Surface capsular polysaccharides play a critical role in protecting several pathogenic microbes against innate host defenses during infection. Little is known about virulence mechanisms of the fish pathogen Streptococcus iniae, though indirect evidence suggests that capsule could represent an important factor. The putative S. iniae capsule operon contains a homologue of the cpsD gene, which is required for capsule polymerization and export in group B Streptococcus and Streptococcus pneumoniae. To elucidate the role of capsule in the S. iniae infectious process, we deleted cpsD from the genomes of two virulent S. iniae strains by allelic exchange mutagenesis to generate the isogenic capsule-deficient ⌬cpsD strains. Compared to wild-type S. iniae, the ⌬cpsD mutants had a predicted reduction in buoyancy and cell surface negative charge. Transmission electron microscopy confirmed a decrease in the abundance of extracellular capsular polysaccharide. Gas-liquid chromatography-mass spectrometry analysis of the S. iniae extracellular polysaccharides showed the presence of L-fucose, D-mannose, D-galactose, D-glucose, D-glucuronic acid, N-acetyl-D-galactosamine, and N-acetyl-D-glucosamine, and all except mannose were reduced in concentration in the isogenic mutant. The ⌬cpsD mutants were highly attenuated in vivo in a hybrid striped bass infection challenge despite being more adherent and invasive to fish epithelial cells and more resistant to cationic antimicrobial peptides than wild-type S. iniae. Increased susceptibility of the S. iniae ⌬cpsD mutants to phagocytic killing in whole fish blood and by a fish macrophage cell line confirmed the role of capsule in virulence and highlighted its antiphagocytic function. In summary, we report a genetically defined study on the role of capsule in S. iniae virulence and provide preliminary analysis of S. iniae capsular polysaccharide sugar components.Streptococcus iniae was first isolated from an Amazon River dolphin (Inia geoffrensis) in the 1970s (38). Though S. iniae infections in humans can occur in the form of cellulitis resulting from a fish handling injury (52), this bacterium is primarily problematic as an aquatic pathogen. Over 30 freshwater and saltwater fish species have demonstrated susceptibility to the disease, including such economically important species as tilapia (39), yellowtail (26), trout (16), and hybrid striped bass (HSB) (17). Common clinical symptoms of S. iniae infection in fish include loss of orientation, lethargy, ulcers, exophthalmia, and erratic swimming (6). Mortality resulting from S. iniae infection is often attributed to meningoencephalitis and is responsible for aquaculture losses measured in the hundreds of millions of dollars annually. S. iniae can also cause significant disease outbreaks in wild fish populations (53). The virulence mechanisms of S. iniae are largely unknown.Our preliminary screening of an S. iniae transposon mutant library in HSB indicated that genes involved in capsule synthesis may be associated with virulence. Among other...
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