) use intranasal inoculation of S. suis following inoculation with Bordetella bronchiseptica or porcine reproductive and respiratory syndrome virus, respectively.…”

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Development of a Multilocus Sequence Typing Scheme for the Pig Pathogen Streptococcus suis : Identification of Virulent Clones and Potential Capsular Serotype Exchange

King

Leigh

Heath³

et al. 2002

J Clin Microbiol

239

285

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Streptococcus suis is an important pathogen of pigs and occasionally causes serious human disease. However, little is known about the S. suis population structure, the clonal relationships between strains, the potential of particular clones to cause disease, and the relevance of serotype as a marker for epidemiology. Here we describe a multilocus sequence typing (MLST) scheme for S. suis developed in order to begin to address these issues. Seven housekeeping gene fragments from each of 294 S. suis isolates obtained from various S. suis diseases and from asymptomatic carriage representing 28 serotypes and nine distinct countries of origin were sequenced. Between 32 and 46 alleles per locus were identified, giving the ability to distinguish >1.6 ؋ 10 11 sequence types (STs). However only 92 STs were identified in this study. Of the 92 STs 18 contained multiple isolates, the most common of which, ST1, was identified on 141 occasions from six countries. Assignment of the STs to lineages resulted in 37 being identified as unique and unrelated STs while the remaining 55 were assigned to 10 complexes. ST complexes ST1, ST27, and ST87 dominate the population; while the ST1 complex was strongly associated with isolates from septicemia, meningitis, and arthritis, the ST87 and ST27 complexes were found to contain significantly higher numbers of lung isolates. In agreement with the observed distribution of disease-causing isolates of S. suis, most isolates previously characterized as of high virulence in porcine infection models belong to ST1, while isolates belonging to other STs appear to be less virulent in general. Finally nine STs were found to contain isolates of multiple serotypes, and many isolates belonging to the same serotypes were found to have very disparate genetic backgrounds. As well as highlighting that the serotype can often be a poor indicator of genetic relatedness between S. suis isolates, these findings suggest that capsular genes may be moving horizontally through the S. suis population.

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“…Thirty colonies from the five pigs challenged with S7c were tested by PCR for the suilysin gene. In all these colonies only the mutant allele was detected, whereas all colonies isolated from P1/7-infected animals had intact suilysin genes (8). Bacterial numbers were assessed semiquantitatively (from a value of 0 for no growth to a value of 4 for heavy growth).…”

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Generation and Characterization of a Defined Mutant of Streptococcus suis Lacking Suilysin

et al. 2001

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A defined allelic-replacement mutant of the sly gene, encoding a thiol-activated cytolysin, from a European isolate of Streptococcus suis serotype 2 was generated and characterized. Unlike the parental strain, it is nonhemolytic, noncytotoxic for cultured macrophage-like cells, avirulent in a mouse infection model, yet only slightly attenuated in a porcine model of systemic infection. Streptococcus suis has been described as the etiological agent for a number of infectious disease syndromes in pigs, including arthritis, septicemia, meningitis, and pneumonia. S. suis produces a secreted hemolysin (suilysin) which has been suggested as playing a role in virulence (2,(4)(5)(6)(7)10). In order to investigate the role played by suilysin in the pathogenesis of a European serotype 2 isolate of S. suis, we generated a defined allelic-replacement mutant of the sly gene and compared the wild-type organism with the sly mutant in a number of assays.Bacterial strains and media. S. suis type 2 strain P1/7 was grown on Columbia agar (Oxoid) containing 10% defibrinated horse blood or in liquid cultures of Todd-Hewitt broth (Oxoid) supplemented with 7% fetal calf serum (FCS) (Gibco). Allelicreplacement mutants of S. suis were maintained on 1 g of erythromycin (Sigma) per ml.Mutagenesis of the suilysin gene from S. suis type 2. An erythromycin resistance gene cassette was introduced into an EcoRV site within a 1,278-bp fragment of the sly gene, contained in the vector pT7-Blue (Stratagene), amplified by PCR using primers suis1 (5Ј-AGCTTGACTTACGAGCCACAAG AG-3Ј) and suis2 (5Ј-CCACCATTCCCAAGCTAATCCTGT-3Ј) with chromosomal DNA from P1/7 as a template. The resulting plasmid, pSUI-erm, contains the erm gene in the same orientation as the sly gene. Plasmid pSUI-erm was introduced into P1/7 by electroporation (22.5 V/cm, 25 F, and 1,000 ⍀). A transformant which had undergone a doublecrossover event, confirmed by Southern hybridization and PCR, with concomitant insertion mutation of the sly gene, was isolated and named S7c.Phenotypic analysis of S7c. Overnight growth of suilysin mutant S7c on Columbia blood agar plates revealed no ␤-hemolysis. Secreted proteins from anaerobically grown overnight cultures of P1/7 and S7c were concentrated 100-fold by ammonium sulfate (50%, wt/vol) precipitation; then 10 l each of these preparations was spotted onto a Columbia horse blood agar plate and incubated at 37°C for 30 min. The proteins from P1/7 show clear zones of hemolysis, whereas there is a complete absence of hemolytic activity in the proteins obtained from S7c (Fig. 1a). The hemolytic activity from the wild-type parental bacteria was enhanced by the addition of ␤-mercaptoethanol, as previously reported (7) (Fig. 1a).The lack of expression of suilysin was confirmed by Western blotting. Supernatants from aerobically grown cultures of both P1/7 and S7C (sterilized using a 0.22-m-pore-size filter and concentrated 25-fold using Amicon filters) were probed with a monoclonal antibody (INT-STS-28-02; A. C. Jacobs, Intervet) raised against purified s...

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Epithelial invasion and cell lysis by virulent strains of Streptococcus suis is enhanced by the presence of suilysin

Cited by 32 publications

References 15 publications

Isolation and characterization of α-enolase, a novel fibronectin-binding protein from Streptococcus suis

Isolation and characterization of α-enolase, a novel fibronectin-binding protein from Streptococcus suis

Development of a Multilocus Sequence Typing Scheme for the Pig Pathogen Streptococcus suis : Identification of Virulent Clones and Potential Capsular Serotype Exchange

Generation and Characterization of a Defined Mutant of Streptococcus suis Lacking Suilysin

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