Dual functioning by the PhoR sensor is a key determinant to Mycobacterium tuberculosis virulence.

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]