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Infection and Immunity, October 2005, p. 6935-6944, Vol. 73, No. 10
0019-9567/05/$08.00+0 doi:10.1128/IAI.73.10.6935-6944.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Departments of Pediatrics,1 Pathology, University of California, San Diego, School of Medicine, La Jolla, California 92093,3 Kent SeaTech Corporation, San Diego, California 921222
Received 14 December 2004/ Returned for modification 11 March 2005/ Accepted 2 June 2005
Streptococcus iniae represents a major health and economic problem in fish species worldwide. Random Tn917 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
PGM mutant cells reaching sizes
3-fold larger than those of the wild type (WT). The
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
PGM 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 pgm gene on a plasmid vector restored antimicrobial peptide resistance and virulence to the
PGM mutant. In analysis of the aborted infectious process, we found that
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
PGM mutant and later challenged with a lethal dose of WT 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
PGM mutant is able to stimulate a protective immune response and may have value as a live attenuated vaccine for aquaculture.
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