Mycobacterium tuberculosis is the etiological agent of tuberculosis (TB) and is responsible for more than 1.7 million deaths and 8 million new infections annually. A state of emergency was recently declared by the World Health Organization (WHO) in Africa where TB has been associated with nearly one-third of all acquired immunodeficiency syndrome (AIDS)-associated deaths. Currently, 2 billion people are predicted to be persistently infected by M. tuberculosis worldwide. The processes that govern long-term infection by M. tuberculosis are complex and multifactorial, and they remain poorly understood. A better understanding of the molecular mechanisms regulating the establishment, maintenance, and reactivation of persistence by M. tuberculosis is urgently needed if TB is to be eradicated. MprAB has been shown to play a role in the establishment and maintenance of persistent infection by M. tuberculosis. mprAB encodes a two-component signaling system that is used to detect, integrate, and coordinate specific survival programs in response to stressful stimuli. Genes regulated by MprAB include mprAB itself, stress-responsive sigma factors sigE and sigB, a putative serine protease pepD, and the DosRST regulon. We hypothesize that the presence of misfolded or altered protein substrates induced in or at the cell surface following exposure of M. tuberculosis to stress activates MprAB and initiates downstream survival programs that include induction of a serine protease to degrade or refold altered protein substrate(s) and genes comprising the DosRST regulon to initiate a general survival response program. In this proposal, two specific aims have been proposed.
The first aim will determine the biochemical function of PepD and its importance in M. tuberculosis physiology and pathogenesis. This will be accomplished by purifying various rPepD derivatives and examining their enzymatic activity. In addition, pepD mutant strains will be constructed and utilized in various in vitro and in vivo assays to investigate the importance of this gene in M. tuberculosis physiology and virulence. Finally, the natural substrate targets of PepD will be identified and verified using proteomic approaches.
The second aim will investigate the role of MprAB in regulation of the DosRST regulon. This will be accomplished by characterizing and comparing promoter regions from MprA and DosR co- regulated genes, and examining the regulation of these genes in various M. tuberculosis derivatives during growth in macrophages in vitro and in a murine model of latent TB. In addition, consequences of mprA and/or dosR gene deletion on M. tuberculosis physiology and virulence will be assessed using in vitro survival assays and in vitro and in vivo model systems of infection. The proposal outlined here is expected to enhance our overall understanding of the factors contributing to M. tuberculosis persistence, including the genetic and environmental factors that regulate this dynamic process. ? ? ?

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
2R01AI051669-06A1
Application #
7257575
Study Section
Bacterial Pathogenesis Study Section (BACP)
Program Officer
Lacourciere, Karen A
Project Start
2001-08-15
Project End
2012-03-31
Budget Start
2007-04-15
Budget End
2008-03-31
Support Year
6
Fiscal Year
2007
Total Cost
$363,851
Indirect Cost
Name
Medical College of Wisconsin
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
937639060
City
Milwaukee
State
WI
Country
United States
Zip Code
53226
Bretl, Daniel J; Bigley, Tarin M; Terhune, Scott S et al. (2014) The MprB extracytoplasmic domain negatively regulates activation of the Mycobacterium tuberculosis MprAB two-component system. J Bacteriol 196:391-406
Bretl, Daniel J; He, Hongjun; Demetriadou, Crystalla et al. (2012) MprA and DosR coregulate a Mycobacterium tuberculosis virulence operon encoding Rv1813c and Rv1812c. Infect Immun 80:3018-33
He, Hongjun; Bretl, Daniel J; Penoske, Renee M et al. (2011) Components of the Rv0081-Rv0088 locus, which encodes a predicted formate hydrogenlyase complex, are coregulated by Rv0081, MprA, and DosR in Mycobacterium tuberculosis. J Bacteriol 193:5105-18
White, Mark J; Savaryn, John P; Bretl, Daniel J et al. (2011) The HtrA-like serine protease PepD interacts with and modulates the Mycobacterium tuberculosis 35-kDa antigen outer envelope protein. PLoS One 6:e18175
Bretl, Daniel J; Demetriadou, Chrystalla; Zahrt, Thomas C (2011) Adaptation to environmental stimuli within the host: two-component signal transduction systems of Mycobacterium tuberculosis. Microbiol Mol Biol Rev 75:566-82
White, Mark J; He, Hongjun; Penoske, Renee M et al. (2010) PepD participates in the mycobacterial stress response mediated through MprAB and SigE. J Bacteriol 192:1498-510
He, Hongjun; Hovey, Raymond; Kane, Jason et al. (2006) MprAB is a stress-responsive two-component system that directly regulates expression of sigma factors SigB and SigE in Mycobacterium tuberculosis. J Bacteriol 188:2134-43
He, Hongjun; Zahrt, Thomas C (2005) Identification and characterization of a regulatory sequence recognized by Mycobacterium tuberculosis persistence regulator MprA. J Bacteriol 187:202-12
Hessner, Martin J; Singh, Vineet K; Wang, Xujing et al. (2004) Utilization of a labeled tracking oligonucleotide for visualization and quality control of spotted 70-mer arrays. BMC Genomics 5:12
Zahrt, Thomas C; Wozniak, Christopher; Jones, Denise et al. (2003) Functional analysis of the Mycobacterium tuberculosis MprAB two-component signal transduction system. Infect Immun 71:6962-70

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