The diarrheal disease cholera poses enormous health, economic, and political burdens worldwide. The causative agent of cholera, Vibrio cholerae, is endemic in aquatic habitats, but it is capable of infecting the human small intestine, causing a massive, life-threatening diarrheal response. To colonize the host and cause disease, V. cholerae must acquire essential nutrients, including iron, in the host environment. The predominant form of iron present in the small intestine is ferrous iron. Thus, it is critical to understand the mechanisms of ferrous iron uptake in V. cholerae. The major ferrous iron transporter in V. cholerae is Feo. The Feo system is widely distributed among all bacterial species, and has important functions in the virulence of several pathogenic species; nevertheless, very little is known about its structure and mechanism of transport. In V. cholerae, the Feo transporter is composed of three proteins, FeoA, FeoB, and FeoC. The membrane-embedded C-terminal domain of FeoB is likely to form the pore for iron transport, and, interestingly, its N-terminal domain has homology to small eukaryotic G proteins, suggesting a novel mechanism of transport. The roles of FeoA and FeoC are unknown. We recently demonstrated that the three Feo proteins associate to form a higher order complex in vivo. This represents the first structural analysis of the mature, membrane- embedded, active Feo complex in vivo. The goal of this proposal is to determine the overall structure of this large complex in order to build and test models for the mechanism of iron transport. In our first specific aim, we will determine the mass and stoichiometry of the native Feo complex. This will lay the groundwork for a structural model of the active Feo transporter.
In aim 2, we will refine this model by delineating the sites of interaction within and between the members of the complex. We will then test the functional significance of these interactions for complex formation and iron transport activity.
In aim 3, we will determine the source of energy for transport through Feo. These studies will significantly advance our knowledge of the structure and function of this important and unique iron transporter. Significantly, all our studies will be carried out using active, membrane-associated Feo complexes in vivo, giving our results an undeniable relevance over the in vitro studies that currently dominate the Feo field. As our previous work shows, V. cholerae is an ideal model organism for the study of Feo. We have already assembled most of the strains and reagents needed, and we, and our collaborators, have the expertise required to carry out the proposed experiments.

Public Health Relevance

Vibrio cholerae, an inhabitant of marine environments, has the ability to colonize the human intestine and cause the severe diarrheal disease cholera. V. cholerae has multiple iron transport systems that allow it to acquire this essential element in both these environments. In this proposal, we will characterize Feo, a ferrous iron transport system that is important for iron acquisition in low oxygen environments, such as the human intestine.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI091957-07
Application #
9383022
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Hall, Robert H
Project Start
2010-12-03
Project End
2021-10-31
Budget Start
2017-11-01
Budget End
2018-10-31
Support Year
7
Fiscal Year
2018
Total Cost
Indirect Cost
Name
University of Texas Austin
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
170230239
City
Austin
State
TX
Country
United States
Zip Code
78759
Peng, Eric D; Payne, Shelley M (2017) Vibrio cholerae VciB Mediates Iron Reduction. J Bacteriol 199:
Stevenson, BegoƱa; Wyckoff, Elizabeth E; Payne, Shelley M (2016) Vibrio cholerae FeoA, FeoB, and FeoC Interact To Form a Complex. J Bacteriol 198:1160-70
Fisher, Carolyn R; Wyckoff, Elizabeth E; Peng, Eric D et al. (2016) Identification and Characterization of a Putative Manganese Export Protein in Vibrio cholerae. J Bacteriol 198:2810-7
Kazi, Misha I; Conrado, Aaron R; Mey, Alexandra R et al. (2016) ToxR Antagonizes H-NS Regulation of Horizontally Acquired Genes to Drive Host Colonization. PLoS Pathog 12:e1005570
Payne, Shelley M; Mey, Alexandra R; Wyckoff, Elizabeth E (2016) Vibrio Iron Transport: Evolutionary Adaptation to Life in Multiple Environments. Microbiol Mol Biol Rev 80:69-90
Peng, Eric D; Wyckoff, Elizabeth E; Mey, Alexandra R et al. (2015) Nonredundant Roles of Iron Acquisition Systems in Vibrio cholerae. Infect Immun 84:511-23
Mey, Alexandra R; Butz, Heidi A; Payne, Shelley M (2015) Vibrio cholerae CsrA Regulates ToxR Levels in Response to Amino Acids and Is Essential for Virulence. MBio 6:e01064
Wyckoff, Elizabeth E; Allred, Benjamin E; Raymond, Kenneth N et al. (2015) Catechol Siderophore Transport by Vibrio cholerae. J Bacteriol 197:2840-9
Weaver, Emily A; Wyckoff, Elizabeth E; Mey, Alexandra R et al. (2013) FeoA and FeoC are essential components of the Vibrio cholerae ferrous iron uptake system, and FeoC interacts with FeoB. J Bacteriol 195:4826-35
Mey, Alexandra R; Craig, Stephanie A; Payne, Shelley M (2012) Effects of amino acid supplementation on porin expression and ToxR levels in Vibrio cholerae. Infect Immun 80:518-28

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