Unraveling the role of an essential two-component system in Mycobacterium tuberculosis ionic signal response and host colonization

Project Summary/Abstract Mycobacterium tuberculosis (Mtb) is remarkably successful in establishing chronic infection in a host, with its ability to transcriptionally respond to its local environment a major factor in host adaptation. The complex, non-uniform ionic milieu within the host serves as a critical source of environmental cues for Mtb. Of particular

note, the encounter of acidic pH (high [H+]) and other abundant ionic signals such as chloride (Cl-), which is sensed in synergy with pH, triggers extensive transcriptional changes within Mtb that are critical for host adaptation. These expression changes are controlled by transcriptional regulators that are critical for Mtb to

mount a coordinated response for continued survival. Mtb contains >200 known or annotated transcription factors (TFs), yet the biological role of many of these TFs remain unknown. We recently identified PrrA as a TF that modulates the transcriptional response of Mtb to both acidic pH and high Cl- concentration. PrrA is an essential TF that comprises 1 of 12 two-component systems in Mtb, and

is phosphorylated not only by its cognate sensor histidine kinase PrrB, but also by multiple serine/threonine protein kinases. This suggests that PrrA may regulate response to multiple environmental signals, and indeed, our data indicate that PrrA also impacts Mtb response to nitric oxide and hypoxia. Although PrrA is essential, its

functional role in Mtb-host interactions is unknown. The proposed studies will address the following questions: (i) What is the regulon of PrrA and what is its impact on global Mtb transcriptional response to conditions reflective of that within a phagosome? (ii) What is the role of PrrA in the context of infection? Both an inducible

overexpression system and a dual inducible protein degradation/gene silencing system will be utilized to uncover the function of PrrA in Mtb infection biology. Further, the C3HeB/FeJ murine infection model, which recapitulates lesions observed in human infections, will be used in combination with the fluorescent Mtb replication reporter

(SSB-GFP) to define the possible differential impact of PrrA on Mtb growth in vivo within sublocations of a caseous necrotic lesion. These studies will provide not just insight into how PrrA enables Mtb to respond to the surrounding ionic milieu in the context of the host, but also open up new avenues for therapeutic targeting.

In addition to shedding critical light on our understanding of Mtb physiology, this study will provide me extensive training. My sponsor Dr. Shumin Tan is dedicated to teaching me the required technical skills to perform my project, as well as other vital skills such as how to effectively communicate my findings with the

scientific community. As described in my training plan, together with my co-sponsor Dr. Ralph Isberg, we have assembled a comprehensive plan with opportunities for me to improve my ability to write, present, and teach. I will also have the opportunity to collaborate with other professors to further diversify my experimental skill set.

This project will allow me to hone all the essential skills, from research conceptualization to communication of findings and teaching, necessary for me to reach my goal of becoming a professor at an academic institute.