The identification of genes controlling mycobacterial persistence and the signals that induce them is of paramount importance in developing new therapeutic agents and improved diagnostics for latent tuberculosis. The genetic mechanisms by which M tuberculosis survives for many years in a stationary state are poorly understood, but are likely to resemble the adaptive mechanisms used by other bacteria. Nutrient starvation and chemical stress are potential inducers of latency. Many bacteria respond to starvation and stress by expressing alternate sigma factors which redirect the cellular RNA polymerase to genes preceded by special promoters and thereby induce dozens of genes essential for survival in the new environment. Sigmas which mediate processes resembling latency, such as the gram-positive sporulation sigmas and the recently identified E. coli stationary phase sigma, are members of the omega70-family for which regions of interspecies conservation have been well-defined. I have shown that degenerate oligonucleotides against four of these conserved regions recognize multiple different fragments of M. tuberculosis chromosomal DNA--suggesting the presence of multiple omega70-hemologues. Moreover, these oligonucleotides amplify fragments of the expected size when used as PCR primers. A computer search of the one-quarter of the M. leprae genome that has already been sequenced reveals at least three potential transcription regulators (two of which are sigma factor homologues). One of these potential regulators shares significant homology with differentiation sigmas from Streptomyces species (close phylogenetic relatives of the mycobacteria) and sporulation sigmas from Bacillus species. This gene might well be a mycobacterial """"""""latency"""""""" sigma. I propose to clone as many mycobacterial sigma-like genes as can be found by a combination of hybridization, PCP, and homology searches of the Mycobacterial Genome Project databank. Northern analysis and gene fusion studies following nutrient depletion in culture will be used to characterize the functions of these sigmas. Complementation of known sigma mutants from other organisms will also be used to better define their functions. Candidate genes which appear to play a role in stationary phase survival will be used as probes for in situ hybridization and RT-PCR of the organisms found in both solid caseous (stationary phase) and liquefied (active growth phase) pulmonary lesions isolated from rabbits infected by the aerosol route. Knockout mutations or alterations of genes controlling latency in the tubercle bacilli might lead to potential vaccine strains which would proliferate at the outset, induce an immune response, but fail to establish persistent infection.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
First Independent Research Support & Transition (FIRST) Awards (R29)
Project #
5R29AI036973-05
Application #
2672408
Study Section
Special Emphasis Panel (SRC (85))
Project Start
1994-09-30
Project End
1999-05-31
Budget Start
1998-08-01
Budget End
1999-05-31
Support Year
5
Fiscal Year
1998
Total Cost
Indirect Cost
Name
Johns Hopkins University
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
045911138
City
Baltimore
State
MD
Country
United States
Zip Code
21218
Winglee, Kathryn; Manson McGuire, Abigail; Maiga, Mamoudou et al. (2016) Whole Genome Sequencing of Mycobacterium africanum Strains from Mali Provides Insights into the Mechanisms of Geographic Restriction. PLoS Negl Trop Dis 10:e0004332
Gupta, Shashank; Tyagi, Sandeep; Bishai, William R (2015) Verapamil increases the bactericidal activity of bedaquiline against Mycobacterium tuberculosis in a mouse model. Antimicrob Agents Chemother 59:673-6
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Maiga, Mariama C; Ahidjo, Bintou Ahmadou; Maiga, Mamoudou et al. (2015) Roflumilast, a Type 4 Phosphodiesterase Inhibitor, Shows Promising Adjunctive, Host-Directed Therapeutic Activity in a Mouse Model of Tuberculosis. Antimicrob Agents Chemother 59:7888-90
Winglee, Kathryn; Lun, Shichun; Pieroni, Marco et al. (2015) Mutation of Rv2887, a marR-like gene, confers Mycobacterium tuberculosis resistance to an imidazopyridine-based agent. Antimicrob Agents Chemother 59:6873-81
Choi, Seong Won; Maiga, Mamoudou; Maiga, Mariama C et al. (2014) Rapid in vivo detection of isoniazid-sensitive Mycobacterium tuberculosis by breath test. Nat Commun 5:4989
Winglee, Kathryn; Eloe-Fadrosh, Emiley; Gupta, Shashank et al. (2014) Aerosol Mycobacterium tuberculosis infection causes rapid loss of diversity in gut microbiota. PLoS One 9:e97048
Lun, Shichun; Miranda, David; Kubler, Andre et al. (2014) Synthetic lethality reveals mechanisms of Mycobacterium tuberculosis resistance to ?-lactams. MBio 5:e01767-14
Choudhary, Eira; Bishai, William; Agarwal, Nisheeth (2014) Expression of a subset of heat stress induced genes of mycobacterium tuberculosis is regulated by 3',5'-cyclic AMP. PLoS One 9:e89759

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