Vibrio anguillarum causes a hemorrhagic septicemia that affects cold- and warm-water adapted fish species. The main goal of this work was to determine the temperature-dependent changes in the virulence factors that could explain the virulence properties of V. anguillarum for fish cultivated at different temperatures. We have found that although the optimal growth temperature is around 25°C, the degree of virulence of V. anguillarum RV22 is higher at 15°C. To explain this result, an RNA-Seq analysis was performed to compare the whole transcriptome profile of V. anguillarum RV22 cultured under low-iron availability at either 25 or 15°C, which would mimic the conditions that V. anguillarum finds during colonization of fish cultivated at warm- or cold-water temperatures. The comparative analysis of transcriptomes at high- and low-iron conditions showed profound metabolic adaptations to grow under low iron. These changes were characterized by a down-regulation of the energetic metabolism and the induction of virulence-related factors like biosynthesis of LPS, production of hemolysins and lysozyme, membrane transport, heme uptake, or production of siderophores. However, the expression pattern of virulence factors under iron limitation showed interesting differences at warm and cold temperatures. Chemotaxis, motility, as well as the T6SS1 genes are expressed at higher levels at 25°C than at 15°C. By contrast, hemolysin RTX pore-forming toxin, T6SS2, and the genes associated with exopolysaccharides synthesis were preferentially expressed at 15°C. Notably, at this temperature, the siderophore piscibactin system was strongly up-regulated. In contrast, at 25°C, piscibactin genes were down-regulated and the vanchrobactin siderophore system seems to supply all the necessary iron to the cell. The results showed that V. anguillarum adjusts the expression of virulence factors responding to two environmental signals, iron levels and temperature. Thus, the relative relevance of each virulence factor for each fish species could vary depending on the water temperature. The results give clues about the physiological adaptations that allow V. anguillarum to cause infections in different fishes and could be relevant for vaccine development against fish vibriosis.

Vibrio anguillarum causes vibriosis, a hemorrhagic septicaemia that affects many cultured marine fish species worldwide. Two catechol siderophores, vanchrobactin and anguibactin, were previously identified in this bacterium. While vanchrobactin is a chromosomally encoded system widespread in all pathogenic and environmental strains, anguibactin is a plasmid-encoded system restricted to serotype O1 strains. In this work, we have characterized, from a serotype O2 strain producing vanchrobactin, a novel genomic island containing a cluster of genes that would encode the synthesis of piscibactin, a siderophore firstly described in the fish pathogen Photobacterium damselae subsp. piscicida. The chemical characterization of this siderophore confirmed that some strains of V. anguillarum produce piscibactin. An in silico analysis of the available genomes showed that this genomic island is present in many of the highly pathogenic V. anguillarum strains lacking the anguibactin system. The construction of single and double biosynthetic mutants for vanchrobactin and piscibactin allowed us to study the contribution of each siderophore to iron uptake, cell fitness, and virulence. Although both siderophores are simultaneously produced, piscibactin constitute a key virulence factor to infect fish, while vanchrobactin seems to have a secondary role in virulence. In addition, a transcriptional analysis of the gene cluster encoding piscibactin in V. anguillarum showed that synthesis of this siderophore is favored at low temperatures, being the transcriptional activity of the biosynthetic genes three-times higher at 18°C than at 25°C. We also show that iron levels and temperature contribute to balance the synthesis of both siderophores.

Summary: The shape of bacteria is maintained by the cell wall. The main component of the cell wall is peptidoglycan ( PG ) that is synthesized by penicillin binding proteins ( PBPs ). The correct positioning of PBPs is essential for the maintenance of cell shape. In the literature, two different models for localization of PBPs have been proposed – localization through interaction with a cytoskeletal structure or localization through the presence of substrate. Here, we show that the localization of PBPs critical for the rod shape of B acillus subtilis is altered when the substrate LipidII is delocalized by treatment of the cells with nisin. Alteration of this localization is only seen in a LipidII ‐dependent manner and is not influenced by dissipation of the membrane potential, a secondary effect of nisin treatment. Our results strongly suggest that the localization of PG synthesis at the periphery of the cell is substrate‐driven, even in bacteria that contain actin‐like MreB cytoskeletal structures.

Piscibactin is a widespread siderophore system present in many different bacteria, especially within the Vibrionaceae family. Previous works showed that most functions required for biosynthesis and transport of this siderophore are encoded by the high-pathogenicity island irp-HPI. In the present work, using Vibrio anguillarum as a model, we could identify additional key functions encoded by irp-HPI that are necessary for piscibactin production and transport and that have remained unknown. Allelic exchange mutagenesis, combined with cross-feeding bioassays and LC-MS analysis, were used to demonstrate that Irp4 protein is an essential component for piscibactin synthesis since it is the thioesterase required for nascent piscibactin be released from the NRPS Irp1. We also show that Irp8 is a MFS-type protein essential for piscibactin secretion. In addition, after passage through the outer membrane transporter FrpA, the completion of ferri-piscibactin internalization through the inner membrane would be achieved by the ABC-type transporter FrpBC. The expression of this transporter is coordinated with the expression of FrpA and with the genes encoding biosynthetic functions. Since piscibactin is a major virulence factor of some pathogenic vibrios, the elements of biosynthesis and transport described here could be additional interesting targets for the design of novel antimicrobials against these bacterial pathogens.

Photobacteriosis is a major threat to marine aquaculture, and the siderophore transporter FrpA represents a promising target for effective subunit vaccine development. In this study, we evaluated Montanide GEL 02 PR and phosphatidylcholine‐based liposomes as aqueous and safer alternatives to Freund's adjuvant for an FrpA‐based subunit vaccine in Solea senegalensis . Fish were immunized with recombinant FrpA formulated with either adjuvant, and induction of FrpA‐specific IgM antibody levels was measured. Vaccine efficacy was subsequently assessed by analysis of innate immune gene expression following early pathogen exposure and by survival analysis after infection challenge. Vaccination with rFrpA–Montanide GEL 02 PR induced the highest levels of specific IgM antibodies and conferred strong protection against photobacteriosis, achieving survival rates comparable to those reported for bacterin‐based vaccines. This formulation also promoted a rapid and coordinated up‐regulation of immune‐related genes, including transferrin ( tf ), lysozyme C1 ( lysC1 ), and complement components C1q and C7. In contrast, rFrpA encapsulated in liposomes elicited weaker immune responses and limited protection. Overall, our results demonstrate that an FrpA‐based subunit vaccine formulated with Montanide GEL 02 PR represents a promising strategy for controlling photobacteriosis in aquaculture.