Tuberculosis constitutes a global health threat of growing proportions. Development of improved strategies for prevention and treatment of tuberculosis will be facilitated by an improved understanding of the cell biology of Mycobacterium tuberculosis. We have established that, whereas phagosomes containing dead M. tuberculosis proceed to phagolysosomes, live M. tuberculosis arrests the maturation of its phagosome at an early endosomal stage. However, the molecular basis underlying this arrested maturation has not been determined. We propose to examine the interaction of the M. tuberculosis phagosome with the host cell by immunoelectron microscopy and by biochemical and proteomic analysis of isolated phagosomes obtained at sequential times after incubation of human macrophages with live or dead M. tuberculosis. We shall determine the host cell protein composition of purified M. tuberculosis phagosomes by metabolic radiolabeling, 1- and 2-D gel electrophoresis, tryptic digestion, and mass spectrometry based proteomics. We shall explore the role of proteins identified by this approach by expressing these molecules as epitope tagged constructs in an adenoviral expression system and determining their localization using immunofluorescence and immunoelectron microscopy. When possible, we shall express dominant positive or negative mutants of these proteins and examine the effect of this on the phenotype of the M. tuberculosis phagosome. As overexpression of various host proteins alters the maturational state of the phagosome, we shall employ destabilized and inducible bacterial GFP expression as direct probes to assess bacterial metabolic activity within phagosomes of various phenotypes. In a similar fashion, we shall also explore the role of known components of the host cell membrane trafficking machinery, including Rab- and Rho- GTPases, and their downstream effectors. Because the lipid composition of the phagosome has an important impact on its capacity to participate in intracellular signaling, interaction with Rab and Rho effector proteins, and interaction with the host cell cytoskeleton, we shall also determine the lipid composition of the M. tuberculosis phagosome by metabolic radiolabeling, chromatographic analysis, and mass spectrometry. These experiments will advance our understanding of the cell biology and pathogenic mechanisms of M. tuberculosis and facilitate the development of new strategies to combat tuberculosis. ? ?

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL077000-12
Application #
6803511
Study Section
Special Emphasis Panel (ZRG1-BM-1 (03))
Program Officer
Peavy, Hannah H
Project Start
1993-09-30
Project End
2008-08-31
Budget Start
2004-09-01
Budget End
2005-08-31
Support Year
12
Fiscal Year
2004
Total Cost
$493,806
Indirect Cost
Name
University of California Los Angeles
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
092530369
City
Los Angeles
State
CA
Country
United States
Zip Code
90095
Horwitz, Lawrence D; Horwitz, Marcus A (2014) The exochelins of pathogenic mycobacteria: unique, highly potent, lipid- and water-soluble hexadentate iron chelators with multiple potential therapeutic uses. Antioxid Redox Signal 21:2246-61
Tullius, Michael V; Harmston, Christine A; Owens, Cedric P et al. (2011) Discovery and characterization of a unique mycobacterial heme acquisition system. Proc Natl Acad Sci U S A 108:5051-6
Lee, Bai-Yu; Jethwaney, Deepa; Schilling, Birgit et al. (2010) The Mycobacterium bovis bacille Calmette-Guerin phagosome proteome. Mol Cell Proteomics 9:32-53
Clemens, Daniel L; Lee, Bai-Yu; Horwitz, Marcus A (2009) Francisella tularensis phagosomal escape does not require acidification of the phagosome. Infect Immun 77:1757-73
Jia, Qingmei; Lee, Bai-Yu; Clemens, Daniel L et al. (2009) Recombinant attenuated Listeria monocytogenes vaccine expressing Francisella tularensis IglC induces protection in mice against aerosolized Type A F. tularensis. Vaccine 27:1216-29
Lee, Bai-Yu; Clemens, Daniel L; Horwitz, Marcus A (2008) The metabolic activity of Mycobacterium tuberculosis, assessed by use of a novel inducible GFP expression system, correlates with its capacity to inhibit phagosomal maturation and acidification in human macrophages. Mol Microbiol 68:1047-60
Clemens, Daniel L; Horwitz, Marcus A (2007) Uptake and intracellular fate of Francisella tularensis in human macrophages. Ann N Y Acad Sci 1105:160-86
Lee, Bai-Yu; Horwitz, Marcus A; Clemens, Daniel L (2006) Identification, recombinant expression, immunolocalization in macrophages, and T-cell responsiveness of the major extracellular proteins of Francisella tularensis. Infect Immun 74:4002-13
Hodges, Yvonne K; Weinberger, Howard D; Stephens, Janet et al. (2006) Desferri-Exochelin, a lipid-soluble, hexadentate iron chelator, effectively removes tissue iron. Transl Res 148:63-71
Horwitz, Marcus A (2005) Recombinant BCG expressing Mycobacterium tuberculosis major extracellular proteins. Microbes Infect 7:947-54

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