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Unique iron coordination in iron-chelating molecule vibriobactin helps Vibrio cholerae evade mammalian siderocalin-mediated immune response.

ABSTRACT: Iron is essential for the survival of almost all bacteria. Vibrio cholerae acquires iron through the secretion of a catecholate siderophore called vibriobactin. At present, how vibriobactin chelates ferric ion remains controversial. In addition, the mechanisms underlying the recognition of ferric vibriobactin by the siderophore transport system and its delivery into the cytoplasm specifically have not been clarified. In this study, we report the high-resolution structures of the ferric vibriobactin periplasmic binding protein ViuP and its complex with ferric vibriobactin. The holo-ViuP structure reveals that ferric vibriobactin does not adopt the same iron coordination as that of other catecholate siderophores such as enterobactin. The three catechol moieties donate five, rather than six, oxygen atoms as iron ligands. The sixth iron ligand is provided by a nitrogen atom from the second oxazoline ring. This kind of iron coordination results in the protrusion of the second catechol moiety and renders the electrostatic surface potential of ferric vibriobactin less negatively polarized compared with ferric enterobactin. To accommodate ferric vibriobactin, ViuP has a deeper subpocket to hold the protrusion of the second catechol group. This structural characteristic has not been observed in other catecholate siderophore-binding proteins. Biochemical data show that siderocalin, which is part of the mammalian innate immune system, cannot efficiently sequester ferric vibriobactin in vitro, although it can capture many catecholate siderophores with high efficiency. Our findings suggest that the unique iron coordination found in ferric vibriobactin may be utilized by some pathogenic bacteria to evade the siderocalin-mediated innate immune response of mammals.

SUBMITTER: Li N

PROVIDER: S-EPMC3308770 | biostudies-literature | 2012 Mar

REPOSITORIES: biostudies-literature

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Unique iron coordination in iron-chelating molecule vibriobactin helps Vibrio cholerae evade mammalian siderocalin-mediated immune response.

Li Ning N Zhang Conggang C Li Bingqing B Liu Xiuhua X Huang Yan Y Xu Sujuan S Gu Lichuan L

The Journal of biological chemistry 20120130 12

Iron is essential for the survival of almost all bacteria. Vibrio cholerae acquires iron through the secretion of a catecholate siderophore called vibriobactin. At present, how vibriobactin chelates ferric ion remains controversial. In addition, the mechanisms underlying the recognition of ferric vibriobactin by the siderophore transport system and its delivery into the cytoplasm specifically have not been clarified. In this study, we report the high-resolution structures of the ferric vibriobac ...[more]

PMID: 22291019

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Project description:Vibrio cholerae is the causative agent of the severe diarrheal disease cholera. V. cholerae thrives within the human host, where it replicates to high numbers, but it also persists within the aquatic environments of ocean and brackish water. To survive within these nutritionally diverse environments, V. cholerae must encode the necessary tools to acquire the essential nutrient iron in all forms it may encounter. A prior study of systems involved in iron transport in V. cholerae revealed the existence of vciB, which, while unable to directly transport iron, stimulates the transport of iron through ferrous (Fe2+) iron transport systems. We demonstrate here a role for VciB in V. cholerae in which VciB stimulates the reduction of Fe3+ to Fe2+, which can be subsequently transported into the cell with the ferrous iron transporter Feo. Iron reduction is independent of functional iron transport but is associated with the electron transport chain. Comparative analysis of VciB orthologs suggests a similar role for other proteins in the VciB family. Our data indicate that VciB is a dimer located in the inner membrane with three transmembrane segments and a large periplasmic loop. Directed mutagenesis of the protein reveals two highly conserved histidine residues required for function. Taken together, our results support a model whereby VciB reduces ferric iron using energy from the electron transport chain.IMPORTANCEVibrio cholerae is a prolific human pathogen and environmental organism. The acquisition of essential nutrients such as iron is critical for replication, and V. cholerae encodes a number of mechanisms to use iron from diverse environments. Here, we describe the V. cholerae protein VciB that increases the reduction of oxidized ferric iron (Fe3+) to the ferrous form (Fe2+), thus promoting iron acquisition through ferrous iron transporters. Analysis of VciB orthologs in Burkholderia and Aeromonas spp. suggest that they have a similar activity, allowing a functional assignment for this previously uncharacterized protein family. This study builds upon our understanding of proteins known to mediate iron reduction in bacteria.

Project description:A 7.5-kbp fragment of chromosomal DNA downstream of the Vibrio cholerae vibriobactin outer membrane receptor, viuA, and the vibriobactin utilization gene, viuB, was recovered from a Sau3A lambda library of O395 chromosomal DNA. By analogy with the genetic organization of the Escherichia coli enterobactin gene cluster, in which the enterobactin biosynthetic and transport genes lie adjacent to the enterobactin outer membrane receptor, fepA, and the utilization gene, fes, the cloned DNA was examined for the ability to restore siderophore synthesis to E. coli ent mutants. Cross-feeding studies demonstrated that an E. coli entF mutant complemented with the cloned DNA regained the ability to synthesize enterobactin and to grow in low-iron medium. Sequence analysis of the cloned chromosomal DNA revealed an open reading frame downstream of viuB which encoded a deduced protein of greater than 2,158 amino acids, homologous to Yersinia sp. HMWP2, Vibrio anguillarum AngR, and E. coli EntF. A mutant with an in-frame deletion of this gene, named vibF, was created with classical V. cholerae strain O395 by in vivo marker exchange. In cross-feeding studies, this mutant was unable to synthesize ferric vibriobactin but was able to utilize exogenous siderophore. Complementation of the mutant with a cloned vibF fragment restored vibriobactin synthesis to normal. The expression of the vibF promoter was found to be negatively regulated by iron at the transcriptional level, under the control of the V. cholerae fur gene. Expression of vibF was not autoregulatory and neither affected nor was affected by the expression of irgA or viuA. The promoter of vibF was located by primer extension and was found to contain a dyad symmetric nucleotide sequence highly homologous to the E. coli Fur binding consensus sequence. A footprint of purified V. cholerae Fur on the vibF promoter, overlapping the Fur binding consensus sequence, was observed using DNase I footprinting. The protein product of vibF is homologous to the multifunctional nonribosomal protein synthetases and is necessary for the biosynthesis of vibriobactin.

Project description:A 74-kDa iron-regulated outer membrane protein of Vibrio cholerae acts as the receptor for the V. cholerae iron-siderophore complex, ferric vibriobactin. MBG14, a mutant of V. cholerae 0395 containing a TnphoA insertion in a gene designated viuA, lacks this 74-kDa outer membrane protein and is unable to bind or utilize exogenous ferric vibriobactin. Introduction of a plasmid containing the complete viuA coding sequence and 513 bp of upstream DNA into MBG14 restored ferric vibriobactin utilization to the mutant. The DNA insert in this plasmid was sequenced, revealing a single open reading frame of 2,061 bp, encoding a deduced protein of 687 amino acids with a predicted molecular mass of 76,417 Da and a predicted initial signal sequence of 37 amino acids. ViuA showed only weak homology to two iron-regulated outer membrane proteins in Escherichia coli, IutA and FecA. Construction of viuA::TnphoA gene fusions allowed study of the regulation of viuA expression by iron. This regulation in E. coli was dependent on the fur gene. Northern (RNA) blot analysis of RNA from wild-type V. cholerae grown in high- and low-iron media revealed a monocistronic viuA message that was negatively regulated by iron at the transcriptional level. Primer extension analysis identified a single transcriptional start site, located 243 bp above the translational start site. The promoter region of viuA contained two interrupted dyad symmetric nucleotide sequences, overlapping the -10 and -35 boxes, each similar to the E. coli Fur binding consensus sequence. Another iron-regulated gene in V. cholerae that is negatively regulated by fur, irgA, requires a positive transcriptional activator (irgB) for expression. However, a strain of V. cholerae mutant in irgB was unaffected in viuA expression. These studies suggest that there is conserved, global coordinate iron regulation in V. cholerae by fur; additional regulatory factors, superimposed upon the fur system, may provide more precise control of individual iron-regulated genes.

Dataset Information

Unique iron coordination in iron-chelating molecule vibriobactin helps Vibrio cholerae evade mammalian siderocalin-mediated immune response.

Publications

Unique iron coordination in iron-chelating molecule vibriobactin helps Vibrio cholerae evade mammalian siderocalin-mediated immune response.

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