ABSTRACT Antibiotics are a cornerstone of modern medicine, but antibiotic resistance threatens the effectiveness of our existing antibacterial arsenal. This phenomenon poses an acute challenge with Mycobacterium tuberculosis, a global pathogen that causes greater mortality than any other bacterium. Nearly half a million drug-resistant tuberculosis infections occurred in 2021, driving an urgent clinical need to develop novel drugs against M. tuberculosis and to rigorously characterize new molecular targets. One promising class of targets are the mycobacterial Clp proteases, which carry out regulated degradation of cytosolic proteins. Although Clp proteases are known to be essential, their cellular roles and the reasons for their essentiality are poorly understood, which in turn constrains efforts to develop Clp-targeting therapeutics. This proposal investigates the connection between arginine phosphorylation and the mycobacterial ClpC1P1P2 Clp protease. In the distantly related species Bacillus subtilis, phosphoarginine (pArg) modifications occur on diverse proteins during proteotoxic stress and are recognized by the ClpCP protease as a degradation signal. We recently reported that pArg modifications also occur in mycobacteria, although the specific conditions that stimulate pArg installation appear to be different than in B. subtilis. Preliminary studies confirm that pArg-bearing proteins are proteolyzed by ClpC1P1P2. Additionally, we find that pArg binds ClpC2, a protein that contributes to antibiotic persistence. We hypothesize that mycobacteria install pArg in response to stress or starvation; that pArg directs ClpC1P1P2 activity and modulates its assembly state; and that ClpC2 is a pArg-sensitive regulator of dormancy and persistence. This proposal seeks to test this overarching hypothesis and interrogate key biochemical aspects of pArg recognition. In Aim 1, we use phosphoproteomics to identify stress conditions that elevate pArg, assess the requirement of pArg on mycobacterial stress tolerance, and identify mycobacterial arginine kinases and phosphatases. In Aim 2, we use in vitro biochemical approaches to test the influence of pArg number and substrate stability on proteolysis by ClpC1, and determine the influence of pArg binding on the ClpC1 activity state. In Aim 3 we assess how pArg-dependent changes in ClpC2 oligomerization influence its function. The outcomes of this proposal will include an expanded understanding of the roles of pArg in mycobacteria, new mechanistic details of how pArg-bearing proteins interact with Clp proteases, and elucidation of the role of ClpC2 in pArg-sensing and persistence. These studies will lay the groundwork for future efforts to test the connection between pArg and M. tuberculosis infection. Moreover, a detailed understanding of pArg-linked proteolytic pathways will improve our ability to screen for new ClpC1-targeting therapeutic leads.

PROJECT NARRATIVE Tuberculosis is a bacterial infectious disease that causes more global death than any other bacterial infection. This proposal investigates how tuberculosis-causing bacteria use phosphoarginine protein modifications to survive stress conditions. Our findings will reveal new information on the biology of these important bacterial pathogens, and will support efforts to develop new antibiotics to combat drug-resistant tuberculosis infections.