Cyclic AMP (cAMP) is known to function as a global regulator of Mycobacterium tuberculosis gene expression. Sequence-based transcriptomic profiling identified the mycobacterial regulon controlled by the cAMP receptor protein, CRP. In this study, we identified a new subset of CRP-associated genes including virulence determinants which are also under the control of a major regulator, PhoP. Our results suggest that PhoP as a DNA binding transcription factor, impacts expression of these genes, and phosphorylated PhoP promotes CRP recruitment at the target promoters. Further, we uncover a distinct regulatory mechanism showing that activation of these genes requires direct recruitment of both PhoP and CRP at their target promoters. The most fundamental biological insight is derived from the inhibition of CRP binding at the regulatory regions in a PhoP-deleted strain owing to CRP-PhoP protein-protein interactions. Based on these results, a model is proposed suggesting how CRP and PhoP function as co-activators of the essential pathogenic determinants. Taken together, these results uncover a novel mode of regulation where a complex of two interacting virulence factors impact expression of virulence determinants. These results have significant implications on TB pathogenesis.

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.

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