Biometals. 30(2):203-216. doi: 10.1007/s10534-017-9994-0.

IutB participates in the ferric-vulnibactin utilization system in Vibrio vulnificus M2799.

Hiroaki Kawano1, Katsushiro Miyamoto1, Miho Negoro1, Eriko Zushi1, Takahiro Tsuchiya1, Tomotaka Tanabe2, Tatsuya Funahashi2, Hiroshi Tsujibo1*

1 Department of Microbiology, Osaka University of Pharmaceutical Sciences, 4-20-1 Nasahara, Takatsuki, Osaka 569-1094, Japan

2 Laboratory of Hygienic Chemistry, College of Pharmaceutical Sciences, Matsuyama University, 4-2 Bunkyo-cho, Matsuyama, Ehime 790-8578, Japan

*Corresponding author

PubMed

 

Abstract

Vibrio vulnificus, an opportunistic pathogen that causes a serious, often fatal, infection in humans, requires iron for its growth. This bacterium utilizes iron from the environment via the vulnibactin-mediated iron uptake system. The mechanisms of vulnibactin biosynthesis, vulnibactin export, and ferric-vulnibactin uptake systems have been reported, whereas the ferric-vulnibactin reduction mechanism in the cell remains unclear. The results of our previous study showed that VuuB, a member of the flavin adenine dinucleotide-containing siderophore-interacting protein family, is a ferric-vulnibactin reductase, but there are other reductases that can complement for the defective vuuB. The aim of this study was to identify these proteins that can complement the loss of function of VuuB. We constructed mutants of genes encoding putative reductases in V. vulnificus M2799, and analyzed their growth under low-iron conditions. Complementation analyses confirmed that IutB, which functions as a ferric-aerobactin reductase, participates in ferric-vulnibactin reduction in the absence of VuuB. This is the first genetic evidence that ferric-vulnibactin is reduced by a member of the ferric-siderophore reductase protein family. In the aerobactin-utilization system, IutB plays a major role in ferric-aerobactin reduction in V. vulnificus M2799, and VuuB and DesB can compensate for the defect of IutB. Furthermore, the expression of iutB and desB was found to be regulated by iron and a ferric uptake regulator.

 

Supplement:

Vibrio vulnificus is a Gram-negative halophilic marine bacterium that can cause gastroenteritis and primary septicemia in humans. The septicemia often occurs after the ingestion of raw oysters or shellfish, and symptoms progress rapidly even after early medical treatment. Primary septicemia tends to occur in immunocompromised patients and those suffering from diseases such as liver cirrhosis, hemochromatosis, and alcoholism. In our previous report, we demonstrated that V. vulnificus M2799, a clinical isolate, shows 100-fold higher lethality in mice than environmental strain JCM3731. So far, several potential V. vulnificus virulence factors have been identified. Among these factors, iron acquisition systems appear to be critical virulence factors. V. vulnificus M2799 acquires iron via the biosynthesis and secretion of the catechol siderophore vulnibactin. Previously, we used proteomic approaches to show that the expression levels of several proteins in V. vulnificus M2799 increased under low iron conditions. Construction of deletion mutants of the genes encoding proteins involved in the vulnibactin-mediated iron uptake system, such as isochorismate synthase (ICS), ferric-vulnibactin reductase (VuuB), the ferric-vulnibactin periplasmic binding protein (FatB), and the ferric-vulnibactin outer membrane receptor protein (VuuA), showed that deletions in ics and vuuA prevented growth under low iron conditions. These results showed that ICS and VuuA are essential for vulnibactin biosynthesis and the ferric-vulnibactin acquisition system, respectively, and are necessary for the growth of V. vulnificus M2799 under low iron conditions. On the other hand, growth was only retarded in the ΔfatB and ΔvuuB mutants, indicating that there must be alternative proteins in V. vulnificus M2799. As such, VatD, which functions as a ferric-aerobactin periplasmic binding protein (PBP), was shown to participate in the ferric-vulnibactin uptake system in the absence of FatB. However, the mechanism of ferric-vulnibactin reduction in the cell remains to be clarified.

We then focused on the ferric-vulnibactin reductase and tried to identify the corresponding protein(s) involved in ferric-vulnibactin reduction that can complement the loss of function of VuuB. Then, we searched for genes encoding putative oxidoreductases located in gene clusters for other iron utilization systems. V. vulnificus M2799 has two hydroxamate siderophore utilization systems: the ferric-aerobactin and ferric-desferrioxamine B utilization systems. The iutB and desB genes were located in gene clusters for the ferric-aerobactin and ferric-desferrioxamine B utilization systems, respectively. The deduced amino acid sequences of IutB and DesB harbored the conserved Cys-Cys-Xaa10-Cys-Xaa2-Cys motif in the C-terminal domain, as part of a 2Fe-2S cluster. Growth of DvuuBDiutB, but not DvuuBDdesB, under low-iron conditions was significantly impaired compared with that of DvuuB but not completely arrested, and growth was restored to the same level as that in DvuuB by the complementation of iutB (Fig. 1). These results indicated that the IutB protein plays a critical role in the vulnibactin-mediated iron acquisition system as a ferric-vulnibactin reductase in the absence of VuuB in M2799 cells.

We propose a model of the ferric-vulnibactin reduction mechanism in V. vulnificus M2799, whereby imported ferric-vulnibactin in the cytoplasm is reduced by VuuB (Fig. 2). In the absence of VuuB, IutB, which is mainly involved in ferric-aerobactin reduction, can function instead of VuuB for efficient iron release from ferric-vulnibactin. In the absence of IutB, VuuB and DesB can compensate for the defect in IutB for efficient iron release from ferric-aerobactin.

 

 

 

Fig. 1  Growth of deletion mutants of vuuB, iutB and desB(A) and complementation analyses (B) under low-iron conditions.

Deletion mutants of genes encoding VuuB, IutB and DesB were cultured in HI-EDDA medium, and OD600 was measured at the indicated times (A). Plasmid pRK415 or pRiutB was transferred into M2799, DvuuB, and DvuuBDiutB strains (B). Data from three independent experiments are shown; vertical lines show standard deviation.

 

 

 

Fig. 2  Schematic representation of ferric-siderophore utilization system of Vibrio vulnificus M2799.