Infections due to Mycobacterium tuberculosis (Mtb) are the leading causes of morbidity and mortality in both HIV infected and immune competent people. Following initial infections Mtb frequently enters a latent or dormant state for extended periods and subsequently, under appropriate conditions or following immune suppression, revives, multiplies and causes a secondary infection. The genetic elements responsible for regulation of Mtb replication and multiplication or the factors that promote latent state in vivo are largely unknown. The two component response regulatory signal transduction systems (2CR) consist of a membrane bound sensor kinase and an intracellular regulator, and may play critical roles in processes responsible for Mtb survival and persistence in vivo. The MtrA-MtrB system is an essential 2CR of Mtb, but the roles, if any, of this system in regulating Mtb multiplication in vivo are unknown. Replication of chromosomal DNA and subsequent cell division are essential for multiplication, and interactions of DnaA, the initiator protein with origin of replication or oriC, result in replication initiation. The genetic elements that regulate initiation of chromosomal DNA replication are unknown. Our proposal focuses on connecting the MtrA-MtrB 2CR system to oriC replication and centers on a hypothesis that MtrA has a role in DNA replication, and that both oriC and DnaA are MtrA targets in vivo. This proposal aims to test the hypothesis that the phosphorylation state of MtrA is affected by growth of Mtb in vivo and this in turn affects the replication and subsequent proliferation of Mtb. Using genetic and molecular biological approaches we will characterize the recombinant strains of Mtb expressing elevated levels of wild type and phosphorylation defective MtrA protein and will evaluate the phosphorylation state of MtrA in Mtb isolated from phagosomes. Furthermore, we will test the hypothesis that Mtb strains expressing elevated levels of MtrA are attenuated for growth in vivo in murine infection model system. Biochemical interactions of recombinant wild type and phosphorylated MtrA with oriC will be examined by surface plasmon resonance technique. These experiments will establish whether oriC replication is a target of MtrA system and will improve our understanding of regulation of Mtb multiplication in vivo. ? ?
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