Tuberculosis (TB) remains the single most important cause of death among adults worldwide. The high-output pathway of nitric oxide (NO) production is recognized to be important in defense against TB in mice, and new evidence supports it's importance in defense against human TB. The pathway's catalyst, inducible NO synthase (iNOS), is present in alveolar macrophages from patients with TB, and NO-resistant clinical strains of M. tuberculosis (Mtb) appear to disseminate more widely in TB-endemic communities. These recent findings direct our attention to genes in Mtb that may confer resistance to reactive nitrogen intermediates (RNI) alone or along with reactive oxygen intermediates (ROI). During the first grant period, we cloned two such genes, noxR1 and ahpC. noxR1 is novel. ahpC itself is well known, but its role in resistance to RNI is novel. The goals of this grant are to study noxR1 in depth as an RNI-resistance gene and to continue the search for others.
The specific aims of the project are as follows: 1) Study the structure and function of noxR1 by site-directed mutagenesis of noxR1, it's expression in E. coli and M. smegmatis, and comparison of their in vitro sensitivity to RNI and ROI and survival in microphages that are wild type (wt), deficient in inducible nitric oxide synthase (inos-I-), deficient in phagocyte oxidase 91-kDa subunit (gp91phox-I-), or deficient in both (PhoxNox-I-); determining NoxR1's mechanism of action and regulation; localizing NoxR1 protein in Mtb; crystallizing recombinant NoxR1 for structural solution by X-ray diffraction. 2) Test the pathophysiologic relevance of NoxR1 by deleting noxR1 in Mtb and testing such derivatives in a murine model of TB; analyzing noxR1 gene sequence and expression in clinical isolates of Mtb varying in RNI- sensitivity. 3) Clone and characterize new RNI/ROI resistance genes by expressing Mtb. Library in E. coli and M. spegmatis; selecting recombinants for survival in wt mice, as well as in the knock-out mice (iNOS, gp91 Phox-I-, and PhoxNOx-I-) and characterizing candidate genes as expressed in E. coli and M. spegmatis for resistance to RNI and ROI in vitro and for survival in macrophages that are wt or deficient in iNOS,gp91phox-I-, or both; studying identified E. coli and M. spegmatis recombinants for survival in vitro in S-nitrosoglutathione, acidified nitrite or peroxynitrite; characterizing candidate genes as expressed in E. coli and M. spegmatis for resistance to RNI and ROI in vitro and for survival in macrophages that are wt or deficient in iNOS,gp91phox, or both. Sequences of the noxR1 region as well as of the newly identified genes will be compared among clinical isolates of Mtb shown to be resistant to RNI and/or ROI in vitro. At the end of 5 years, these studies may yield better understanding of Mtb factors that contribute to resistance to antimicrobial effector molecules naturally produced by human macrophages, and may provide opportunities to discover targets and strategies for new therapies.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL051967-08
Application #
6330073
Study Section
Special Emphasis Panel (ZRG5-BM-2 (13))
Program Officer
Peavy, Hannah H
Project Start
1993-09-30
Project End
2002-11-30
Budget Start
2000-12-01
Budget End
2002-11-30
Support Year
8
Fiscal Year
2001
Total Cost
$238,142
Indirect Cost
Name
University of California Berkeley
Department
Public Health & Prev Medicine
Type
Schools of Public Health
DUNS #
124726725
City
Berkeley
State
CA
Country
United States
Zip Code
94704
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Firmani, Marcia A; Riley, Lee W (2002) Reactive nitrogen intermediates have a bacteriostatic effect on Mycobacterium tuberculosis in vitro. J Clin Microbiol 40:3162-6
Stewart, G R; Ehrt, S; Riley, L W et al. (2000) Deletion of the putative antioxidant noxR1 does not alter the virulence of Mycobacterium tuberculosis H37Rv. Tuber Lung Dis 80:237-42
Murray, H W; Nathan, C F (1999) Macrophage microbicidal mechanisms in vivo: reactive nitrogen versus oxygen intermediates in the killing of intracellular visceral Leishmania donovani. J Exp Med 189:741-6
Ruan, J; St John, G; Ehrt, S et al. (1999) noxR3, a novel gene from Mycobacterium tuberculosis, protects Salmonella typhimurium from nitrosative and oxidative stress. Infect Immun 67:3276-83
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Chen, L; Xie, Q W; Nathan, C (1998) Alkyl hydroperoxide reductase subunit C (AhpC) protects bacterial and human cells against reactive nitrogen intermediates. Mol Cell 1:795-805
Karupiah, G; Chen, J H; Mahalingam, S et al. (1998) Rapid interferon gamma-dependent clearance of influenza A virus and protection from consolidating pneumonitis in nitric oxide synthase 2-deficient mice. J Exp Med 188:1541-6
Karupiah, G; Chen, J H; Nathan, C F et al. (1998) Identification of nitric oxide synthase 2 as an innate resistance locus against ectromelia virus infection. J Virol 72:7703-6
Ehrt, S; Shiloh, M U; Ruan, J et al. (1997) A novel antioxidant gene from Mycobacterium tuberculosis. J Exp Med 186:1885-96

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