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 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.

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.