Worldwide, tuberculosis (TB) is the chief opportunistic infection in HIV-infected hosts, as well as the leading cause of death from infectious disease in the population as a whole. The macrophage is the cell responsible for either allowing Mycobacterium tuberculosis (Mtb) to proliferate, or for forcing it into nonreplicative persistence. In the first award period we generated single and double knockout mice whose phenotypes proved the indispensability of two antimicrobial mechanisms of macrophages: production of reactive nitrogen intermediates (RNI) and reactive oxygen intermediates (ROI). We and collaborators showed that Mtb proliferates at its fastest known rate in mice deficient in inducible nitric oxide synthase (iNOS), the source of immunologically induced RNI; that chemotherapy fails to cure mice that lack iNOS; and that iNOS is present in human macrophages from lungs of TB patients. Others confirmed iNOS expression in TB patients' lung macrophages, showed that these cells make large amounts of RNI, and found that human alveolar macrophages can use iNOS to kill mycobacteria. The fact that Mtb nonetheless proliferates to cause TB suggests that disease-causing Mtb may express RNI resistance mechanisms, analogous to known ROI resistance mechanisms like SOD and catalase. In fact, in the first award period we cloned four genes, each of which confers both RNI- and ROI-resistance on bacterial hosts or whose knock-out makes them sensitive to RNI and ROI. Two of these are novel genes found only in Mtb: nitrogen oxides and oxygen intermediates resistance (NOXR)-1 and -3. The other two are widespread, but their role in resistance to RNI was unknown: alkyl hydroperoxide reductase subunit C (AhpC) and peptidyl methionyl sulfoxide reductase (msrA). The goal of this application is to learn how AhpC, NOXR3 and msrA inactivate RNI and ROI, and what overall contribution these gene products make to the ability of Mtb to overcome the host's immune defenses. We will focus first on AhpC, recently shown to be a virulence factor for Mycobacterium bovis, and analyze the impact of mutations, its biochemical mechanism and its expression in Mtb. We will knock out AhpC in Mtb and use wild type, AhpC-deficient and reconstituted Mtb to infect wild type mice and mice genetically deficient in production of RNI, ROI or both. Similar analyses will be done on NOXR3 and msrA. These studies will answer if inhibition of one or more of these gene products might sensitize Mtb to the diminished nitrosative and oxidative attack mounted by the HIV- infected host, and thereby improve the success of residual immunity and chemotherapy.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
1R01HL061241-01A1
Application #
2875500
Study Section
Special Emphasis Panel (ZRG1-AARR-4 (01))
Project Start
1999-03-01
Project End
2004-02-29
Budget Start
1999-03-01
Budget End
2000-02-29
Support Year
1
Fiscal Year
1999
Total Cost
Indirect Cost
Name
Weill Medical College of Cornell University
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
201373169
City
New York
State
NY
Country
United States
Zip Code
10065
Bryk, Ruslana; Arango, Nancy; Venugopal, Aditya et al. (2010) Triazaspirodimethoxybenzoyls as selective inhibitors of mycobacterial lipoamide dehydrogenase . Biochemistry 49:1616-27
Lee, Warren L; Gold, Benjamin; Darby, Crystal et al. (2009) Mycobacterium tuberculosis expresses methionine sulphoxide reductases A and B that protect from killing by nitrite and hypochlorite. Mol Microbiol 71:583-93
Hisert, Katherine B; MacCoss, Michael; Shiloh, Michael U et al. (2005) A glutamate-alanine-leucine (EAL) domain protein of Salmonella controls bacterial survival in mice, antioxidant defence and killing of macrophages: role of cyclic diGMP. Mol Microbiol 56:1234-45
Darwin, K Heran; Nathan, Carl F (2005) Role for nucleotide excision repair in virulence of Mycobacterium tuberculosis. Infect Immun 73:4581-7
Schnappinger, Dirk; Ehrt, Sabine; Voskuil, Martin I et al. (2003) Transcriptional Adaptation of Mycobacterium tuberculosis within Macrophages: Insights into the Phagosomal Environment. J Exp Med 198:693-704
Nathan, Carl (2003) Specificity of a third kind: reactive oxygen and nitrogen intermediates in cell signaling. J Clin Invest 111:769-78
Darwin, K Heran; Ehrt, Sabine; Gutierrez-Ramos, Jose-Carlos et al. (2003) The proteasome of Mycobacterium tuberculosis is required for resistance to nitric oxide. Science 302:1963-6
Shi, Shuangping; Nathan, Carl; Schnappinger, Dirk et al. (2003) MyD88 primes macrophages for full-scale activation by interferon-gamma yet mediates few responses to Mycobacterium tuberculosis. J Exp Med 198:987-97
Bryk, R; Lima, C D; Erdjument-Bromage, H et al. (2002) Metabolic enzymes of mycobacteria linked to antioxidant defense by a thioredoxin-like protein. Science 295:1073-7
Ehrt, S; Schnappinger, D; Bekiranov, S et al. (2001) Reprogramming of the macrophage transcriptome in response to interferon-gamma and Mycobacterium tuberculosis: signaling roles of nitric oxide synthase-2 and phagocyte oxidase. J Exp Med 194:1123-40

Showing the most recent 10 out of 12 publications