Tuberculosis (TB) continues to kill millions of people around the world. New tools to prevent and treat this disease are urgently needed. Iron is an essential metal for all forms of life and most bacterial pathogens including mycobacteria must import iron from its host to survive. Hence iron acquisition pathways are well studied in mycobacteria as their components are essential to mycobacterial viability. Thus far, it is thought that iron uptake in mycobacteria is orchestrated by mycobactins that are capable of removing iron from human transferrin. However in humans, transferrin iron accounts for less than 1% of the body's total iron whereas heme iron can represent greater than 80%. Thus one may speculate that mycobacteria are capable of acquiring iron from human heme sources. Recent studies, which focus on mycobactin deficient mutants of Mycobacterium tuberculosis (Mtb) and BCG, suggest that there is a novel heme acquisition pathway in mycobacteria. Interestingly, BCG has an attenuated heme uptake pathway compared to Mtb. In addition, an Mtb proteome-wide approach has been undertaken in our laboratory to identify potential proteins involved in heme acquisition. We propose a putative pathway where heme is sequestered from human hemoglobin by a secreted hemophore, transferred across the membrane by heme transporters, and broken-down by cytosolic heme-degrading protein to release iron. This research will shed light on the molecular mechanism of heme transfer from humans to bacteria. In addition, we will investigate the affect each gene within this proposed pathway, has on mycobacterial heme uptake in vivo.
The specific aims of this proposal are as follows: 1) Biophysical and biochemical investigation of the novel mycobacterial hemophore. 2) Explore the mechanism of heme transfer from host hemoglobin to hemophore to heme transporter. 3) Identification and characterization of other proteins involved in heme uptake. 4) Investigation into mycobacterial heme uptake system in Mtb. My proposed research focuses on gaining a comprehensive understanding of this novel mycobacterial heme uptake system on both molecular (single protein) and cellular levels. Interestingly, most of the proteins involved in this pathway have no close non- mycobacterial, protein sequence homologs, and additionally, the hemophore has a novel three-dimensional fold. With this in mind, the heme uptake pathway provides a number of good protein targets for the development of therapeutics against TB.

Public Health Relevance

Tuberculosis (TB) continues to kill millions of people worldwide and is caused by the bacterial pathogen Mycobacterium tuberculosis (Mtb). Iron is essential for all living species, and the TB community thought that Mtb acquired its iron in vivo solely via its iron siderophores;thus our identification of a novel heme acquisition pathway whereby iron is obtained via the uptake of human heme breaks this paradigm. In this proposal, we will characterize this mycobacterial specific heme uptake pathway at a molecular and cellular level, which will open new avenues for anti-TB drug development.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI081161-05
Application #
8463104
Study Section
Macromolecular Structure and Function A Study Section (MSFA)
Program Officer
Boyce, Jim P
Project Start
2009-06-15
Project End
2014-05-31
Budget Start
2013-06-01
Budget End
2014-05-31
Support Year
5
Fiscal Year
2013
Total Cost
$309,794
Indirect Cost
$102,502
Name
University of California Irvine
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
046705849
City
Irvine
State
CA
Country
United States
Zip Code
92697
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Contreras, Heidi; Chim, Nicholas; Credali, Alfredo et al. (2014) Heme uptake in bacterial pathogens. Curr Opin Chem Biol 19:34-41
Chim, Nicholas; Johnson, Parker M; Goulding, Celia W (2014) Insights into redox sensing metalloproteins in Mycobacterium tuberculosis. J Inorg Biochem 133:118-26
Graves, Amanda B; Morse, Robert P; Chao, Alex et al. (2014) Crystallographic and spectroscopic insights into heme degradation by Mycobacterium tuberculosis MhuD. Inorg Chem 53:5931-40
Owens, Cedric P; Chim, Nicholas; Graves, Amanda B et al. (2013) The Mycobacterium tuberculosis secreted protein Rv0203 transfers heme to membrane proteins MmpL3 and MmpL11. J Biol Chem 288:21714-28
Owens, Cedric P; Chim, Nicholas; Goulding, Celia W (2013) Insights on how the Mycobacterium tuberculosis heme uptake pathway can be used as a drug target. Future Med Chem 5:1391-403
Matsui, Toshitaka; Nambu, Shusuke; Ono, Yukari et al. (2013) Heme degradation by Staphylococcus aureus IsdG and IsdI liberates formaldehyde rather than carbon monoxide. Biochemistry 52:3025-7
Nambu, Shusuke; Matsui, Toshitaka; Goulding, Celia W et al. (2013) A new way to degrade heme: the Mycobacterium tuberculosis enzyme MhuD catalyzes heme degradation without generating CO. J Biol Chem 288:10101-9
Owens, Cedric P; Du, Jing; Dawson, John H et al. (2012) Characterization of heme ligation properties of Rv0203, a secreted heme binding protein involved in Mycobacterium tuberculosis heme uptake. Biochemistry 51:1518-31
Ekworomadu, MarCia T; Poor, Catherine B; Owens, Cedric P et al. (2012) Differential function of lip residues in the mechanism and biology of an anthrax hemophore. PLoS Pathog 8:e1002559

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