The role of ClpC2 in mycobacterial stress response
Date
2025
Authors
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Journal ISSN
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Publisher
University of Delaware
Abstract
Bacteria of the pathogenic Mycobacterium genus are significant for their ability to cause disease and, indeed, to thrive in the hostile and challenging host environments. This sturdiness is especially characteristic of Mycobacterium tuberculosis (Mtb), the causative agent of human tuberculosis (TB). The resilience and ability of Mtb to withstand nutrient deprivation as well as oxidative, thermal, and proteotoxic stress are attributed to its complex stress response mechanisms, which have also been implicated in its tolerance to antibiotic treatment. (Dawan & Ahn, 2022; Moukendza Koundi et al., 2024) Moreover, the substantial global burden of TB cases tolerant to available antibiotics poses a serious public health threat globally. Consequently, efforts to develop new drugs are urgently needed to combat this public health crisis. ☐ Clp proteases, a class of ATP-dependent enzymes that plays a central role in bacterial protein homeostasis and quality control, have emerged as key regulators of bacterial pathways, including stress responses in a wide cross-section of bacterial pathogens. (Lupoli et al., 2018) Whereas Clp proteases are dispensable in many bacteria, these enzymes are absolutely essential for viability in mycobacteria, making them attractive novel drug targets against drug-resistant M. tuberculosis. (d’Andrea et al., 2022; Hoi et al., 2023; Lupoli et al., 2018; Schmitz & Sauer, 2014) ☐ Previous work in the Schmitz lab has centered around characterizing and understanding the role of ClpC1, an unfoldase component of mycobacterial Clp proteases, in substrate recognition and proteolytic regulation. ClpC1 is a particularly appealing target because it is absolutely essential for mycobacterial survival and it lacks a direct ortholog in humans. (Choules et al., 2019; Lunge et al., 2020; Schmitz & Sauer, 2014; Weinhäupl et al., 2022a) Several naturally occurring antibiotics, including rufomycin and cyclomarin A, are known to potently dysregulate ClpC1 activity and kill Mtb cells in culture, further validating ClpC1 as a viable drug target. (Choules et al., 2019; Taylor et al., 2023) However, our limited understanding of ClpC1’s physiological roles and range of substrates in M. tuberculosis complicates efforts to develop clinically viable ClpC1-targeting drugs. Recent studies in the Schmitz lab and others have expanded our understanding of ClpC1 physiology by establishing that mycobacteria post-translationally phosphorylate arginine residues of some cytosolic proteins (Ogbonna et al., 2022; Suskiewicz et al., 2019), and that these phosphoarginine modifications (pArg) are directly recognized by the N-terminal domain of ClpC1 as markers for proteolysis. (Hoi et al., 2023; Taylor et al., 2023) However, the details and regulation of arginine phosphorylation in mycobacteria remains poorly understood and is an area of active research. ☐ Interestingly, mycobacteria also possess a homolog of ClpC1, termed ClpC2, which is not known to be directly involved in proteolysis. ClpC2 bears strong sequence and structural homology to the N-terminal domain of ClpC1, sharing ~40% sequence identity. (Taylor et al., 2023). ClpC2 has been found to be a transcriptional regulator that represses transcription of the clpC2 promoter (Taylor et al., 2023). Interestingly, some ClpC1-targeting antibiotics, including rufomycin, bind to ClpC2, block its interaction with DNA, and cause strong ClpC2 overexpression. The accumulated ClpC2 serves as a molecular sponge by sequestering antibiotics from ClpC1 (Hoi et al., 2023a; Taylor et al., 2023). Recent evidence from the Schmitz lab demonstrates that ClpC2, like ClpC1, binds phosphoarginine, and that pArg similarly blocks DNA operator binding and relieves repression by ClpC2. Based on these initial studies, our overarching hypothesis is that ClpC2 functions as a pArg-sensor that responds to cellular pArg levels and regulates downstream transcriptional programs during stress. ☐ This project tests aspects of this hypothesis by addressing two major gaps in our understanding of the physiological interplay between pArg and ClpC2 in the model mycobacterium Mycolicibacterium smegmatis. In Chapter 3, we use RNA-seq to identify transcripts that are differentially expressed upon antibiotic-induced dysregulation of ClpC2 or deletion of the clpC2 coding region. Results of this experiment show that ClpC2 only regulates its own operon. Unexpectedly, these data also suggest a ClpC2-independent connection between antibiotic-induced dysregulation ClpC1 and transcriptional regulation of other genes. ☐ Overall, these studies provide new information on the behavior of ClpC2 and expand our understanding of the role of phosphoarginine in mycobacteria. Our findings may aid efforts to develop ClpC1-targeting therapeutics that avoid cross-targeting ClpC2. Moreover, our results provide new unexpected insight into the essential role of ClpC1 in mycobacteria.
Description
Keywords
Mycobacterial, Phosphoarginine, Stress response, Mycobacterium tuberculosis, Human tuberculosis