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

Fabián Galvis,Lucía Ageitos,Carlos Jiménez,Miguel Balado,Jaime Rodríguez,Manuel L Lemos,Juan L Barja

doi:10.3389/fcimb.2021.750567

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.

Highlights

Bacteria belonging to the genus Vibrio (Vibrios) are ubiquitously distributed in the marine environment and are a dominant fraction of bivalve microbiota (Vezzulli et al, 2018)
The main virulence factors characterized to date in bacteria pathogenic for marine bivalve molluscs are those related to motility, chemotaxis and, in a major extent, to extracellular products
The V. neptunius pathogenicity is not yet well known, some studies made with V. coralliilyticus could shed some light to the V. neptunius virulence factors, since these are two close-related bacteria (Thompson et al, 2003; Sawabe et al, 2013; Dubert et al, 2017)

Summary

Introduction

Bacteria belonging to the genus Vibrio (Vibrios) are ubiquitously distributed in the marine environment and are a dominant fraction of bivalve microbiota (Vezzulli et al, 2018). Mollusc hemolymph is a critical site for the host immune response (Potgieter et al, 2015). Some Vibrios are part of the resident microbiota of bivalves as they persist in the hemolymph in the absence of an environmental source of population (Potgieter et al, 2015; Lokmer et al, 2016; Vezzulli et al, 2018; Zhang et al, 2018). The microbiota benefits the host as it boosts the immune system, and promotes reproduction, nutrition and defence mechanisms (Engel et al, 2002; McFall-Ngai et al, 2013; Cahill et al, 2016; Utermann et al, 2018). Under unfavourable conditions some bacteria are responsible for disease outbreaks. Vibriosis is a serious epizootic disease caused by some Vibrio spp. that has become the most important limiting factor of the intensive fish and shellfish mariculture industry worldwide (Paillard et al, 2004; Toranzo et al, 2005; Travers et al, 2015; Dubert et al, 2017)

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