Low salinity activates a virulence program in the generalist marine pathogen Photobacterium damselae subsp. damselae.

Facultative marine bacterial pathogens sense environmental signals so that the expression of virulence factors is upregulated on entry into hosts and downregulated during the free-living lifestyle in the environment. In this study, we utilized transcriptome sequencing to compare the transcriptional profiles of Photobacterium damselae subsp. damselae, a generalist pathogen that causes disease in diverse marine animals and fatal infections in humans at NaCl concentrations that mimic the free-living lifestyle or host internal milieu, respectively. We here show that NaCl concentration constitutes a major regulatory signal that shapes the transcriptome and uncover 1,808 differentially expressed genes (888 upregulated and 920 downregulated in response to low-salt conditions). Growth at 3% NaCl, a salinity that mimics the free-living lifestyle, upregulated genes involved in energy production, nitrogen metabolism, transport of compatible solutes, utilization of trehalose and fructose, and carbohydrate and amino acid metabolism with strong upregulation of the arginine deiminase system (ADS). In addition, we observed a marked increase in resistance to antibiotics at 3% NaCl. On the contrary, the low salinity conditions (1% NaCl) that mimic those encountered in the host triggered a virulence gene expression profile that maximized the production of the type 2 secretion system (T2SS)-dependent cytotoxins damselysin, phobalysin P, and a putative PirAB-like toxin, observations that were corroborated by the analysis of the secretome. Low salinity also upregulated the expression of iron-acquisition systems, efflux pumps, and other functions related to stress response and virulence. The results of this study greatly expand our knowledge of the salinity-responsive adaptations of a generalist and versatile marine pathogen. IMPORTANCE Pathogenic Vibrionaceae species experience continuous shifts of NaCl concentration in their life cycles. However, the impact of salinity changes in gene regulation has been studied in a small number of Vibrio species. In this study, we analyzed the transcriptional response of Photobacterium damselae subsp. damselae (Pdd), a generalist and facultative pathogen, to changes in salinity, and demonstrate that growth at 1% NaCl in comparison to 3% NaCl triggers a virulence program of gene expression, with a major impact in the T2SS-dependent secretome. The decrease in NaCl concentration encountered by bacteria on entry into a host is proposed to constitute a regulatory signal that upregulates a genetic program involved in host invasion and tissue damage, nutrient scavenging (notably iron), and stress responses. This study will surely inspire new research on Pdd pathobiology, as well as on other important pathogens of the family Vibrionaceae and related taxa whose salinity regulons still await investigation.

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.

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.

The two-component system RstAB regulates production of a polysaccharide capsule with a role in virulence in the marine pathogen Photobacterium damselae subsp. damselae.

The marine bacterium Photobacterium damselae subsp. damselae (Pdd) causes disease in marine animals and humans. Previous studies demonstrated that mutation of the two-component system RstAB strongly impacts virulence of this pathogen, but the RstAB regulon has not been thoroughly elucidated. We here compared the transcriptomes of Pdd RM-71 and ΔrstA and ΔrstB derivatives using RNA-seq. In accordance with previous studies, RstAB positively regulated cytotoxins Dly, PhlyP and PhlyC. This analysis also demonstrated a positive regulation of outer membrane proteins, resistance against antimicrobials and potential virulence factors by this system. Remarkably, RstAB positively regulated two hitherto uncharacterised gene clusters involved in the synthesis of a polysaccharide capsule. Presence of a capsular layer in wild-type cells was confirmed by transmission electron microscopy, whereas rstA and rstB mutants were non-capsulated. Mutants for capsule synthesis genes, wza and wzc exhibited acapsular phenotypes, were impaired in resistance against the bactericidal action of fish serum and mucus, and were strongly impaired in virulence for fish, indicating a major role of capsule in virulence. Collectively, this study demonstrates that RstAB is a major positive regulator of key virulence factors including a polysaccharide capsule essential for full virulence in a pathogenic Photobacterium.

A Secreted NlpC/P60 Endopeptidase from Photobacterium damselae subsp. piscicida Cleaves the Peptidoglycan of Potentially Competing Bacteria.

Peptidoglycan (PG) is a major component of the bacterial cell wall, forming a mesh-like structure enwrapping the bacteria that is essential for maintaining structural integrity and providing support for anchoring other components of the cell envelope. PG biogenesis is highly dynamic and requires multiple enzymes, including several hydrolases that cleave glycosidic or amide bonds in the PG. This work describes the structural and functional characterization of an NlpC/P60-containing peptidase from Photobacterium damselae subsp. piscicida (Phdp), a Gram-negative bacterium that causes high mortality of warm-water marine fish with great impact for the aquaculture industry. PnpA ( PhotobacteriumNlpC-like protein A) has a four-domain structure with a hydrophobic and narrow access to the catalytic center and specificity for the γ-d-glutamyl-meso-diaminopimelic acid bond. However, PnpA does not cleave the PG of Phdp or PG of several Gram-negative and Gram-positive bacterial species. Interestingly, it is secreted by the Phdp type II secretion system and degrades the PG of Vibrio anguillarum and Vibrio vulnificus This suggests that PnpA is used by Phdp to gain an advantage over bacteria that compete for the same resources or to obtain nutrients in nutrient-scarce environments. Comparison of the muropeptide composition of PG susceptible and resistant to the catalytic activity of PnpA showed that the global content of muropeptides is similar, suggesting that susceptibility to PnpA is determined by the three-dimensional organization of the muropeptides in the PG.IMPORTANCE Peptidoglycan (PG) is a major component of the bacterial cell wall formed by long chains of two alternating sugars interconnected by short peptides, generating a mesh-like structure that enwraps the bacterial cell. Although PG provides structural integrity and support for anchoring other components of the cell envelope, it is constantly being remodeled through the action of specific enzymes that cleave or join its components. Here, it is shown that Photobacterium damselae subsp. piscicida, a bacterium that causes high mortality in warm-water marine fish, produces PnpA, an enzyme that is secreted into the environment and is able to cleave the PG of potentially competing bacteria, either to gain a competitive advantage and/or to obtain nutrients. The specificity of PnpA for the PG of some bacteria and its inability to cleave others may be explained by differences in the structure of the PG mesh and not by different muropeptide composition.

Diverse Horizontally-Acquired Gene Clusters Confer Sucrose Utilization to Different Lineages of the Marine Pathogen Photobacterium damselae subsp. damselae.

The ability to metabolize sucrose is a variable trait within the family Vibrionaceae. The marine bacterium Photobacterium damselae subsp. damselae (Pdd), pathogenic for marine animals and humans, is generally described as negative for sucrose utilization (Scr-). Previous studies have reported sucrose-utilizing isolates (Scr+), but the genetic basis of this variable phenotype remains uncharacterized. Here, we carried out the genome sequencing of five Scr+ and two Scr- Pdd isolates and conducted a comparative genomics analysis with sixteen additional Pdd genomes sequenced in previous studies. We identified two different versions of a four-gene cluster (scr cluster) exclusive of Scr+ isolates encoding a PTS system sucrose-specific IIBC component (scrA), a fructokinase (scrK), a sucrose-6-phosphate hydrolase (scrB), and a sucrose operon repressor (scrR). A scrA deletion mutant did not ferment sucrose and was impaired for growth with sucrose as carbon source. Comparative genomics analyses suggested that scr clusters were acquired by horizontal transfer by different lineages of Pdd and were inserted into a recombination hot-spot in the Pdd genome. The incongruence of phylogenies based on housekeeping genes and on scr genes revealed that phylogenetically diverse gene clusters for sucrose utilization have undergone extensive horizontal transfer among species of Vibrio and Photobacterium.

Exposure of the Opportunistic Marine Pathogen Photobacterium damselae subsp. damselae to Human Body Temperature Is a Stressful Condition That Shapes the Transcriptome, Viability, Cell Morphology, and Virulence.

Photobacterium damselae subsp. damselae (Pdd), an important pathogen for marine animals, is also an opportunistic human pathogen that can cause fatal necrotizing fasciitis. The regulatory changes triggered by the temperature shift experienced by this marine pathogen upon entering the human body, are completely unknown. Here we report an RNA-seq approach combined with phenotypical assays to study the response of Pdd to cultivation at 37°C in comparison to 25°C. We found that cultivation of a Pdd highly virulent strain for fish and mice, RM-71, at 37°C, initially enhanced bacterial growth in comparison to 25°C as evidenced by the increase in optical density. However, cells were found to undergo a progressive loss of viability after 6 h cultivation at 37°C, and no viable cells could be detected from 30 h cultures at 37°C. In contrast, at 25°C, viable cell counts achieved the highest values at 30 h cultivation. Cells grown at 25°C showed normal rod morphology by scanning electron microscopy analysis whereas cells grown at 37°C exhibited chain-like structures and aberrant long shapes suggesting a defect in daughter cell separation and in septum formation. Cells grown at 37°C also exhibited reduced tolerance to benzylpenicillin. Using a RNA-seq approach we discovered that growth at 37°C triggered a heat-shock response, whereas genes involved in motility and virulence were repressed including iron acquisition systems, the type two secretion system, and damselysin toxin, a major virulence factor of Pdd. Human isolates did not exhibit advantage growing at 37°C compared to fish isolates, and comparative genomics did not reveal gene markers specific of human isolates, suggesting that any Pdd genotype existing in the marine environment might potentially cause disease in humans. Altogether, these data indicate that the potential of Pdd to cause disease in humans is an accidental condition rather than a selected trait, and that human body temperature constitutes a stressful condition for Pdd. This study provides the first transcriptome profile of Pdd exposed at human body temperature, and unveils a number of candidate molecular targets for prevention and control of human infections caused by this pathogen.

Highly Transferable pAQU-Related Plasmids Encoding Multidrug Resistance Are Widespread in the Human and Fish Pathogen Photobacterium damselae subsp. damselae in Aquaculture Areas in the Black Sea.

The marine bacterium Photobacterium damselae subsp. damselae is a pathogen that causes disease in diverse marine animals, and is also a serious opportunistic human pathogen that can cause fatal infections. Strains of this pathogen isolated from diseased European sea bass in aquaculture facilities in the Turkish coast of the Black Sea were found to exhibit reduced sensitivity to multiple antimicrobials. Selected representative strains were subjected to complete genome sequencing and plasmid characterization. It was found that multidrug resistant (MDR) isolates harboured large conjugative plasmids sharing part of their sequence backbone with pAQU-group plasmids, hitherto reported exclusively in China and Japan. Four new pAQU-group versions of plasmids were identified in the present study, containing distinct combinations of the resistance determinants tetB, floR, sul2, qnrVC, dfrA and strAB. Conjugative transfer of pPHDD2-OG2, a representative plasmid of 170,998 bp, occurred at high frequencies (2.2 × 10-2 transconjugants per donor cell), to E. coli and to pathogenic P. damselae subsp. damselae and subsp. piscicida strains. Upon transfer, pPHDD2-OG2 conferred reduced susceptibility to a number of antimicrobials to the recipient strains. Comparative genomics analysis of host strains suggested that these MDR plasmids of the pAQU-group were acquired by different genetic lineages of Pdd. This study provides evidence that P. damselae subsp. damselae isolated from diseased fish constitute a reservoir for conjugative MDR pAQU-group plasmids in the Mediterranean basin, and have the potential to spread to diverse bacterial species.

The RstAB System Impacts Virulence, Motility, Cell Morphology, Penicillin Tolerance and Production of Type II Secretion System-Dependent Factors in the Fish and Human Pathogen Photobacterium damselae subsp. damselae.

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.

Draft Genome Sequences of Photobacterium damselae subsp. piscicida SNW-8.1 and PP3, Two Fish-Isolated Strains Containing a Type III Secretion System.

Here, we report the draft genome sequences of two strains of the fish pathogen Photobacterium damselae subsp. piscicida, isolated from Salmo salar (SNW-8.1) and Seriola quinqueradiata (PP3). The identification of a type III secretion system in the two genomes furthers our understanding of the pathobiology of this subspecies.

Molecular Epidemiology of Photobacterium damselae subsp. damselae Outbreaks in Marine Rainbow Trout Farms Reveals Extensive Horizontal Gene Transfer and High Genetic Diversity.

The marine bacterium Photobacterium damselae subsp. damselae is a pathogen for a variety of marine animals, as well as for humans, and is nowadays considered an emerging pathogen for fish of importance in marine aquaculture. Recent studies have suggested that outbreaks in fish farms are caused by multiclonal populations of this subspecies that exist in the environment. Here, we report the study of a collection of 31 strains isolated during the course of disease outbreaks in marine rainbow trout farms in Denmark in 1994, 1995, and 2006, respectively. A phylogenetic analysis based on the toxR gene sequence, and the screening of virulence-related genes uncovered a high genetic heterogeneity, even among strains isolated from the same fish farm at the same time. Moreover, comparative analysis of the whole genome sequences of four selected strains revealed a large number of differentially occurring genes, which included virulence genes, pPHDD1 plasmid, polysaccharide synthesis gene clusters, CRISPR-Cas systems and putative new mobile genetic elements. This study provides sound evidence that P. damselae subsp. damselae outbreaks in Danish rainbow trout farms were caused by multiclonal populations and that horizontal gene transfer constitutes a strong driving force in the generation of intraspecific diversity in this pathogen.

Photobacterium damselae subsp. damselae, a generalist pathogen with unique virulence factors and high genetic diversity.

Photobacterium damselae subsp. damselae 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 Vibrio species, it has unique features that differentiate it from related toxins. Dly phospholipase constitutes a singular trait of P. damselae subsp. damselae among the Vibrionaceae, although related toxins are found in members of the Aeromonadaceae 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.

Transcriptome changes in response to temperature in the fish pathogen Photobacterium damselae subsp. damselae: Clues to understand the emergence of disease outbreaks at increased seawater temperatures.

The marine bacterium Photobacterium damselae subsp. damselae (Pdd) is a generalist and facultative pathogen that causes disease in a wide range of marine animals including fish species of importance in aquaculture. Disease outbreaks in fish farms have been correlated with an increased water temperature during summer months. In this study, we have used RNA sequencing to analyze the transcriptome of Pdd RM-71 cultured at two different temperatures, which simulated temperature conditions experienced during free swimming lifestyle at mid latitudes in winter months (15°C) and during outbreaks in aquaculture in warm summer months (25°C). The enhanced bacterial growth of Pdd observed at 25°C in comparison to 15°C suggests that an elevated seawater temperature contributes to the build-up of a sufficient bacterial population to cause disease. In comparison to growth at 15°C, growth at 25°C resulted in the upregulation of genes involved in DNA synthesis, nutrient uptake, chemotaxis, flagellar motility, secretion systems and antimicrobial resistance. Plasmid-encoded virulence factors, which include a putative adhesin/invasin OmpU, a transferrin receptor and a serum resistance protein, were also upregulated. Transcription factor RpoS, genes involved in cold shock response, modulation of cell envelope and amino acid metabolism, as well as genes of yet unknown function were downregulated at 25°C. Notably, the gene encoding damselysin cytotoxin (Dly) was among the most highly transcribed genes at the two assayed temperatures, at levels comparable to the most highly expressed housekeeping genes. This study contributes to our understanding of the regulatory networks and biology of a generalist marine bacterial pathogen, and provides evidence that temperature regulates multiple physiological and virulence-related functions in Pdd.

Cytotoxin- and Chemotaxis-Genes Cooperate to Promote Adhesion of Photobacterium damselae subsp. damselae.

Photobacterium damselae subsp. damselae (Pdd) is an emerging pathogen of marine animals that sometimes causes serious infections in humans. Two related pore forming toxins, phobalysins P and C, and damselysin, a phospholipase D, confer strong virulence of Pdd in mice. Because infections by Pdd are typically caused following exposure of wounds to sea water we investigated how salinity impacts toxin activity, swimming, and association of Pdd with epithelial cells. These activities were low when bacteria were pre-cultured in media with 3.5% NaCl, the global average salinity of sea water. In contrast, lower salinity increased swimming of wild type Pdd peaking at 2% NaCl, hemolysis, and association with epithelial cells peaking at 1-1.5%. Previously, we have found that hemolysin genes enhance the association of Pdd with epithelial cells, but the underlying mechanisms have remained ill-defined. We here searched for potential links between hemolysin-production, chemotaxis and association of Pdd with target cells at varying salt concentrations. Unexpectedly, disruption of chemotaxis regulator cheA not only affected bacterial swimming and association with epithelial cells at intermediate to low salinity, but also reduced the production of plasmid-encoded phobalysin (PhlyP). The results thus reveal unforeseen links between chemotaxis regulators, a pore forming toxin and the association of a marine bacterium with target cells.

The Apoptogenic Toxin AIP56 Is Secreted by the Type II Secretion System of Photobacterium damselae subsp. piscicida.

AIP56 (apoptosis-inducing protein of 56 kDa) is a key virulence factor of Photobacterium damselae subsp. piscicida (Phdp), the causative agent of a septicaemia affecting warm water marine fish species. Phdp-associated pathology is triggered by AIP56, a short trip AB toxin with a metalloprotease A domain that cleaves the p65 subunit of NF-κB, an evolutionarily conserved transcription factor that regulates the expression of inflammatory and anti-apoptotic genes and plays a central role in host responses to infection. During infection by Phdp, AIP56 is systemically disseminated and induces apoptosis of macrophages and neutrophils, compromising the host phagocytic defence and contributing to the genesis of pathology. Although it is well established that the secretion of AIP56 is crucial for Phdp pathogenicity, the protein secretion systems operating in Phdp and the mechanism responsible for the extracellular release of the toxin remain unknown. Here, we report that Phdp encodes a type II secretion system (T2SS) and show that mutation of the EpsL component of this system impairs AIP56 secretion. This work demonstrates that Phdp has a functional T2SS that mediates secretion of its key virulence factor AIP56.

Secreted Citrate Serves as Iron Carrier for the Marine Pathogen Photobacterium damselae subsp damselae.

Photobacterium damselae subsp damselae (Pdd) is a Vibrionaceae that has a wide pathogenic potential against many marine animals and also against humans. Some strains of this bacterium acquire iron through the siderophore vibrioferrin. However, there are virulent strains that do not produce vibrioferrin, but they still give a strong positive reaction in the CAS test for siderophore production. In an in silico search on the genome sequences of this type of strains we could not find any ORF which could be related to a siderophore system. To identify genes that could encode a siderophore-mediated iron acquisition system we used a mini-Tn10 transposon random mutagenesis approach. From more than 1,400 mutants examined, we could isolate a mutant (BP53) that showed a strong CAS reaction independently of the iron levels of the medium. In this mutant the transposon was inserted into the idh gene, which encodes an isocitrate dehydrogenase that participates in the tricarboxylic acid cycle. The mutant did not show any growth impairment in rich or minimal media, but it accumulated a noticeable amount of citrate (around 7 mM) in the culture medium, irrespective of the iron levels. The parental strain accumulated citrate, but in an iron-regulated fashion, being citrate levels 5-6 times higher under iron restricted conditions. In addition, a null mutant deficient in citrate synthase showed an impairment for growth at high concentrations of iron chelators, and showed almost no reaction in the CAS test. Chemical analysis by liquid chromatography of the iron-restricted culture supernatants resulted in a CAS-positive fraction with biological activity as siderophore. HPLC purification of that fraction yielded a pure compound which was identified as citrate from its MS and NMR spectral data. Although the production of another citrate-based compound with siderophore activity cannot be ruled out, our results suggest that Pdd secretes endogenous citrate and use it for iron scavenging from the cell environment.

Genomic analysis of the marine fish pathogen Photobacterium damselae subsp. piscicida: Insertion sequences proliferation is associated with chromosomal reorganisations and rampant gene decay.

Photobacterium damselae subsp. piscicida (Pdp) is an intracellular fish pathogen that causes photobacteriosis, a disease proven deadly in farmed fish worldwide. This work focuses on the analysis of genome sequences, chromosomes structure and gene contents of two strains from Sparus aurata (DI21) and Solea senegalensis (L091106-03H), isolated on the Spanish Atlantic coast. The comparative genomic analysis revealed that DI21 and L091106-03H share 98% of their genomes, including two virulence plasmids: pPHDP70 encoding siderophore piscibactin synthesis and pPHDP10 encoding the apoptotic toxin AIP56. Both genomes harbour a surprisingly large number of IS elements accounting for 12-17% of the total genome, representing an IS density of 0.15 elements per kb, one of the highest IS density values in a bacterial pathogen. This massive proliferation of ISs is responsible for the generation of a high number of pseudogenes that caused extensive loss of biological functions. Pseudogene formation is one of the main features of Pdp genome that explains most of the ecological and phenotypic differences with respect to its sibling subspecies P. damselae subsp. damselae and to other Vibrionaceae. Evidence was also found proving the existence of two chromosomal configurations depending on the origin of the strains: an European and an Asian/American types of genome organisation, reinforcing the idea of the existence of two geographically-linked clonal lineages in Pdp. In short, our study suggests that the host-dependent lifestyle of Pdp allowed massive IS proliferation and gene decay processes, which are major evolutionary forces in the shaping of the Pdp genome.

rstB Regulates Expression of the Photobacterium damselae subsp. damselae Major Virulence Factors Damselysin, Phobalysin P and Phobalysin C.

The marine pathogenic bacterium Photobacterium damselae subsp. damselae causes septicemia in marine animals and in humans. The pPHDD1 plasmid-encoded hemolysins damselysin (Dly) and phobalysin P (PhlyP), and the chromosome-encoded hemolysin phobalysin C (PhlyC) constitute its main virulence factors. However, the mechanisms by which expression of these three hemolysins is regulated remain unknown. Here we report the isolation of a mini-Tn10 transposon mutant which showed a strong impairment in its hemolytic activity. The transposon disrupted a putative sensor histidine kinase gene vda_000600 (rstB), which together with vda_000601 (rstA) is predicted to encode a putative two-component regulatory system. This system showed to be homologous to the Vibrio cholerae CarSR/VprAB and Escherichia coli RstAB systems. Reconstruction of the mutant by allelic exchange of rstB showed equal impairment in hemolysis, and complementation with a plasmid expressing rstAB restored hemolysis to wild-type levels. Remarkably, we demonstrated by promoter expression analyses that the reduced hemolysis in the rstB mutant was accompanied by a strong decrease in transcription activities of the three hemolysin genes dly (damselysin), hlyApl (phobalysin P) and hlyAch (phobalysin C). Thus, RstB, encoded in the small chromosome, regulates plasmid and chromosomal virulence genes. We also found that reduced expression of the three virulence genes correlated with a strong decrease in virulence in a sea bass model, demonstrating that RstB constitutes a master regulator of the three P. damselae subsp. damselae hemolysins and plays critical roles in the pathogenicity of this bacterium. This study represents the first evidence of a direct role of a RstAB-like system in the regulation of bacterial toxins.

A proteomic analysis of the iron response of Photobacterium damselae subsp. damselae reveals metabolic adaptations to iron levels changes and novel potential virulence factors.

Photobacterium damselae subsp. damselae (Pdd) is a marine bacterium that can infect numerous species of marine fish as well as other species including humans. Low iron availability is one of the signs that bacterial pathogens can detect in order to begin colonizing their host, and the reduction of iron levels is a nonspecific host defense strategy that prevents bacterial proliferation. In this work a proteomic approach was used to study the gene expression adaptations of a Pdd strain in response to iron availability. A comparative analysis of induced proteins in both high- and low-iron conditions showed profound cellular metabolic adaptations that result, for instance, in amino acid requirement. It also provided important information about the changes that occur in the energetic metabolism induced by the surrounding iron levels, allowing for the identification of novel potential virulence factors. Among others, genes involved in the synthesis and transport of a vibrioferrin-like siderophore were identified for the first time. In addition to plasmid pPHDD1-encoded Dly and HlyA hemolysins, a pPHDD1-borne operon, which may encode a transferrin receptor, was also found. This operon identification suggests that this virulence plasmid could encode so-far unknown additional virulence factors other than hemolysins.

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

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.