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Characterization of virulence factors in the aquatic pathogen Streptococcus iniae

  • Author(s): Locke, Jeffrey Brian
  • et al.
Abstract

Streptococcus iniae has emerged as a leading pathogen of intensive finfish aquaculture operations worldwide, though an understanding of its virulence mechanisms and effective therapeutic strategies are lacking. The roles of four putative S. iniae virulence factors: Streptolysin S (SLS), capsular polysaccharide, M-like protein, and C5a peptidase, were investigated through targeted allelic exchange mutagenesis coupled with in vitro and in vivo models of bacterial pathogenesis. Highly attenuated mutants were then evaluated as live vaccine candidates. SLS is a potent, broad-spectrum, secreted cytolysin first characterized in group A Streptococcus. Allelic replacement of the sagA gene in the S. iniae SLS operon generated a mutant that was significantly less cytotoxic to a variety of fish cell types. Reduced cytotoxicity in vivo towards brain cells in particular may account for the mutant's high level of attenuation in a hybrid striped bass (HSB) challenge model. Capsular polysaccharide serves as a protective extracellular coating in many pathogenic streptococci. Targeted allelic replacement of the S. iniae cpsD capsule biosynthesis gene generated a mutant that was completely attenuated in HSB even at a dose 1,000X the lethal WT dose, likely due to its high susceptibility to phagocytic clearance, as demonstrated with a fish macrophage cell line. Through pyrosequencing of the S. iniae genome we identified gene homologues to classical surface-anchored streptococcal virulence factors: M-like protein (simA) and C5a peptidase (scpI). Analysis of the corresponding allelic mutants in HSB and zebrafish revealed that only simA contributes significantly to S. iniae pathogenesis. In vitro cell-based analyses indicated that SiMA, like other M family proteins, contributes to adherence and invasion and provides resistance to phagocytic killing. The immunoprotective capacity of the [Delta]sagA, [Delta]cpsD, and [Delta]simA attenuated mutant strains was compared with a killed bacterin vaccine in HSB through injection and immersion delivery. The [Delta]cpsD, [Delta]simA, and bacterin vaccines met vaccination safety criteria and were effective in generating high levels of immune protection through injection, however the [Delta]simA mutant was the only vaccine candidate to provide 100% protection through both injection and immersion delivery. Characterization of these four genes has contributed significantly to our understanding of S. iniae virulence mechanisms and demonstrates the application of pathogenesis research to vaccine development

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