Photobacterium damselae subsp. damselae Major Virulence Factors Dly, Plasmid-Encoded HlyA, and Chromosome-Encoded HlyA Are Secreted via the Type II Secretion System

Photobacterium damselae subsp. damselae is a marine bacterium that causes septicemia in marine animals and in humans. Previously, we had determined a major role of pPHDD1 plasmid-encoded Dly (damselysin) and HlyA (HlyApl) and the chromosome-encoded HlyA (HlyAch) hemolysins in virulence. However, the mechanisms by which these toxins are secreted remain unknown. In this study, we found that a mini-Tn10 transposon mutant in a plasmidless strain showing an impaired hemolytic phenotype contained an insertion in epsL, a component of a type II secretion system (T2SS). Reconstruction of the mutant by allelic exchange confirmed the specific involvement of epsL in HlyAch secretion. In addition, mutation of epsL in a pPHDD1-harboring strain caused an almost complete abolition of hemolytic activity against sheep erythrocytes, indicating that epsL plays a major role in secretion of the plasmid-encoded HlyApl and Dly. This was further demonstrated by analysis of different combinations of hemolysin gene mutants and by strain-strain complementation assays. We also found that mutation of the putative prepilin peptidase gene pilD severely affected hemolysis, which dropped at levels inferior to those of epsL mutants. Promoter expression analyses suggested that impairment of hemolysin secretion in epsL and pilD mutants might constitute a signal that affects hemolysin and T2SS gene expression at the transcriptional level. In addition, single epsL and pilD mutations caused a drastic decrease in virulence for mice, demonstrating a major role of T2SS and pilD in P. damselae subsp. damselae virulence.