Synergistic and Additive Effects of Chromosomal and Plasmid-Encoded Hemolysins Contribute to Hemolysis and Virulence in Photobacterium damselae subsp. damselae

Abstract

ABSTRACT Photobacterium damselae subsp. damselae causes infections and fatal disease in marine animals and in humans. Highly hemolytic strains produce damselysin (Dly) and plasmid-encoded HlyA (HlyA pl ). These hemolysins are encoded by plasmid pPHDD1 and contribute to hemolysis and virulence for fish and mice. In this study, we report that all the hemolytic strains produce a hitherto uncharacterized chromosome-encoded HlyA (HlyA ch ). Hemolysis was completely abolished in a single hlyA ch mutant of a plasmidless strain and in a dly hlyA pl hlyA ch triple mutant. We found that Dly, HlyA pl , and HlyA ch are needed for full hemolytic values in strains harboring pPHDD1, and these values are the result of the additive effects between HlyA pl and HlyA ch , on the one hand, and of the synergistic effect of Dly with HlyA pl and HlyA ch , on the other hand. Interestingly, Dly-producing strains produced synergistic effects with strains lacking Dly production but secreting HlyA, constituting a case of the CAMP ( C hristie, A tkins, and M unch- P etersen) reaction. Environmental factors such as iron starvation and salt concentration were found to regulate the expression of the three hemolysins. We found that the contributions, in terms of the individual and combined effects, of the three hemolysins to hemolysis and virulence varied depending on the animal species tested. While Dly and HlyA pl were found to be main contributors in the virulence for mice, we observed that the contribution of hemolysins to virulence for fish was mainly based on the synergistic effects between Dly and either of the two HlyA hemolysins rather than on their individual effects.