n-hexanoyl-l-homoserine-lactone and Fish-Diseases

Four strains of N-hexanoyl-L-homoserine lactone (AHL)-degrading Pseudomonas spp., named PsDAHP1, PsDAHP2, PsDAHP3, and PsDAHP4 were isolated and identified from the intestine of Fenneropenaeus indicus. PsDAHP1 showed the highest AHL-degrading activity among the four isolates. PsDAHP1 inhibited biofilm-forming exopolysaccharide and altered cell surface hydrophobicity of virulent green fluorescent protein (GFP)-tagged Vibrio parahaemolyticus DAHV2 (GFP-VpDAHV2). Oral administration of PsDAHP1 significantly reduced zebrafish mortality caused by GFP-VpDAHV2 challenge, and inhibited colonisation of GFP-VpDAHV2 in the gills and intestine of zebrafish as evidence by confocal laser scanning microscope and selective plating. Furthermore, zebrafish receiving PsDAHP1-containing feed had increased phagocytic cells of its leucocytes, increased serum activities of superoxide dismutase and lysozyme. The results suggest that Pseudomonas aeruginosa PsDAHP1 could protect zebrafish from V. parahaemolyticus infection by inhibiting biofilm formation and enhancing defence mechanisms of the fish.

Topics: 4-Butyrolactone; Analysis of Variance; Animals; Biofilms; Fish Diseases; Green Fluorescent Proteins; Hydrophobic and Hydrophilic Interactions; Microscopy, Confocal; Penaeidae; Pseudomonas aeruginosa; Species Specificity; Vibrio Infections; Vibrio parahaemolyticus; Zebrafish

Vibrio anguillarum, which causes terminal hemorrhagic septicemia in fish, was previously shown to possess a LuxRI-type quorum-sensing system (vanRI) and to produce N-(3-oxodecanoyl)homoserine lactone (3-oxo-C10-HSL). However, a vanI null mutant still activated N-acylhomoserine lactone (AHL) biosensors, indicating the presence of an additional quorum-sensing circuit in V. anguillarum. In this study, we have characterized this second system. Using high-pressure liquid chromatography in conjunction with mass spectrometry and chemical analysis, we identified two additional AHLs as N-hexanoylhomoserine lactone (C6-HSL) and N-(3-hydroxyhexanoyl)homoserine lactone (3-hydroxy-C6-HSL). Quantification of each AHL present in stationary-phase V. anguillarum spent culture supernatants indicated that 3-oxo-C10-HSL, 3-hydroxy-C6-HSL, and C6-HSL are present at approximately 8.5, 9.5, and 0.3 nM, respectively. Furthermore, vanM, the gene responsible for the synthesis of these AHLs, was characterized and shown to be homologous to the luxL and luxM genes, which are required for the production of N-(3-hydroxybutanoyl)homoserine lactone in Vibrio harveyi. However, resequencing of the V. harveyi luxL/luxM junction revealed a sequencing error present in the published sequence, which when corrected resulted in a single open reading frame (termed luxM). Downstream of vanM, we identified a homologue of luxN (vanN) that encodes a hybrid sensor kinase which forms part of a phosphorelay cascade involved in the regulation of bioluminescence in V. harveyi. A mutation in vanM abolished the production of C6-HSL and 3-hydroxy-C6-HSL. In addition, production of 3-oxo-C10-HSL was abolished in the vanM mutant, suggesting that 3-hydroxy-C6-HSL and C6-HSL regulate the production of 3-oxo-C10-HSL via vanRI. However, a vanN mutant displayed a wild-type AHL profile. Neither mutation affected either the production of proteases or virulence in a fish infection model. These data indicate that V. anguillarum possesses a hierarchical quorum sensing system consisting of regulatory elements homologous to those found in both V. fischeri (the LuxRI homologues VanRI) and V. harveyi (the LuxMN homologues, VanMN).

Topics: 4-Butyrolactone; Amino Acid Sequence; Animals; Bacterial Proteins; Chromatography, High Pressure Liquid; Chromatography, Thin Layer; Cloning, Molecular; Escherichia coli; Fish Diseases; Gene Deletion; Genes, Bacterial; Homoserine; Lactones; Mass Spectrometry; Molecular Sequence Data; Mutagenesis, Site-Directed; Oncorhynchus mykiss; Open Reading Frames; Polymerase Chain Reaction; Sequence Homology, Amino Acid; Signal Transduction; Vibrio; Virulence