Manuel L. Lemos

Vibrio anguillarum is a major pathogen impacting Solea senegalensis aquaculture. While host–pathogen competition for iron drives disease progression, the in vivo molecular evidence linking iron homeostasis and immune responses in fish remains limited. Here, we integrated metabolomic and proteomic analyses to characterize the splenic response of S. senegalensis during experimental vibriosis. Multiomics integration revealed disruption of iron homeostasis alongside coordinated immunometabolic reprogramming. We detected the bacterial siderophore piscibactin directly in spleen tissue of sole exhibiting advanced stages of vibriosis, providing in vivo evidence of siderophore-mediated iron acquisition. Infection-driven metabolic shifts in lysine degradation, nucleotide metabolism, and mitochondrial energy pathways were correlated with heightened cellular proliferation and metabolic stress. Integrative analysis identified metabolites including 2-aminoadipic acid, saccharopine, and methylsuccinic acid, together with iron-regulatory proteins (e.g., STEAP4) as candidate biomarkers. These findings enhance our understanding of vibriosis-associated immunometabolic responses and provide a systems-level framework for future disease monitoring and management in aquaculture.

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.

The irp -HPI genomic island encodes the salicylate-type siderophore piscibactin, a key virulence factor in several pathogenic Vibrio species. To advance our understanding of the siderophore metabolome (siderome) in Vibrio anguillarum , we combined high-resolution metabolomics with genetic and phenotypic analyses of biosynthetic mutants. LC-MS/MS and Feature-Based Molecular Networking (FBMN) revealed the presence of known siderophores, vanchrobactin and piscibactin/photoxenobactin-like siderophores, alongside two non-reported catecholate analogs of the formed siderophores, 2-hydroxypiscibactin (2-OH-Pcb) and 2-hydroxyphotoxenobactin E (2-OH-PxbE). Functional characterization of mutants defective in piscibactin or vanchrobactin production demonstrated biosynthetic and uptake-level interactions between these systems. We show that the salicylate-activating enzyme Irp5 is a promiscuous aryl acid adenylation enzyme capable of activating 2,3-dihydroxybenzoate (DHBA) and driving the synthesis of catecholate-piscibactin/photoxenobactin and fluorinated analogs through precursor-directed biosynthesis. Our data support photoxenobactin E as the terminal product of the piscibactin/photoxenobactin pathway in V. anguillarum . Comparative genomics revealed frequent co-occurrence of irp -HPI with other siderophore systems, especially catecholate-type systems such as vanchrobactin. These findings provide new insight into the molecular basis of virulence in Vibrionaceae and highlight the ecological and evolutionary significance of siderophore system interplay, which may enhance metabolic diversity, promote environmental adaptability, and preserve virulence under mutational pressure. IMPORTANCE: Iron acquisition is critical for bacterial survival and virulence, especially under the iron-limited conditions encountered in host environments. This study uncovers unexpected siderophore diversification within the piscibactin pathway, including two novel catecholate analogs and fluorinated derivatives produced via precursor-directed biosynthesis. We identify Irp5 as a versatile salicylate-activating enzyme that drives this metabolic flexibility. Moreover, the piscibactin and vanchrobactin systems interact at both biosynthetic and uptake levels, forming a resilient and adaptable iron acquisition network that supports virulence. These findings advance our understanding of siderophore-mediated iron acquisition and illustrate how enzymatic promiscuity and system interplay can be leveraged for synthetic biology and antimicrobial development. Given the widespread distribution of irp -HPI in Vibrionaceae , including human pathogens, our study provides a foundation for the rational design of antimicrobial strategies.

Siderophore production is a key fitness trait for bacteria, particularly in iron-limited environments such as marine ecosystems and host tissues. In this study, we identify the tenABECDC2D2hp1-4 gene cluster as responsible for siderophore biosynthesis in Tenacibaculum maritimum, confirmed through genome analysis and a tenCD-inactivated mutant. This cluster, highly similar to the desferrioxamine biosynthesis system in Streptomyces coelicolor, features a unique tenCD duplication/fusion, essential for siderophore formation. Additionally, accessory genes (hp1-4) encode functions such as nitroreductase and N-acetyltransferase, likely contributing to siderophore diversification. LC/MS analysis of T. maritimum cultures revealed the production of 20 amphiphilic, acylated desferrioxamine-like siderophores. These findings provide new insights into the genetic and metabolic versatility of marine pathogens in iron acquisition.

Hydroxamate siderophores are key virulence factors in multiple pathogens, and their structures present interesting scaffolds for potential biotechnological applications such as the design of novel antimicrobials. However, their variable and scarce production, their low stability, and occurrence as complex mixtures in natural samples complicate their analysis. Herein, we present a new strategy, named XAD-LC/MS-FBMN-IMS, which enables the analysis of the holo-hydroxamate siderophore composition from microbial cultures by integrating traditional XAD resin extraction, modern Feature-Based Molecular Networking (FBMN) tools, and a novel direct infusion ion mobility mass spectrometry validation approach. Using ferrioxamine as a model, XAD resins demonstrated high efficiency in extracting hydroxamate siderophore metal complexes when compared to other sorbents. This strategy was applied for detecting the siderophores produced by Tenacibaculum maritimum, a relevant pathogenic bacterium in fish aquaculture. This analysis unveiled three families of hydroxamate siderophores, including three known desferrioxamine derivatives along with 17 new putative acyl-desferrioxamine-like structures. Overall, the XAD-LC/MS-FBMN-IMS strategy significantly enhances siderophore and metallophore investigation in microbial cultures, improving our understanding of their production and roles in bacteria and fungi, ultimately facilitating their biotechnological applications.

Tenacibaculum maritimum is a Gram-negative marine fish pathogen. Here, we present the complete genome sequence of strain SP9.1, representing the emerging serotype O4. Selected as a model for pathogenesis studies, this genome provides valuable insights into the genetic basis of virulence and adaptation, complementing existing data for serotype O1.

This chapter provides an overview of the current understanding of iron acquisition mechanisms in Aeromonas. Two mechanisms, heme utilization and siderophore synthesis, have been extensively studied. All Aeromonas species can use heme to get iron, a mechanism facilitated by the production of hemolysins that release heme-containing molecules from host tissues. The predominant siderophore produced by most Aeromonas spp. is amonabactin, comprising a family of four different compounds. Amonabactins are internalized via FstC, an outer membrane transporter (OMT) characterized by a broad ligand plasticity. In addition to amonabactin, A. salmonicida also produces the siderophore acinetobactin, which is transported through FstB. Notably, both siderophores are concurrently produced, sharing part of the biosynthetic pathways. OMTs involved in iron acquisition hold a potential utility as tools for identification and as antigens for novel vaccines. Furthermore, synthetic derivatives of siderophores could serve as promising candidates for the development of novel antimicrobials, leveraging their specific internalization through OMTs.

Photoxenobactin E (1) is a natural product with an unusual thiocarboxylic acid terminus recently isolated from an entomopathogenic bacterium. The biosynthetic gene cluster associated with photoxenobactin E, and other reported derivatives, is very similar to that of piscibactin, the siderophore responsible for the iron uptake among bacteria of the Vibrionaceae family, including potential human pathogens. Here, the reisolation of 1 from the fish pathogen Vibrio anguillarum RV22 cultured under iron deprivation, its ability to chelate Ga(III), and the full NMR spectroscopic characterization of the Ga(III)-photoxenobactin E complex are presented. Our results show that Ga(III)-photoxenobactin E in solution exists in a thiol-thione tautomeric equilibrium, where Ga(III) is coordinated through the sulfur (thiol form) or oxygen (thione form) atoms of the thiocarboxylate group. This report represents the first NMR study of the chemical exchange between the thiol and thione forms associated with thiocarboxylate-Ga(III) coordination, including the kinetics of the interconversion process associated with this tautomeric exchange. These findings show significant implications for ligand design as they illustrate the potential of the thiocarboxylate group as a versatile donor for hard metal ions such as Ga(III).