Infections due to Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, are the leading causes of morbidity and mortality in both HIV infected and immune competent people. A major factor contributing to virulence of Mtb is its intrinsic slow growth rate and the ability to maintain a non-replicative persistent (NRP) state. Mtb employs several two-component regulatory signal transduction systems (2CRS) for its optimal survival upon infection, and the MtrAB is the only essential 2CRS. DNA replication is essential for multiplication of organisms and is believed to be regulated at the initiation step. The genetic elements responsible for the regulation of Mtb replication and or the factors that promote NRP state in vivo are largely unknown. DnaA protein initiates this process at a unique site on the genome called oriC. Our recent data indicate that the MtrA response regulator (RR) binds to oriC and the dnaA promoter. Our proposal focuses on the regulation of initiation of DnaA mediated oriC replication by MtrA RR in synchronously replicating cells. The novel Mtb dnaA cold sensitive mutants that we have generated will be used to obtain synchronously replicating population. We hypothesize that Mtb DnaA mediated oriC replication is regulated in part by the MtrA RR in a phosphorylation dependent manner. A consequence of this regulation will determine if, upon infection, DNA replication will continue resulting in bacterial multiplication or will stall/ shut-down promoting metabolically active, but NRP state. We will evaluate the contribution of the MtrAB signal transduction pathway on oriC replication by biochemical, genetic and physiological approaches. In the Specific Aim 1 we will evaluate MtrA binding to oriC both in the absence and presence of DnaA, and the consequence of these interactions on the oriC replication. In the Specific Aim 2, we will construct and characterize constitutively active MtrA~P (MtrA~P*) in an effort to evaluate the role of MtrA in Mtb proliferation. In the Specific Aim 3, we will evaluate the MtrA occupancy of oriC under synchronous replication conditions in cells producing MtrA~P*. Our proposed experiments will define how the DnaA mediated oriC replication is regulated by the MtrAB system. It is hoped that these experiments will define how the DNA replication process in Mtb is regulated during active replicative and the NRP growth states, hence will improve our ability to control Mtb infections.

Public Health Relevance

Tuberculosis remains a major public health crisis despite being a curable disease. Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, is the leading cause of morbidity and mortality in both HIV infected and immune competent people. The characteristic features of Mtb are its slow growth and the ability to maintain a non-replicative persistent state. Mtb employees several two-component regulatory systems (2CRS) of signal transduction for its optimal survival in vivo and the MtrAB system is the only essential 2CRS. DNA replication is essential for cell duplication and subsequent proliferation. Our research proposal focuses on understanding how DNA replication, one of the important factors contributing to persistence, is regulated upon infection by the MtrAB system. This knowledge will ultimately improve our ability to control Mtb infections.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI084734-04
Application #
8323346
Study Section
Special Emphasis Panel (ZRG1-AARR-H (02))
Program Officer
Jacobs, Gail G
Project Start
2009-09-22
Project End
2014-08-31
Budget Start
2012-09-01
Budget End
2014-08-31
Support Year
4
Fiscal Year
2012
Total Cost
$345,357
Indirect Cost
$100,423
Name
University of Texas Health Center at Tyler
Department
Type
Organized Research Units
DUNS #
800772337
City
Tyler
State
TX
Country
United States
Zip Code
75708
Purushotham, Gorla; Sarva, Krishna B; Blaszczyk, Ewelina et al. (2015) Mycobacterium tuberculosis oriC sequestration by MtrA response regulator. Mol Microbiol 98:586-604
Plocinska, Renata; Martinez, Luis; Gorla, Purushotham et al. (2014) Mycobacterium tuberculosis MtrB sensor kinase interactions with FtsI and Wag31 proteins reveal a role for MtrB distinct from that regulating MtrA activities. J Bacteriol 196:4120-9
Plocinski, P; Martinez, L; Sarva, K et al. (2013) Mycobacterium tuberculosis CwsA overproduction modulates cell division and cell wall synthesis. Tuberculosis (Edinb) 93 Suppl:S21-7
Satsangi, Akash T; Pandeeti, Emmanuel P; Sarva, Krishna et al. (2013) Mycobacterium tuberculosis MtrAY102C is a gain-of-function mutant that potentially acts as a constitutively active protein. Tuberculosis (Edinb) 93 Suppl:S28-32
Plocinski, P; Arora, N; Sarva, K et al. (2012) Mycobacterium tuberculosis CwsA interacts with CrgA and Wag31, and the CrgA-CwsA complex is involved in peptidoglycan synthesis and cell shape determination. J Bacteriol 194:6398-409
Plocinska, Renata; Purushotham, Gorla; Sarva, Krishna et al. (2012) Septal localization of the Mycobacterium tuberculosis MtrB sensor kinase promotes MtrA regulon expression. J Biol Chem 287:23887-99
Maloney, Erin; Madiraju, Sai Chandana; Rajagopalan, Malini et al. (2011) Localization of acidic phospholipid cardiolipin and DnaA in mycobacteria. Tuberculosis (Edinb) 91 Suppl 1:S150-5
Plocinski, P; Ziolkiewicz, M; Kiran, M et al. (2011) Characterization of CrgA, a new partner of the Mycobacterium tuberculosis peptidoglycan polymerization complexes. J Bacteriol 193:3246-56
Al Zayer, Maha; Stankowska, Dorota; Dziedzic, Renata et al. (2011) Mycobacterium tuberculosis mtrA merodiploid strains with point mutations in the signal-receiving domain of MtrA exhibit growth defects in nutrient broth. Plasmid 65:210-8
Vadrevu, Indumathi S; Lofton, Hava; Sarva, Krishna et al. (2011) ChiZ levels modulate cell division process in mycobacteria. Tuberculosis (Edinb) 91 Suppl 1:S128-35

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