Chromosome-Encoded Hemolysin, Phospholipase, and Collagenase in Plasmidless Isolates of Photobacterium damselae subsp. damselae Contribute to Virulence for Fish

Abstract

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.

Antibacterial potential of phytochemicals alone or in combination with antimicrobials against fish pathogenic bacteria

“…The production of exotoxins, known as haemolysins, is a characteristic of bacteria highly pathogenic for fish (Vences et al 2017). The production of exotoxins, known as haemolysins, is a characteristic of bacteria highly pathogenic for fish (Vences et al 2017).…”

“…Among the several virulence factors of bacteria, the ability to cause haemolysis is one of the most known. The production of exotoxins, known as haemolysins, is a characteristic of bacteria highly pathogenic for fish (Vences et al 2017). For this reason, we evaluated the capability of the phytochemicals to inhibit the haemolysis caused by the b-haemolytic strain isolated from fish.…”

Alternative dietary protein and water temperature influence the skin and gut microbial communities of yellowtail kingfish (Seriola lalandi)

“…Analysis of the sequence variants within OTU 1,622 indicates that this likely represents the species P. damselae, but does not distinguish between different strains of this species which are reported to differ in their pathogenicity (Vences et al, 2017). Detection of the plpV virulence gene in all digesta samples for which OTU 1,622 was abundant (>20% of relative abundance) indicates that this OTU is potentially a representative of pathogenic P. dameselae.…”

“…A region of the plpV gene was amplified using primers described by Vences et al (2017). PCR amplification was performed on 1:50 dilutions of extracted DNA using MyFi Mix (Bioline, Eveleigh, NSW, Australia) with a primer concentration of 400 nM in a final volume of 30 µL.…”

Health assessment of the cleaner fish ballan wrasse Labrus bergylta from the British south-west coast

“…Moreover, bioinformatic analysis of this strain re vealed that it does not possess the highly virulent pPHDD1 plasmid, or the plasmid-coded phobalysin P (PhylP) and damselysin (Dly) toxins (Rivas et al 2011(Rivas et al , 2013. The alternative chromosome-encoded toxins, phobalysin C (PhylC) and novel phospholipase of Vibronaceae (PlpV), have been recently described as virulence factors, responsible for pathogenic potential in non-pPHDD1 harbouring strains, causing haemolysis and virulence in sea bass Dicentrarchus labrax (Vences et al 2017). Both of these chromosomally encoded toxins were present in this strain, as shown by the presence of genes hlyA ch and plpV.…”