Novel Role of the Lipopolysaccharide O1 Side Chain in Ferric Siderophore Transport and Virulence of Vibrio anguillarum

Abstract

From a library of approximately 20,000 transposon mutants, we have identified mutants affected in chromosomal genes involved in synthesis of the siderophore anguibactin, as well as in ferric anguibactin utilization. Genetic and sequence analyses of one such transport-defective mutant revealed that the transposon insertion occurred in an open reading frame (ORF) with homology to rmlC, a dTDP-rhamnose biosynthetic gene. This ORF resides within a cluster of four ORFs, all of which are predicted to function in the biosynthesis of this O side chain precursor. The same phenotype was seen in a mutant obtained by allelic exchange in rmlD, another ORF in this dTDP-rhamnose biosynthetic cluster. This mutation could be complemented with the wild-type rmlD gene, restoring both production of the O1 antigen side chain and ferric anguibactin transport. Presence of the O1 side chain was crucial for the resistance of Vibrio anguillarum to the bactericidal action of nonimmune serum from the fish host. Surprisingly, further analysis demonstrated that these mutations were pleiotropic, leading to a dramatic decrease in the levels of FatA, the outer membrane protein receptor for ferric anguibactin transport, and a concomitant reduction in iron transport. Thus, our results in this work demonstrate that the lipopolysaccharide O1 side chain is required for the operation of two critical virulence factors in V. anguillarum: serum resistance and anguibactin-mediated iron transport. These factors allow V. anguillarum to survive in serum and multiply in the iron-limiting milieu of the host vertebrate.

Complete Genome Sequence of the Marine Fish Pathogen Vibrio anguillarum Harboring the pJM1 Virulence Plasmid and Genomic Comparison with Other Virulent Strains of V. anguillarum and V. ordalii

“…Strains from the Pacific coast of the United States and Japan harbor a pJM1 plasmid very similar to pJM1 in 775 and produce similar levels of anguibactin, whereas strains originated in the U.S. Atlantic coast and in Spain, Scotland, and the Scandinavian countries carry pJM1-like plasmids that possess several extra insertion elements and a mutation in the angR gene (71,74). This mutation results in higher anguibactin production levels than those in Pacific coast strains, although this difference was not translated into an enhanced virulence phenotype and no major differences were found by DNA hybridization and biochemical studies (72,82). These findings suggest that all O1 strains carrying a pJM1-like plasmid, independent of plasmid type and geographical isolation, are very similar.…”

“…Furthermore, LPS genes such as rmlD (wbhC) and rmlC (wbhD) are essential for stability of the outer membrane ferric anguibactin-specific receptor FatA in the 531A strain of V. anguillarum, which carries the pJM1-like plasmid pJHC1. Thus, the LPS genes are necessary for ferric anguibactin transport and virulence (82). Until now, the rjg gene has not been described in V. anguillarum; however, in this work we located the entire locus in Chr1 of V. anguillarum 775, which included the gmhD and rjg genes surrounding the O-antigen cluster genes ( Fig.…”

Characterization of EmpA protease in Vibrio anguillarum M3

“…The disease is still a major obstacle for aquaculture industry at present. A few of virulence determinants of V. anguillarum have been reported, including iron-acquiring system (Crosa, 1980;Stork et al, 2002;Welch et al, 2005), serum resistance (Boesen et al, 1998), colonization and invasion of the host (Croxatto et al, 2007), and production of hemolysins and protease (Hirono et al, 1996;Denkin et al, 2004;Rock et al, 2006). Extracellular proteases are considered putative virulence factors in several pathogens, including V. cholera (Silva et al, 2006), V. vulnificus (Wang et al, 2008), V. harveyi (Lee et al, 1999), V. fisheri (Stevens et al, 1997), V. anguillarum (Norqvist et al, 1990;Milton et al, 1992) and Pseudomonas aeruginosa (Zhang et al, 2007).…”

Vibrio Iron Transport: Evolutionary Adaptation to Life in Multiple Environments

“…The siderophore receptors are typically localized to the outer membrane without the need for accessory factors. However, the V. anguillarum anguibactin receptor FatA (100, 101) requires O-antigen side-chain biosynthesis for outer membrane localization (102). The phenotype of the O-antigen mutants was similar to that of a fatA mutant.…”