How does M. tuberculosis Rv2623 regulate mycobacterial growth?

Abstract

M. tuberculosis, the causative agent of tuberculosis, is an important pathogen with 8.8 million new infections in 2010. After M. tuberculosis bacteria are inhaled, they are phagocytosed by alveolar macrophages and trigger an innate immune response. M. tuberculosis responds to host-induced stresses including hypoxia, nitric oxide, cytokines and chemokines by slow growth, altered metabolism to utilize available carbon sources, and induction of the dormancy response (dosR) regulon. Among the dosR regulon genes up-regulated is the universal stress protein (USP) Rv2623. Previous studies in our laboratory characterized Rv2623 as growth-regulatory USP protein and its growth regulatory nature depends on its ATP binding capacity as ATP-binding deficient mutants did not regulate M. tuberculosis growth.;USP proteins are involved in mediating protein-protein interactions. In order to determine potential interaction partners, we conducted a yeast-two-hybrid screen. Among the potential interaction partners was Rv1747, an ABC transporter with two (forkhead associated) FHA domains. We confirmed this interaction by co-immunoprecipitation. FHA domains preferentially bind phosphorylated threonine residues. The five solvent accessible threonine residues (90, 103, 212, 237, and 280) of Rv2623 were mutated to alanine and analyzed for growth phenotype. Over-expression studies of the mutants in M. smegmatis indicated the T237A mutant reversed the over-expression phenotype seen with wildtype Rv2623 over-expression. This suggests a mechanism of Rv2623 action, where the phosphorylated Rv2623 binds to Rv1747 and alters its activity.;The Deltarv2623 strain has a settling phenotype in culture. Deletion of M. tuberculosis WhiB3 elicits the same phenotype, and an altered lipid profile. We examined the effect of Rv2623 expression on M. tuberculosis lipid profiles, but do not have a consistent phenotype to report. This is likely due to the limitation of the TLC technique used. We examined cell morphology and the cell wall using Scanning Electron Microscopy and Transmission Electron microscopy and preliminary analyses indicate no apparent differences in cell shape or cell wall associated with the presence of Rv2623. This is in contrast to gene expression profiling. Itraq proteomic experiments compared M. smegmatis pmv261 (empty vector) to pmv261