Functional Genomics Reveals Extended Roles of the Mycobacterium tuberculosis Stress Response Factor σ ^H

Abstract

Mycobacterium tuberculosis is one of the most successful pathogens of humankind. During infection, M. tuberculosis must cope with and survive against a variety of different environmental conditions. Sigma factors likely facilitate the modulation of the pathogen's gene expression in response to changes in its extracellular milieu during infection. sigma(H), an alternate sigma factor encoded by the M. tuberculosis genome, is induced by thiol-oxidative stress, heat shock, and phagocytosis. In response to these conditions, sigma(H) induces the expression of sigma(B), sigma(E), and the thioredoxin regulon. In order to more effectively characterize the transcriptome controlled by sigma(H), we studied the long-term effects of the induction of sigma(H) on global transcription in M. tuberculosis. The M. tuberculosis isogenic mutant of sigma(H) (Delta-sigma(H)) is more susceptible to diamide stress than wild-type M. tuberculosis. To study the long-term effects of sigma(H) induction, we exposed both strains to diamide, rapidly washed it away, and resumed culturing in diamide-free medium (post-diamide stress culturing). Analysis of the effects of sigma(H) induction in this experiment revealed a massive temporal programming of the M. tuberculosis transcriptome. Immediately after the induction of sigma(H), genes belonging to the functional categories "virulence/detoxification" and "regulatory proteins" were induced in large numbers. Fewer genes belonging to the "lipid metabolism" category were induced, while a larger number of genes belonging to this category were downregulated. sigma(H) caused the induction of the ATP-dependent clp proteolysis regulon, likely mediated by a transcription factor encoded by Rv2745c, several members of the mce1 virulence regulon, and the sulfate acquisition/transport network.

ClgR regulation of chaperone and protease systems is essential for Mycobacterium tuberculosis parasitism of the macrophage

“…A homologue of ClgR exists in M. tuberculosis and its expression is highly upregulated in stress conditions such as heat shock (Stewart et al, 2002) and during macrophage infection (Schnappinger et al, 2003). Two recent studies of M. tuberculosis and Mycobacterium smegmatis suggest that ClgR also regulates expression of ClpP1/ClpP2 and ClpC1 in mycobacteria (Barik et al, 2010;Mehra & Kaushal, 2009). A wider examination of the ClgR regulon has not been made prior to the present study.…”

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

“…were used to compare transcriptome-wide responses in Mtb and the mutant to stress caused by hypoxia and reaeration. Detailed protocols for array procedures have been described earlier (19,22,23). Genes were considered to have a perturbed expression level if they exhibited a 2-fold higher or lower expression in the mycobacterial strain (wild-type or mutant) at a given time point relative to control samples in each of the three biological replicate arrays and in every technical replicate spot on each array.…”

“…Bacterial RNA Extraction-25 ml of culture was used to extract RNA upon cell lysis via the TRIzol bead beater method and phenol extraction (22). RNA concentrations were quantified using a Nanodrop 2000 (NanoDrop Technologies).…”

“…For RT-PCR, RNA was treated with DNase as previously described (17,22,24). RNA was reverse-transcribed following the manufacturers' instructions using the High Capacity RNA-to-cDNA kit (Applied Biosystems) (17,22,24). RT PCR was performed as per the manufacturers' instructions using Power SYBR Green PCR Master Mix (Applied Biosystems) and as previously described (17,22,24).…”

“…Data can be retrieved using the GEO platform number GPL18320. For RT-PCR, RNA was treated with DNase as previously described (17,22,24). RNA was reverse-transcribed following the manufacturers' instructions using the High Capacity RNA-to-cDNA kit (Applied Biosystems) (17,22,24).…”