The long-term goal of this research is to improve our understanding of the pathogenesis of tuberculosis. This research will address this long-term goal by examining the role of sigma factors in the regulation of mycobacterial gene expression. A clearer picture of the molecular biology of mycobacterial gene expression may provide insight into mechanisms that these organisms use to survive the hostile intracellular environment of the host cell. Sigma factors whose activity is essential under conditions relevant to in vivo replication and survival may also provide a means to identify regulated genes that are essential for mycobacterial virulence. A better understanding of mycobacterial gene expression and disease pathogenesis at the molecular level will provide opportunities to develop new approaches to treatment and prevention of mycobacterial infection and disease. The long-term goals of this research will be approached through three specific aims. First, sigma factor genes of M. tuberculosis will be cloned and sequenced. Dominant negative mutations will then be created in these cloned sigma factor genes by site-directed or PCR mutagenesis, focusing on the -10 recognition region of these sigma factors. Homologues in M. fortuitum of these M. tuberculosis sigma factor genes will be cloned, and M. fortuitum strains with null mutations in these genes will be created by allelic exchange. These M. tuberculosis and M. fortuitum mutants will be used to examine the role of specific sigma factors on patterns of mycobacterial gene expression by two-dimensional gel electrophoresis. Second, these mutant sigma factor genes in M. tuberculosis and M. fortuitum will be used to examine the role of sigma factor function on replication and survival a) in culture under various conditions, b) after uptake by macrophages in vitro, and c) in mice in vivo. Third, initial characterization of the promoters of genes whose expression is regulated by specific sigma factors will be undertaken by identifying a limited number of these regulated genes. Consensus promoter elements in these genes will be determined through searching of the M. tuberculosis sequence database and through cloning and sequencing these genes.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
1R01AI037901-01
Application #
2074791
Study Section
Special Emphasis Panel (SRC (49))
Project Start
1995-06-01
Project End
2000-05-31
Budget Start
1995-06-01
Budget End
1996-05-31
Support Year
1
Fiscal Year
1995
Total Cost
Indirect Cost
Name
Children's Hospital Boston
Department
Type
DUNS #
076593722
City
Boston
State
MA
Country
United States
Zip Code
02115
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Ymele-Leki, Patrick; Cao, Shugeng; Sharp, Jared et al. (2012) A high-throughput screen identifies a new natural product with broad-spectrum antibacterial activity. PLoS One 7:e31307
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Song, Taeksun; Song, Seung-Eun; Raman, Sahadevan et al. (2008) Critical role of a single position in the -35 element for promoter recognition by Mycobacterium tuberculosis SigE and SigH. J Bacteriol 190:2227-30
Park, Sang Tae; Kang, Choong-Min; Husson, Robert N (2008) Regulation of the SigH stress response regulon by an essential protein kinase in Mycobacterium tuberculosis. Proc Natl Acad Sci U S A 105:13105-10
Williams, Diana L; Pittman, Tana L; Deshotel, Mike et al. (2007) Molecular basis of the defective heat stress response in Mycobacterium leprae. J Bacteriol 189:8818-27
Raman, Sahadevan; Puyang, Xiaoling; Cheng, Tan-Yun et al. (2006) Mycobacterium tuberculosis SigM positively regulates Esx secreted protein and nonribosomal peptide synthetase genes and down regulates virulence-associated surface lipid synthesis. J Bacteriol 188:8460-8
Hahn, Mi-Young; Raman, Sahadevan; Anaya, Mauricio et al. (2005) The Mycobacterium tuberculosis extracytoplasmic-function sigma factor SigL regulates polyketide synthases and secreted or membrane proteins and is required for virulence. J Bacteriol 187:7062-71
Raman, Sahadevan; Hazra, Rohan; Dascher, Christopher C et al. (2004) Transcription regulation by the Mycobacterium tuberculosis alternative sigma factor SigD and its role in virulence. J Bacteriol 186:6605-16

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