Streptococcus iniae phosphoglucomutase is a virulence factor an a target for vaccine development

Streptococcus iniae represents a major health and economic problem in fish species worldwide. Random Trt917 mutagenesis and high-throughput screening in a hybrid striped bass (HSB) model of meningoencephalitis identified attenuated S. iniae mutants. The Tn917 insertion in one mutant disrupted an S. iniae homologue of a phosphoglucomutase (pgm) gene. Electron microscopy revealed a decrease in capsule thickness and cell wall rigidity, with Delta PGM mutant cells reaching sizes similar to 3-fold larger than those of the wild type (WT). The Delta PGM mutant was cleared more rapidly in HSB blood and was more sensitive to killing by cationic antimicrobial peptides including moronecidin from HSB. In vivo, the Delta PGM mutant was severely attenuated in HSB, as intraperitoneall challenge with 1,000 times the WT lethal dose produced only 2.5% mortality. Reintroduction of an intact copy of the S. iniae pgm gene on a plasmid vector restored antimicrobial peptide resistance and virulence to the Delta PGM mutant. In analysis of the aborted infectious process, we found that Delta PGM mutant organisms initially disseminated to the blood, brain, and spleen but were eliminated by 24 h without end organ damage. Ninety to 100% of fish injected with the Delta PGM mutant and later challenged with a lethal dose of W S. iniae survived. We conclude that the pgm gene is required for virulence in S. iniae, playing a role in normal cell wall morphology, surface capsule expression, and resistance to innate immune clearance mechanisms. An S. iniae Delta PGM mutant is able to stimulate a protective immune response and may have value as a live attenuated vaccine for aquaculture.