M. tuberculosis encounters reductive stress under acidic pH. To investigate the mechanism of coupled stress response, we show that PhoP plays a major role in mycobacterial redox stress response.
PhoP-PhoR empowersM. tuberculosisto adapt to diverse environmental conditions, and remains essential for virulence. Although PhoP and PhoR have been structurally characterized, the signal(s) that this TCS responds to remains unknown. In this study, we show that PhoR is a sensor of acidic pH/high salt conditions, which activate PhoP via phosphorylation. Transcriptomic studies uncover that acidic pH-inducible expression of PhoP regulon is significantly inhibited in a PhoR-deletedM. tuberculosis. Using genome-wide screening we further identify a non-canonical mechanism of PhoP phosphorylation by the sensor kinase PrrB. To investigate how phosphorylation of PhoP is regulated, we discovered that PhoR functions as a phosphatase. Our results identify the motif/residues responsible for contrasting kinase/phosphatase dual functioning of PhoP, and collectively determine the homeostatic regulation of intra-mycobacterial P~PhoP which controls the final output of PhoP regulon. Together, these data uncover that PhoR plays a central role in mycobacterial adaptation to low pH conditions within the host macrophage phagosome. Consistent with these results a PhoR-deletedM. tuberculosisremains significantly attenuated in macrophages and animal models.
Survival of M. tuberculosis within the host macrophages requires presence of the virulence regulator PhoP, but the underlying mechanism is yet to be understood. We discovered a signalling pathway which controls mycobacterial cAMP-inducible gene expression and cAMP homeostasis. We show that the level of intra-mycobacterial cAMP, one of the most widely used second messengers, is regulated by the virulence regulator PhoP, which recruits the cAMP responsive protein, CRP. We provide evidence to show that PhoP -dependent repression of cAMP specific phosphodiesterase Rv0805, which degrades cAMP hydrolytically, accounts for mycobacterial cAMP homeostasis. In keeping with these findings, genetic manipulation to inactivate PhoP-Rv0805-cAMP pathway leads to disruption of cAMP homeostasis, increased stress sensitivity and most critically, reduced mycobacterial survival in macrophages and animal models. Together, PhoP-dependent cAMP inducible gene expression and cAMP homeostasis represent a molecular checkpoint during intra-phagosomal survival and growth program of mycobacteria.
The main purpose of this study is to understand how mycobacteria can sense numerous stress conditions and mount an appropriate stress response. Recent studies suggest that at low pH M. tuberculosis encounters reductive stress, and in response, modulates redox homeostasis by utilizing the phoPR regulatory system. However, the mechanism of integrated regulation of stress response remains unknown. To probe how PhoP contributes to redox stress response, we find that a PhoP-depleted M. tuberculosis shows a significantly enhanced susceptibility to redox stress relative to the WT bacilli. In keeping with these results, PhoP was shown to contribute to mycothiol redox state. Because SigH, one of the alternative sigma factors of mycobacteria, is known to control expression of redox inducible genes, we probed whether previously-reported PhoP-SigH interaction accounts for mycobacterial redox stress response. We had shown that under acidic conditions PhoP functions in maintaining pH homeostasis via its interaction with SigE. In striking contrast, here we show that under redox stress, direct recruitment of SigH, but not PhoP-SigH interaction, controls expression of mycobacterial thioredoxin genes, a major mycobacterial anti-oxidant system. Together, these unexpected results uncover novel stress-specific enhanced or reduced interaction events of sigma factors and PhoP, as the underlying mechanisms of an adaptive programme, which couples low pH conditions and mycobacterial thiol redox homeostasis.
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