Expression of a Subset of Heat Stress Induced Genes of Mycobacterium tuberculosis Is Regulated by 3',5'-Cyclic AMP

Abstract

Mycobacterium tuberculosis (Mtb) secretes excess of a second messenger molecule, 3',5'-cyclic AMP (cAMP), which plays a critical role in the survival of Mtb in host macrophages. Although Mtb produces cAMP in abundance, its exact role in the physiology of mycobacteria is elusive. In this study we have analyzed the expression of 16 adenylate cyclases (ACs) and kinetics of intracellular cAMP levels in Mtb during in vitro growth under the regular culture conditions, and after exposure to different stress agents. We observed a distinct expression pattern of these ACs which is correlated with intracellular cAMP levels. Interestingly cAMP levels are significantly elevated in Mtb following heat stress, whereas other stress conditions such as oxidative, nitrosative or low pH do not affect intracellular cAMP pool in vitro. A significant increase in expression by >2-fold of five ACs namely Rv1647, Rv2212, Rv1625c, Rv2488c and Rv0386 after heat stress further suggested that cAMP plays an important role in controlling Mtb response to heat stress. In the light of these observations, effect of exogenous cAMP on global gene expression profile was examined by using microarrays. The microarray gene expression analysis demonstrated that cAMP regulates expression of a subset of heat stress-induced genes comprising of dnaK, grpE, dnaJ, and Rv2025c. Further we performed electrophoretic mobility shift assay by using cAMP-receptor protein of Mtb (CRPM), which demonstrated that CRPM specifically recognizes a sequence −301 AGCGACCGTCAGCACG −286 in 5'-untranslated region of dnaK.

The adenylyl cyclase Rv2212 modifies the proteome and infectivity of Mycobacterium bovis BCG

“…Our results suggest that hsp60-driven Rv2212 induces a phenotype resembling stressed mycobacteria, where gene regulation of chaperones might induce the capacity of adaptation to macrophage infection, mediated by proteins related with correct folding and metabolic pathways. In agreement with this proposal, it has just been shown that dnaK, grpE, dnaJ, and Rv2025c are regulated by cAMP in M. tuberculosis (Choudhary et al 2014). We acknowledge that other proteins we found differentially expressed in response to Rv2212 in BCG have not previously been shown to respond directly to CRP, therefore indicating either cAMP produced by Rv2212 activity might be modifying primary targets to then affect BfrB, GroEL2, BCG0191, and PepA, or that these proteins respond to general stress.…”

The Mycobacterium tuberculosis Clp Gene Regulator Is Required for in Vitro Reactivation from Hypoxia-induced Dormancy

“…rpoB encodes for DNA-dependent RNA polymerase, and mutations within rpoB can result in rifampicin resistance (44,45). dnaK encodes heat shock protein that is the most abundant protein within Mtb and plays a crucial role in preventing partially misfolded proteins from aggregating by binding to hydrophobic residues, which then allows these proteins to refold (46). As such, coordinated transcriptional and proteomic regulation of these known and proposed Clp protease substrates is crucial to the transition from a metabolically active to dormant state.…”

Regulation of the ahpC Gene Encoding Alkyl Hydroperoxide Reductase in Mycobacterium smegmatis

“…Recently it was demonstrated in M. tuberculosis that increased cellular levels of cAMP by heat stress or exogenous dibutyryl cAMP treatment led to upregulation of some heat stress-induced genes that have the Crp-binding sequences in their control regions, strongly indicating that the cellular cAMP level correlates with expression levels of the Crp regulon [66]. In this study, we observed both reduced expression of ahpC in a PDE-overexpressed strain of M. smegmatis relative to the control strain and an increase in cellular levels of cAMP under oxidative stress conditions.…”