Photobacterium damselae subsp. damselae is a pathogen of marine animals, including fish of importance in aquaculture. The virulence plasmid pPHDD1, characteristic of highly hemolytic isolates, encodes the hemolysins damselysin (Dly) and phobalysin (PhlyP). Strains lacking pPHDD1 constitute the vast majority of the isolates from fish outbreaks, but genetic studies to identify virulence factors in plasmidless strains are scarce. Here, we show that the chromosome I-encoded hemolysin PhlyC plays roles in virulence and cell toxicity in pPHDD1-negative isolates of this pathogen. By combining the analyses of whole genomes and of gene deletion mutants, we identified two hitherto uncharacterized chromosomal loci encoding a phospholipase (PlpV) and a collagenase (ColP). PlpV was ubiquitous in the subspecies and exerted hemolytic activity against fish erythrocytes, which was enhanced in the presence of lecithin. ColP was restricted to a fraction of the isolates and was responsible for the collagen-degrading activity in this subspecies. Consistent with the presence of signal peptides in PlpV and ColP sequences, mutants for the type II secretion system (T2SS) genes epsL and pilD exhibited impairments in phospholipase and collagenase activities. Sea bass virulence experiments and cell culture assays demonstrated major contributions of PhlyC and PlpV to virulence and toxicity. IMPORTANCE This study constitutes genetic and genomic analyses of plasmidless strains of an emerging pathogen in marine aquaculture, Photobacterium damselae subsp. damselae. To date, studies on the genetic basis of virulence were restricted to the pPHDD1 plasmid-encoded toxins Dly and PhlyP. However, the vast majority of the recent isolates of this pathogen from fish farm outbreaks lack this plasmid. Here we demonstrate that the plasmidless strains produce two hitherto uncharacterized ubiquitous toxins encoded in chromosome I, namely, the hemolysin PhlyC and the phospholipase PlpV. We report the main roles of these two toxins in fish virulence and in cell toxicity. Our results constitute the basis for a better understanding of the virulence of a widespread marine pathogen.

The RstB histidine kinase of the two component system RstAB positively regulates the expression of damselysin (Dly), phobalysin P (PhlyP) and phobalysin C (PhlyC) cytotoxins in the fish and human pathogen Photobacterium damselae subsp. damselae , a marine bacterium of the family Vibrionaceae . However, the function of the predicted cognate response regulator RstA has not been studied so far, and the role of the RstAB system in other cell functions and phenotypes remain uninvestigated. Here, we analyzed the effect of rstA and rstB mutations in cell fitness and in diverse virulence-related features. Both rstA and rstB mutants were severely impaired in virulence for sea bream and sea bass fish. Mutants in rstA and rstB genes were impaired in hemolysis and in Dly-dependent phospholipase activity but had intact PlpV-dependent phospholipase and ColP-dependent gelatinase activities. rstA and rstB mutants grown at 0.5% NaCl exhibited impaired swimming motility, enlarged cell size and impaired ability to separate after cell division, whereas at 1% NaCl the mutants exhibited normal phenotypes. Mutation of any of the two genes also impacted tolerance to benzylpenicillin. Notably, rstA and rstB mutants showed impaired secretion of a number of type II secretion system (T2SS)-dependent proteins, which included the three major cytotoxins Dly, PhlyP and PhlyC, as well as a putative delta-endotoxin and three additional uncharacterized proteins which might constitute novel virulence factors of this pathogenic bacterium. The analysis of the T2SS-dependent secretome of P. damselae subsp. damselae also led to the identification of RstAB-independent potential virulence factors as lipoproteins, sialidases and proteases. The RstAB regulon included plasmid, chromosome I and chromosome II-encoded genes that showed a differential distribution among isolates of this subspecies. This study establishes RstAB as a major regulator of virulence and diverse cellular functions in P. damselae subsp. damselae .

The iron uptake mechanisms based on siderophore synthesis used by the fish pathogen Aeromonas salmonicida subsp. salmonicida are still not completely understood, and the precise structure of the siderophore(s) is unknown. The analysis of genome sequences revealed that this bacterium possesses two gene clusters putatively involved in the synthesis of siderophores. One cluster is a candidate to encode the synthesis of acinetobactin, the siderophore of the human pathogen Acinetobacter baumannii, while the second cluster shows high similarity to the genes encoding amonabactin synthesis in Aeromonas hydrophila. Using a combination of genomic analysis, mutagenesis, biological assays, chemical purification, and structural determination procedures, here we demonstrate that most A. salmonicida subsp. salmonicida strains produce simultaneously the two siderophores, acinetobactin and amonabactin. Interestingly, the synthesis of both siderophores relies on a single copy of the genes encoding the synthesis of the catechol moiety (2,3-dihydroxybenzoic acid) and on one encoding a phosphopantetheinyl transferase. These genes are present only in the amonabactin cluster, and a single mutation in any of them abolishes production of both siderophores. We could also demonstrate that some strains, isolated from fish raised in seawater, produce only acinetobactin since they present a deletion in the amonabactin biosynthesis gene amoG. Our study represents the first evidence of simultaneous production of acinetobactin and amonabactin by a bacterial pathogen and reveals the plasticity of bacterial genomes and biosynthetic pathways. The fact that the same siderophore is produced by unrelated pathogens highlights the importance of these systems and their interchangeability between different bacteria.

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...

subsp. causes vibriosis in a variety of marine animals, including fish species of importance in aquaculture. It also may cause wound infections in humans that can progress into a fatal outcome. Two major virulence factors are encoded within the large conjugative plasmid pPHDD1: the phospholipase-D damselysin (Dly) and the pore-forming toxin Phobalysin P (PhlyP). The two toxins exert hemolytic and cytolytic activity in a synergistic manner. Albeit PhlyP has close homologues in many species, it has unique features that differentiate it from related toxins. Dly phospholipase constitutes a singular trait of subsp. among the , although related toxins are found in members of the Fish farm outbreaks can also be caused by plasmidless strains. Such observation led to the characterization of two ubiquitous, chromosome-encoded toxins with lesser cytolytic activity: the pore forming-toxin Phobalysin C (PhlyC) and the phospholipase-hemolysin PlpV. Special attention deserves the high genetic diversity of this pathogen, with a number of strain-specific features including the cell envelope polysaccharide synthesis clusters. Fish outbreaks are likely caused by multiclonal populations which contain both plasmidless and pPHDD1-harbouring isolates, and not by well-adapted clonal complexes. Still, among such a genetic heterogeneity, it is feasible to identify conserved weak points in the biology of this bacterium: the two-component regulatory system RstAB (CarSR) was found to be necessary for maximal production of virulence factors and its inactivation severely impaired virulence.