Activation of the MprBA two-component system in Mycobacterium smegmatis under acidic and respiration-inhibitory conditions and crosstalk between MprA and KdpD.

Abstract

The MprBA two-component system (TCS) plays a critical role in Mycobacterium tuberculosis persistence and forms a signaling pathway with the alternative sigma factors SigE and SigB. Here, we demonstrate that the MprBA-SigE-SigB signaling pathway is activated in Mycobacterium smegmatis under multiple stress conditions associated with respiratory inhibition, including genetic inactivation of the aa₃ cytochrome c oxidase, treatment with the bcc₁ complex inhibitor Q203, hypoxia, high ionic strength, and acidic pH. Strikingly, acidic pH elicited the strongest activation of MprA-dependent sigE expression, suggesting that MprB integrates multiple stress signals beyond respiratory dysfunction. Deletion of mprB resulted in constitutive activation of MprA rather than inactivation, as evidenced by elevated sigE expression, increased MprA phosphorylation, and enhanced rifampicin resistance in an mprB mutant. Using CRISPRi-mediated repression and genetic deletion, we identified the KdpD histidine kinase, a component of the potassium homeostasis KdpDE TCS, as required for phosphorylating and activating MprA in the absence of MprB. These findings reveal unexpected crosstalk between KdpD and MprA. Expression of a truncated MprB lacking the N-terminal sensory domain abolished sigE expression under both neutral and acidic conditions, indicating that this domain is essential for sensing environmental signals and that the truncated protein behaves as a phosphatase-dominant form under the conditions tested. Growth analysis revealed that MprA, SigE, and SigB are required for optimal growth under acidic conditions, demonstrating the importance of the MprBA-SigE-SigB pathway for survival of M. smegmatis under acidic stress.IMPORTANCEThe MprBA TCS contributes to mycobacterial stress adaptation and persistence by regulating SigE- and SigB-mediated stress response pathways. Here, we show that the MprBA TCS is activated under respiration-inhibitory and acidic conditions in Mycobacterium smegmatis, suggesting that this pathway responds to environmental cues relevant to host infection. We also identify an unexpected regulatory relationship between the MprBA and KdpDE systems: in the absence of MprB, MprA can be phosphorylated and activated by the noncognate histidine kinase KdpD. These findings suggest that MprB phosphatase activity is critical for maintaining low basal MprA activity and preventing inappropriate KdpD-mediated activation under non-stress conditions.