Vibrio neptunius Produces Piscibactin and Amphibactin and Both Siderophores Contribute Significantly to Virulence for Clams

Abstract

Vibrio neptunius is an inhabitant of mollusc microbiota and an opportunistic pathogen causing disease outbreaks in marine bivalve mollusc species including oysters and clams. Virulence of mollusc pathogenic vibrios is mainly associated with the production of extracellular products. However, siderophore production is a common feature in pathogenic marine bacteria but its role in fitness and virulence of mollusc pathogens remains unknown. We previously found that V. neptunius produces amphibactin, one of the most abundant siderophores in marine microbes. In this work, synthesis of the siderophore piscibactin was identified as the second siderophore produced by V. neptunius. Single and double mutants in biosynthetic genes of each siderophore system, piscibactin and amphibactin, were constructed in V. neptunius and their role in growth ability and virulence was characterized. Although the High Pathogenicity Island encoding piscibactin is a major virulence factor in vibrios pathogenic for fish, the V. neptunius wild type did not cause mortality in turbot. The results showed that amphibactin contributes more than piscibactin to bacterial fitness in vitro. However, infection challenges showed that each siderophore system contributes equally to virulence for molluscs. The V. neptunius strain unable to produce any siderophore was severely impaired to cause vibriosis in clams. Although the inactivation of one of the two siderophore systems (either amphibactin or piscibactin) significantly reduced virulence compared to the wild type strain, the ability to produce both siderophores simultaneously maximised the degree of virulence. Evaluation of the gene expression pattern of each siderophore system showed that they are simultaneously expressed when V. neptunius is cultivated under low iron availability in vitro and ex vivo. Finally, the analysis of the distribution of siderophore systems in genomes of Vibrio spp. pathogenic for molluscs showed that the gene clusters encoding amphibactin and piscibactin are widespread in the Coralliilyticus clade. Thus, siderophore production would constitute a key virulence factor for bivalve molluscs pathogenic vibrios.

Metabolomics Approaches to Dereplicate Natural Products from Coral-Derived Bioactive Bacteria

“…Piscibactin has been reported from the fish pathogen Photobacterium damselae subsp. piscicida and from V. neptunius , where the production of piscibactin was shown to correlate with the pathogenicity of V. neptunius toward molluscs . Previous in silico analyses of Vibrio spp.…”

“…genomes revealed that V. coralliilyticus has the genomic potential to produce piscibactin. 58,59 Indeed, six of the seven isolates in which piscibactin was detected in this study were identified as V. coralliilyticus, two of which are the putative coral pathogens OfT6−21 and OfT7−21. Thus, our data show that Vibrio spp.…”

“…piscicida 57 and from V. neptunius, where the production of piscibactin was shown to correlate with the pathogenicity of V. neptunius toward molluscs. 58 Previous in silico analyses of Vibrio spp. genomes revealed that V. coralliilyticus has the genomic potential to produce piscibactin.…”

Structural Requirements for Ga3+ Coordination in Synthetic Analogues of the Siderophore Piscibactin Deduced by Chemical Synthesis and Density Functional Theory Calculations

“…Alternatively, a very highly stereoselective synthesis of the synepimer β-hydroxy acid 14 was carried out by diastereoselective reduction of tetramic acid 20 (Scheme 3). 35 Thus, the synthesis of the tetramic acid core was performed by the condensation of N-protected amino acid 10 (see Scheme 2) with Meldrum's acid in the presence of N-ethyl-N′- (3dimethylaminopropyl) In a parallel procedure, the trityl-protected (S)-α-azidocysteine methyl ester (25) was obtained from D-cysteine methyl ester (23) in two steps following a described method. 36,37 Thus, the thiol group of D-cysteine methyl ester (23) was protected with trityl to give amine 24, which was subjected to a diazo transfer reaction, using the electrophilically activated azide TfN 3 , NaHCO 3 as the base, and CuSO 4 as the catalyst, to afford azide 25 in a very good yield (86%) (Scheme 3).…”

Identification of Key Functions Required for Production and Utilization of the Siderophore Piscibactin Encoded by the High-Pathogenicity Island irp-HPI in Vibrionaceae

“…piscicida [ 6 ], Vibrio anguillarum [ 7 ] or V. ordalii [ 8 ]. Piscibactin production is also necessary for full virulence of the bivalve mollusks pathogen V. neptunius, and irp -HPI is present in the genome of other mollusks Vibrio pathogens of the Coralliilyticus clade [ 9 ]. Furthermore, irp -HPI is widespread among many other Vibrio spp., including human pathogens like V. cholerae [ 10 ].…”