Unraveling the link between serine/threonine protein kinase and two-component system regulation of environment-mediated Mycobacterium tuberculosis growth arrest

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PROJECT SUMMARY/ABSTRACT A marked feature of Mycobacterium tuberculosis (Mtb) infection is heterogeneity that encompasses several aspects, including in bacterial replication status and in the local microenvironment. This heterogeneity exists not just temporally, but also spatially even within a single lesion, as revealed at the single bacterium level by an integrated imaging system that combines the use of fluorescent reporter Mtb strains, a murine infection model that recapitulates hallmark caseous necrotic lesions, and confocal imaging. While environmental signals such as nitric oxide (NO) are known to be able to drive Mtb into growth arrest, how Mtb coordinates its replication with environmental cue response remains largely unknown. Further, the interplay between the two key systems that play central roles in Mtb signal transduction, namely serine/threonine protein kinases (STPKs) and two- component systems (TCSs), is also poorly understood. To this end, we recently uncovered the essential transcription factor PrrA, part of the PrrAB TCS, as (i) a regulator of Mtb response to multiple environmental cues, including NO, and (ii) a TCS whose function is significantly modulated by STPK phosphorylation, with consequent effects on Mtb replication status in response to NO. Aim 1 of this proposal thus seeks to elucidate the global transcriptional impact of STPK regulation of PrrA on the adaptive entry of Mtb into a non-replicating state upon extended NO exposure, utilizing a PrrA STPK phosphoablative mutant. A bacterial-two-hybrid approach will further be undertaken to uncover the STPKs responsible for phosphorylation of PrrA. Aim 2 focuses on understanding the functional consequences of STPK regulation of PrrA on Mtb replication status during infection in vivo, with single bacterium resolution. This will exploit the use of a replication reporter- expressing STPK phosphoablative PrrA mutant with our integrated imaging system, to delineate how STPK regulation of PrrA may differentially influence Mtb growth in disparate lesion sublocations, and reveal its relation to local NO conditions. This project is conceptually innovative in its focus on the connection between STPKs and TCSs in Mtb, and between Mtb environmental response and replication regulation. By laying the groundwork for revealing key connecting nodes in these understudied concepts, these studies will provide insight into facets of Mtb infection biology critical for bacterial colonization success, and open new avenues of study targeted at understanding and exploiting these vital aspects for therapeutic purposes.