Streptococcus iniaePhosphoglucomutase Is a Virulence Factor and a Target for Vaccine Development

ABSTRACTStreptococcus iniaerepresents a major health and economic problem in fish species worldwide. Random Tn917mutagenesis and high-throughput screening in a hybrid striped bass (HSB) model of meningoencephalitis identified attenuated S. iniaemutants. The Tn917insertion in one mutant disrupted an S. iniaehomologue of a phosphoglucomutase (pgm) gene. Electron microscopy revealed a decrease in capsule thickness and cell wall rigidity, with ΔPGM mutant cells reaching sizes ∼3-fold larger than those of the wild type (WT). The ΔPGM 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 ΔPGM mutant was severely attenuated in HSB, as intraperitoneal challenge with 1,000 times the WT lethal dose produced only 2.5% mortality. Reintroduction of an intact copy of the S. iniae pgmgene on a plasmid vector restored antimicrobial peptide resistance and virulence to the ΔPGM mutant. In analysis of the aborted infectious process, we found that ΔPGM 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 ΔPGM mutant and later challenged with a lethal dose of WT S. iniaesurvived. We conclude that the pgmgene 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. iniaeΔPGM mutant is able to stimulate a protective immune response and may have value as a live attenuated vaccine for aquaculture.