Chromosome-Encoded Hemolysin, Phospholipase, and Collagenase in Plasmidless Isolates of Photobacterium damselae subsp. damselae Contribute to Virulence for Fish.
Photobacterium damselaesubsp.damselaeis 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) genesepsLandpilDexhibited 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.IMPORTANCEThis study constitutes genetic and genomic analyses of plasmidless strains of an emerging pathogen in marine aquaculture,Photobacterium damselaesubsp.damselaeTo 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.
Departamento de Microbioloxía e Parasitoloxía, Instituto de Acuicultura, Universidade de Santiago de Compostela, Santiago de Compostela, Galicia, Spain.
Ana
A
Vences
Departamento de Microbioloxía e Parasitoloxía, Instituto de Acuicultura, Universidade de Santiago de Compostela, Santiago de Compostela, Galicia, Spain.|Institut für Medizinische Mikrobiologie und Hygiene, Universitätsmedizin der Johannes Gutenberg-Universität, Mainz, Germany.
Amable J
AJ
Rivas
Departamento de Microbioloxía e Parasitoloxía, Instituto de Acuicultura, Universidade de Santiago de Compostela, Santiago de Compostela, Galicia, Spain.
Manuel L
ML
Lemos
Institut für Medizinische Mikrobiologie und Hygiene, Universitätsmedizin der Johannes Gutenberg-Universität, Mainz, Germany.
Matthias
M
Husmann
Departamento de Microbioloxía e Parasitoloxía, Instituto de Acuicultura, Universidade de Santiago de Compostela, Santiago de Compostela, Galicia, Spain cr.osorio@usc.es.
Carlos R
CR
Osorio
D000818:Animals; D001492:Bass / Q000382:microbiology; D002876:Chromosomes, Bacterial / Q000235:genetics* / Q000378:metabolism; D017364:Collagenases / Q000235:genetics / Q000378:metabolism*; D005393:Fish Diseases / Q000382:microbiology*; D016905:Gram-Negative Bacterial Infections / Q000382:microbiology / Q000662:veterinary*; D006460:Hemolysin Proteins / Q000235:genetics / Q000378:metabolism; D010740:Phospholipases / Q000235:genetics / Q000378:metabolism*; D010776:Photobacterium / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism / Q000472:pathogenicity*; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D014774:Virulence
D006460:Hemolysin Proteins; D010740:Phospholipases; D017364:Collagenases
PhlyC; PhlyP; Photobacterium damselae; collagenase; damselysin; hemolysin; hlyA; lecithinase; phobalysin; phospholipase