Regulation of the SigH stress response regulon by an essential protein kinase in Mycobacterium tuberculosis

Abstract

SigH is a key regulator of an extensive transcriptional network that responds to oxidative, nitrosative, and heat stresses in Mycobacterium tuberculosis, and this sigma factor is required for virulence in animal models of infection. SigH is negatively regulated by RshA, its cognate anti-sigma factor, which functions as a stress sensor and redox switch. While RshA provides a direct mechanism for sensing stress and activating transcription, bacteria use several types of signal transduction systems to sense the external environment. M. tuberculosis encodes several serine-threonine protein kinase signaling molecules, 2 of which, PknA and PknB, are essential and have been shown to regulate cell morphology and cell wall synthesis. In this work, we demonstrate that SigH and RshA are phosphorylated in vitro and in vivo by PknB. We show that phosphorylation of RshA, but not SigH, interferes with the interaction of these 2 proteins in vitro. Consistent with this finding, negative regulation of SigH activity by RshA in vivo is partially relieved in strains in which pknB is over-expressed, resulting in increased resistance to oxidative stress. These findings demonstrate an interaction between the signaling pathways mediated by PknB and the stress response regulon controlled by SigH. The intersection of these apparently discrete regulatory systems provides a mechanism by which limited activation of the SigH-dependent stress response in M. tuberculosis can be achieved. Coordination of the PknB and SigH regulatory pathways through phosphorylation of RshA may lead to adaptive responses that are important in the pathogenesis of M. tuberculosis infection.anti-sigma factor ͉ sigma factor ͉ transcription regulation ͉ phosphorylation S igH, an alternative sigma factor of Mycobacterium tuberculosis and other mycobacterial species, is a central regulator of the response to oxidative, nitrosative, and heat stresses. SigH directly regulates both effectors of the response to these stresses and additional transcription regulators that control expression of a broad range of stress response genes (1-4). The SigHdependent activation of this extensive stress response regulon is critical for M. tuberculosis virulence, as a sigH mutant is highly attenuated in the mouse model of infection (5).SigH activity is regulated at the transcriptional level via autoregulation of the sigH promoter, and posttranslationally via interaction with its cognate anti-sigma factor, RshA. This protein is a member of the Zinc-associated anti-sigma (ZAS) family, several members of which, including RshA, have been shown to function as redox switch proteins (3, 6-8). The interaction of RshA with SigH is disrupted under oxidizing conditions, allowing SigH to associate with core RNA polymerase and activate transcription of stress response genes and additional transcription regulators. The autoregulation of the sigH promoter results in rapid, strong induction of the SigH regulon following oxidative stress, which is maintained until redox homeostasis is reestablished and th...

Interaction of Mycobacterium tuberculosis Elongation Factor Tu with GTP Is Regulated by Phosphorylation

“…The pathogenic bacterium Mycobacterium tuberculosis has 11 Ser/Thr protein kinases (STPKs), one Ser/Thr phosphatase (PstP), two Tyr phosphatases (16), and one Tyr kinase (9). PknB, an essential STPK of M. tuberculosis (42), has been associated with a large number of events relating to cell division and growth (18,23,25,31), metabolism (8,32,33), cell wall synthesis (20,51,52), and stress response (10,35). M. tuberculosis survives various stress conditions during disease establishment and has the ability to enter into dormancy.…”

Differential Expression of sigH Paralogs during Growth and under Different Stress Conditions in Mycobacterium smegmatis

“…Often, these include a transmembrane protein with an extracytoplasmic sensory domain and an intracellular inhibitory domain functioning as an anti-sigma factor that binds and inhibits its cognate sigma factor (16). SigH activity is transcriptionally regulated at its autoregulated promoter and posttranslationally via interaction with its cognate anti-sigma factor, RshA, whose gene resides in the same operon (13). The presence of cupin domain protein genes downstream of sigH1, sigH5, and sigH7 is noteworthy.…”

“…SigH regulates the transcriptional network that responds to oxidative, nitrosative, and heat stresses in mycobacteria (13,17). An M. smegmatis sigH mutant showed increased sensitivity to oxidative stress by cumene hydroperoxide but no defect in survival at 53°C or oxidative stress due to hydrogen peroxide (5).…”

“…Similar survival instincts of the sigH mutant and the wild type suggest the presence of multiple mechanisms to counter these stresses in this species. Although the regulatory mechanism of sigH expression and sigH's role have been worked out (13,16,22), the multiplicity of its paralogous circuit in M. smegmatis remains to be analyzed. Determining the conditions under which the expression of sigH paralogs are induced or repressed is vital to the understanding of their possible function and role in the regulation of gene expression in M. smegmatis in response to different environmental stimuli.…”

Autoregulatory Characteristics of a Bacillus anthracis Serine/Threonine Kinase

“…Although the family of bacterial STKs is homologous across several genera of bacteria, each microbe has coopted its STK to regulate processes critical to the particular parent organism. Collectively, prokaryotic STKs have been reported to regulate growth and virulence, as well as stress responses (29), gene expression (15,34), development (24,28), biofilm formation (18), and metabolism (6,33), in an organism-specific manner. Furthermore, a recent study by Shakir et al characterized a serine/threonine kinase-phosphatase pair important for survival of B. anthracis within cultured macrophages (37).…”