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

Abstract

b 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 (HlyA pl ) and the chromosome-encoded HlyA (HlyA ch ) 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 HlyA ch 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 HlyA pl 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.T he marine bacterium Photobacterium damselae subsp. damselae is considered a primary pathogen of a wide range of marine animals, including wild and cultivated fish, causing losses of economical importance in marine aquaculture (1-3). In addition, this pathogen is of special concern for humans, since it can cause a highly severe necrotizing fasciitis that may lead to a fatal outcome (4, 5). The majority of the reported infections in humans have their primary origin in wounds exposed to seawater (6), inflicted during fish and fishing tool handling (7), while practicing aquatic sports (8), and occasionally after consumption of seafood (9).Highly hemolytic strains of this pathogen harbor the virulence plasmid pPHDD1, which encodes the hemolysins damselysin (Dly) and HlyA (HlyA pl ) (10). In addition, all of the hemolytic P. damselae subsp. damselae strains encode a third hemolysin, chromosome-encoded HlyA (HlyA ch ), in chromosome I (11, 12). Dly is a potent phospholipase D cytotoxin with hemolytic activity that removes choline phosphate head groups from sphingomyelin (13, 14), whereas HlyA pl and HlyA ch are predicted to be pore-forming toxins (11). HlyA ch and HlyA pl show 92% identity in their amino acid sequences, but HlyA pl is twice more active against sheep erythrocytes than HlyA ch (11). When the two HlyA hemolysins are produced by a P. damselae subsp. damselae strain, their activities demonstrated to exert an additive effect i...

Phobalysin, a Small β-Pore-Forming Toxin of Photobacterium damselae subsp. damselae

“…The bacterial strains and plasmids used in this study are listed in Table 1. Culture conditions and conjugative matings used in this study have been described previously (9,14,15). The Staphylococcus aureus strains have been described previously (24).…”

“…Deletion of the P. damselae subsp. damselae prepilin peptidase gene pilD severely affects hemolysin secretion and prevents pilus formation (15). In addition to pilD, the gene encoding TadV, another prepilin peptidase, which belongs to the tight adherence (tad) cluster carried on pPHDD1, is involved in the production of type IV pili (54).…”

“…P. damselae subsp. damselae hemolysins are secreted via the type II secretion system (T2SS), which was also shown to be required for virulence and hemolysis (15). In contrast to Dly, HlyA has not been characterized so far.…”

Bacterial Sphingomyelinases and Phospholipases as Virulence Factors

“…P. damselae subsp. damseale secretes a hemolytic SMase D, designated damselysin (Table 3), through a type II secretion system (67). This enzyme is structurally unrelated to the enzymes described in the previous sections, and it is also catalytically active against PC and phosphatidylethanolamine (PE) (67).…”

“…damseale secretes a hemolytic SMase D, designated damselysin (Table 3), through a type II secretion system (67). This enzyme is structurally unrelated to the enzymes described in the previous sections, and it is also catalytically active against PC and phosphatidylethanolamine (PE) (67). Damselysin is encoded on a conjugative plasmid and contributes to the virulence of P. damselae in experimental infections in fish and mice (68).…”