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PhoP-PhoR, one of the 12 two-component systems (TCSs) that empower M. tuberculosis to sense and adapt to diverse environmental conditions, remains essential for virulence, and therefore, represents a major target to develop novel anti-TB therapies. Although both PhoP and PhoR have been structurally characterized, the signal(s) that this TCS responds to remains unknown. Here, we show that PhoR is a sensor of acidic pH/high salt conditions, which subsequently activate PhoP via phosphorylation. In keeping with this, transcriptomic data uncover that acidic pH- inducible expression of PhoP regulon is significantly inhibited in a PhoR-deleted M. tuberculosis. Strikingly, a set of PhoP regulon genes displayed a low pH-dependent activation even in the absence of PhoR, suggesting the presence of non-canonical mechanism(s) of PhoP activation. Using genome-wide interaction-based screening coupled with phosphorylation assays, we identify a non-canonical mechanism of PhoP phosphorylation by the sensor kinase PrrB. To investigate how level of P~PhoP is regulated, we discovered that in addition to its kinase activity PhoR functions as a phosphatase of P~PhoP. Our subsequent results identify the motif/residues responsible for kinase/phosphatase dual functioning of PhoR. Collectively, these results uncover that contrasting kinase and phosphatase functions of PhoR determine the homeostatic mechanism of regulation of intra-mycobacterial P~PhoP which controls the final output of the PhoP regulon. Together, these results connect PhoR to pH-dependent activation of PhoP with downstream functioning of the regulator. Thus, PhoR plays a central role in mycobacterial adaptation to low pH conditions within the host macrophage phagosome, and a PhoR-deleted M. tuberculosis remains significantly attenuated in macrophages and animal models. Author summary: Virulence-associated PhoP-PhoR of M. tuberculosis represents an attractive target to develop anti-tubercular therapy, but to date, the signal(s) that this regulatory system responds to remains unknown. We discovered that acidic pH and high salt conditions activate PhoP using a PhoR-dependent mechanism. Thus, pH inducible PhoP regulon expression is significantly impacted in a PhoR-depleted M. tuberculosis H37Rv. Our subsequent investigations reveal that homeostatic mechanism of regulation of P~PhoP relies on kinase/phosphatase dual functioning of PhoR, which determines mycobacterial pH homeostasis by controlling the final output of the PhoP regulon. Unexpectedly, global regulatory studies uncover that there can be PhoR-independent mechanism(s) of in vivo activation of PhoP. While probing for a non-canonical mechanism, we demonstrate that the sensor kinase (SK) PrrB phosphorylates PhoP. These results connect two SKs with signal-dependent activation of PhoP. [ABSTRACT FROM AUTHOR]

Mycobacterium tuberculosis retains the ability to establish an asymptomatic latent infection. A fundamental question in mycobacterial physiology is to understand the mechanisms involved in hypoxic stress, a critical player in persistence. Here, we show that the virulence regulator PhoP responds to hypoxia, the dormancy signal, and effectively integrates hypoxia with nitrogen metabolism. We also provide evidence to demonstrate that both under nitrogen limiting conditions and during hypoxia, phoP locus controls key genes involved in nitrogen metabolism. Consistently, under hypoxia a ΔphoP strain shows growth attenuation even with surplus nitrogen, the alternate electron acceptor, and complementation of the mutant restores bacterial growth. Together, our observations provide new biological insights into the role of PhoP in integrating nitrogen metabolism with hypoxia by the assistance of the hypoxia regulator DosR. The results have significant implications on the mechanism of intracellular survival and growth of the tubercle bacilli under a hypoxic environment within the phagosome. [ABSTRACT FROM AUTHOR]

A hallmark feature of Mycobacterium tuberculosis pathogenesis lies in the ability of the pathogen to survive within macrophages under a stressful environment. Thus, coordinated regulation of stress proteins is critically important for an effective adaptive response of M. tuberculosis, the failure of which results in elevated immune recognition of the tubercle bacilli with reduced survival during chronic infections. Here, we show that virulence regulator PhoP impacts the global regulation of heat shock proteins, which protect M. tuberculosis against stress generated by macrophages during infection. Our results identify that in addition to classical DNA-protein interactions, newly discovered protein-protein interactions control complex mechanisms of expression of heat shock proteins, an essential pathogenic determinant of M. tuberculosis. While the C-terminal domain of PhoP binds to its target promoters, the N-terminal domain of the regulator interacts with the C-terminal end of the heat shock repressors. Remarkably, our findings delineate a regulatory pathway which involves three major transcription factors, PhoP, HspR, and HrcA, that control in vivo recruitment of the regulators within the target genes and regulate stress-specific expression of heat shock proteins via protein-protein interactions. The results have implications on the mechanism of regulation of PhoP-dependent stress response in M. tuberculosis. [ABSTRACT FROM AUTHOR]

The ability to sense acid stress and mount an appropriate adaptive response by Mycobacterium tuberculosis, which adapts a long-term residence in the macrophage phagosome, remains one of the critical features that defines mycobacterial physiology and its intracellular location. To understand the mechanistic basis of adaptation of the intracellular pathogen, we studied global regulation of M. tuberculosis gene expression in response to acid stress. Although recent studies indicate a role for the virulence-associated phoP locus in pH-driven adaptation, in this study, we discovered a strikingly novel regulatory mechanism, which controls acid-stress homeostasis. Using mycobacterial protein fragment complementation and in vitro interaction analyses, we demonstrate that PhoP interacts with acid-inducible extracytoplasmic SigE (one of the 13 M. tuberculosis sigma factors) to regulate a complex transcriptional program. Based on these results, we propose a model to suggest that PhoP-SigE interaction represents a major requirement for the global acid stress response, absence of which leads to strongly reduced survival of the bacilli under acidic pH conditions. These results account for the significant growth attenuation of the phoP mutant in both cellular and animal models, and unravel the underlying global mechanism of how PhoP induces an adaptive program in response to acid stress. [ABSTRACT FROM AUTHOR]