Latency in the human host is a unique aspect of the pathogenesis of M. tuberculosis infection with important implications for prevention, treatment and elimination strategies. The understanding of latency is limited, though recent insights into the adaptation of M. tuberculosis to the environment of the macrophage, and to oxygen limitation may have important implications for latency in the human host. The hypothesis of this research is that gene expression that is regulated in concert with cell division is likely to play an important role in the transition of bacterial physiology to the latent state, and in the emergence of the organism from the latent state. The research proposed in this R21 application is designed to begin to characterize the role of two serine/threonine kinases, PknA and PknB, in the regulation of M. tuberculosis gene expression. These kinases have the structure of signal transduction kinases with predicted extracellular receptor domains and intracellular kinase domains. Their location near the bacterial origin of replication in an operon containing genes involved in cell wall biosynthesis suggests that their expression is linked to cell division. To begin to investigate the function of these regulatory genes in the context of active replication and latency, and their potential as targets for therapeutic intervention, two specific aims are proposed: 1) To undertake an initial functional characterization of pknA and pknB, including the effects of these regulatory proteins on growth, viability and patterns of gene expression and 2) To identify protein targets of the kinase domains of the serine/threonine kinases PknA and PknB. Approaches used in this research include regulated expression of pknA and pknB wild type and kinase-inactive (dominant negative) constructs, expression library cloning, and two-dimensional protein gel electrophoresis. The goal of this research is to gain initial insight into the function of these genes to provide a framework for future long-term investigation of their role in M. tuberculosis infection and latency.
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