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Streptococcus iniae Capsule Impairs Phagocytic Clearance and Contributes to Virulence in Fish

Jeffrey B. Locke,^1,2, Kelly M. Colvin,^4, Anup K. Datta,³ Silpa K. Patel,¹ Nandita N. Naidu,³ Melody N. Neely,⁵ Victor Nizet,^1,2,3 and John T. Buchanan^1,4*

Department of Pediatrics, Division of Pharmacology and Drug Discovery,¹ Center for Marine Biotechnology and Biomedicine, Scripps Institution of Oceanography,² Glycotechnology Core Resource, Glycobiology Research and Training Center, University of California, San Diego, La Jolla, California 92093,³ Kent SeaTech Corporation, San Diego, California 92121,⁴ Department of Immunology and Microbiology, Wayne State School of Medicine, Detroit, Michigan 48201⁵

Received 31 July 2006/ Accepted 2 November 2006

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.

Published ahead of print on 10 November 2006.

These authors contributed equally to the research presented in this publication.

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