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.

Molecular Connectivity between Extracytoplasmic Sigma Factors and PhoP Accounts for Coupled Mycobacterial Stress Response.

Mycobacterium tuberculosis encounters numerous stress conditions within the host, but how it is able to mount a coupled stress response remains unknown. Growing evidence suggests that under acidic pH, M. tuberculosis modulates redox homeostasis. In an attempt to dissect the mechanistic details of responses to multiple stress conditions, here we studied the significance of connectivity of extracytoplasmic sigma factors with PhoP. We show that PhoP impacts the mycothiol redox state, and the H37Rv ΔphoP deletion mutant strain displays a significantly higher susceptibility to redox stress than the wild-type bacilli. To probe how the two regulators PhoP and redox-active sigma factor SigH contribute to redox homeostasis, we show that SigH controls expression of redox-active thioredoxin genes, a major mycobacterial antioxidant system, and under redox stress, SigH, but not PhoP, is recruited at the target promoters. Consistent with these results, interaction between PhoP and SigH fails to impact redox-dependent gene expression. This is in striking contrast to our previous results showing PhoP-dependent SigE recruitment within acid-inducible mycobacterial promoters to maintain pH homeostasis. Our subsequent results demonstrate reduced PhoP-SigH interaction in the presence of diamide and enhanced PhoP-SigE interaction under low pH. These contrasting results uncover the underlying mechanism of the mycobacterial adaptive program, coupling low pH with maintenance of redox homeostasis. IMPORTANCE 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. We observed a strong correlation of phoP-dependent redox-active expression of thioredoxin genes, a major mycobacterial antioxidant system. Further probing of functioning of regulators revealed that while PhoP controls pH homeostasis via its interaction with SigE, direct recruitment of SigH, but not PhoP-SigH interaction, controls expression of thioredoxin genes. These strikingly contrasting results showing enhanced PhoP-SigE interaction under acidic pH and reduced PhoP-SigH interaction under redox conditions uncover the underlying novel mechanism of the mycobacterial adaptive program, coupling low pH with maintenance of redox homeostasis.

Efficient assembly of a synthetic attenuated SARS-CoV-2 genome in Saccharomyces cerevisiae using multi-copy yeast vectors.

Saccharomyces cerevisiae has been demonstrated to be an excellent platform for the multi-fragment assembly of large DNA constructs through its powerful homologous recombination ability. These assemblies have invariably used the stable centromeric single copy vectors. However, many applications of these assembled genomes would benefit from assembly in a higher copy number vector for improved downstream extraction of intact genomes from the yeast. A review of the literature revealed that large multi-fragment assemblies did not appear to have been attempted in multicopy vectors. Therefore, we devised a toolkit that would enable one to seamlessly transition with the same assembling fragments between a single copy and a multicopy vector. We evaluated the assembly of a 28 kb attenuated SARSCoV- 2 genome (lacking the N gene) from 10 fragments in both single copy and multicopy vector systems. Our results reveal that assembly was comparably efficient in the two vector systems. The findings should add to the synthetic biology toolkit of S. cerevisiae and should enable researchers to utilize any of these vector systems depending on their downstream applications.

Mycobacterium tuberculosis PhoP integrates stress response to intracellular survival by regulating cAMP level.

Survival of Mycobacterium tuberculosis within the host macrophages requires the bacterial virulence regulator PhoP, but the underlying reason remains unknown. 3',5'-Cyclic adenosine monophosphate (cAMP) is one of the most widely used second messengers, which impacts a wide range of cellular responses in microbial pathogens including M. tuberculosis. Herein, we hypothesized that intra-bacterial cAMP level could be controlled by PhoP since this major regulator plays a key role in bacterial responses against numerous stress conditions. A transcriptomic analysis reveals that PhoP functions as a repressor of cAMP-specific phosphodiesterase (PDE) Rv0805, which hydrolyzes cAMP. In keeping with these results, we find specific recruitment of the regulator within the promoter region of rv0805 PDE, and absence of phoP or ectopic expression of rv0805 independently accounts for elevated PDE synthesis, leading to the depletion of intra-bacterial cAMP level. Thus, genetic manipulation to inactivate PhoP-rv0805-cAMP pathway decreases cAMP level, stress tolerance, and intracellular survival of the bacillus.