A Highly Unstable and Elusive Plasmid That Encodes the Type III Secretion System Is Necessary for Full Virulence in the Marine Fish Pathogen Photobacterium damselae subsp. piscicida

Abstract

The marine bacterium Photobacterium damselae subsp. piscicida (Pdp) causes photobacteriosis in fish and important financial losses in aquaculture, but knowledge of its virulence factors is still scarce. We here demonstrate that an unstable plasmid (pPHDPT3) that encodes a type III secretion system (T3SS) is highly prevalent in Pdp strains from different geographical origins and fish host species. We found that pPHDPT3 undergoes curing upon in vitro cultivation, and this instability constitutes a generalized feature of pPHDPT3-like plasmids in Pdp strains. pPHDPT3 markers were detected in tissues of naturally-infected moribund fish and in the Pdp colonies grown directly from the fish tissues but were undetectable in a fraction of the colonies produced upon the first passage of the primeval colonies on agar plates. Notably, cured strains exhibited a marked reduction in virulence for fish, demonstrating that pPHDPT3 is a major virulence factor of Pdp. The attempts to stabilize pPHDPT3 by insertion of antibiotic resistance markers by allelic exchange caused an even greater reduction in virulence. We hypothesize that the existence of a high pressure to shed pPHDPT3 plasmid in vitro caused the selection of clones with off-target mutations and gene rearrangements during the process of genetic modification. Collectively, these results show that pPHDPT3 constitutes a novel, hitherto unreported virulence factor of Pdp that shows a high instability in vitro and warn that the picture of Pdp virulence genes has been historically underestimated, since the loss of the T3SS and other plasmid-borne genes may have occurred systematically in laboratories for decades.

Characterization and Vaccine Potential of Outer Membrane Vesicles from Photobacterium damselae subsp. piscicida

“…Additionally, it was also reported that Phdp secretes PnpA, a peptidoglycan hydrolase that can degrade the peptidoglycan of potential Phdp competitors [ 24 ], although the role of this protein in Phdp virulence remains unknown. In addition to these two factors, which are secreted by the type II secretion system (T2SS) of Phdp [ 24 , 25 ], it was recently reported that most isolates of Phdp harbor a type III secretion system (T3SS) [ 26 , 27 ], which is encoded in an unstable virulence-associated plasmid [ 28 ]. Although it is likely that the T3SS is involved in Phdp virulence, its specific functions remain undisclosed [ 28 ].…”

“…In addition to these two factors, which are secreted by the type II secretion system (T2SS) of Phdp [ 24 , 25 ], it was recently reported that most isolates of Phdp harbor a type III secretion system (T3SS) [ 26 , 27 ], which is encoded in an unstable virulence-associated plasmid [ 28 ]. Although it is likely that the T3SS is involved in Phdp virulence, its specific functions remain undisclosed [ 28 ].…”

Transforming the untransformable with knockout minicircles: High‐efficiency transformation and vector‐free allelic exchange knockout in the fish pathogen Photobacterium damselae

“…When a high‐efficiency transformation method is not available, an allelic exchange vector may be delivered by conjugation (Ferrières et al, 2010 ; Strand et al, 2014 ). To date, conjugative transfer of pir/RP4 suicide vectors from SM10/S17‐1 λ pir donor strains of Escherichia coli was the only method used to deliver the allelic exchange DNA in Pdp KO mutagenesis (Abushattal et al, 2022 ; Naka et al 2005 , 2007 ; Osorio et al, 2015 ). These donors contain the chromosomally integrated RP4::Mu conjugative transfer cluster (Simon et al, 1983 ), which was repeatedly shown to introduce off‐target mutations via Mu phage integration into the allelic‐exchange vector and/or the recipient's DNA, as well as Mu phage‐mediated and RP4‐mediated transfer and illegitimate integration of E. coli genes into the target host (Ferrières et al, 2010 ; Strand et al, 2014 ).…”