Student Theses and Dissertations
Date of Award
2024
Document Type
Thesis
Degree Name
Doctor of Philosophy (PhD)
Thesis Advisor
Jeremy M. Rock
Abstract
Despite progress over the last century, Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), remains the leading cause of death from an infectious disease worldwide. The high global burden of Mtb, alongside its protracted treatment regimen and insidious rise of multi-drug resistance, underscores the need for non-canonical drug targets and pathways to improve antitubercular chemotherapy. Mtb’s success as a pathogen is linked to its ability to infect and evade clearance by the human immune system despite a wide range of noxious host stressors. Along with transcriptional responses, the maintenance of protein homeostasis is critical to this adaptability and pathogenicity. Previous transposon insertion sequencing (TnSeq) screens, in conjunction with our laboratory’s work on mycobacterial genetic vulnerability, identified the caseinolytic protease (Clp) system, a central component of the proteostasis network, as essential and highly vulnerable in Mtb. As an attractive, emergent anti-tubercular target, a deeper mechanistic understanding of the Clp complex in vivo is instrumental in refining ongoing drug discovery efforts and improving our understanding of Clp’s role in Mtb pathogenesis. The Clp system in Mtb is comprised of a two-tiered proteolytic barrel of homoheptameric ClpP1 and ClpP2 rings that degrades protein substrates delivered by homohexameric AAA+ unfoldases, ClpX and ClpC1. In contrast to other organisms, all components of the Clp system in Mtb are highly vulnerable and essential for growth in axenic culture and mouse models of infection. Due to the technical challenges associated with studying essential genes in mycobacteria, few bona-fide substrates of the Clp system have been identified. Furthermore, because of these challenges, correlations between seminal biochemical and structural studies with in vivo function are scarce. With the development of an inducible and tunable CRISPR interference (CRISPRi) platform in our laboratory, we were uniquely positioned to systematically investigate key questions in mycobacterial Clp biology, including which processes are regulated by the Clp system and contribute to its essentiality. By complementing knockdown of endogenous clp components with CRISPRi-resistant alleles encoding biochemically functional mutations, we unexpectedly found that only the proteolytic activity of ClpP1, but not ClpP2, is essential for Clp substrate degradation, Mtb growth, and murine infection. Our observations not only support a revised model of the Mtb Clp system, where ClpP2 essentiality stems from scaffolding chaperone binding while ClpP1 provides the essential proteolytic activity of the complex, but also hold important implications for the current development of inhibitors toward this therapeutic target. Through a combination of methods including co-immunoprecipitation coupled with mass spectrometry (co-IP/MS) and in vivo substrate accumulation experiments, several new ClpC1 substrates and interactors were identified in the non-pathogenic, Mtb model organism M. smegmatis (Msmeg). RbpA, an essential transcription factor, was highly enriched and found to be regulated post-translationally by ClpC1 through recognition of an N-terminal degron sequence. Such hits contribute to the short list of documented mycobacterial Clp substrates and directly implicate the ClpC1 chaperone in transcriptional regulation. Fluorescent degradation assays successfully identified novel C-terminal ClpC1 regulated degrons in a higher throughput manner, with certain degron sequences degraded more strongly than others. We found that ClpC1 interactors span broad biological roles emphasizing the central role of this complex in mycobacterial proteostasis. To better understand the processes underlying clpC1 essentiality, a genetic interaction screen was undertaken in a hypomorphic clpC1 knockdown background. Genes were identified and validated through growth assays that were either sensitized to knockdown, termed collateral vulnerabilities, or experienced enhanced fitness, positive interactors. Among the major collateral vulnerabilities were genes involved in cell envelope biosynthetic processes, consistent with morphological defects observed by microscopy upon clp disruption. Positive interactors were diverse in function and likely have complex interactions with clpC1. A highly vulnerable gene and strong positive interactor, gatD, involved in lipid II and peptidoglycan biosynthesis, was found to be a putative novel ClpC1 substrate through recognition of a C-terminal degron. A plate suppressor screen found that suppression of the succinate dehydrogenase, sdh, or succinyl-CoA synthase, suc, operons partially rescues an otherwise lethal strong clpC1 knockdown strain, suggesting ClpC1 may be involved in cellular respiration. That repression of no single gene fully rescues the strong growth defect imparted by strong clpC1 knockdown suggests clpC1 essentiality likely stems from pleiotropic functions in the bacterium, in contrast to findings of clp essentiality in the model bacterium Caulobacter crescentus. Taken together, the results presented in this thesis deepen our understanding of the mycobacterial Clp system in vivo. By fine tuning the expression of endogenous clp genes, the functional essentiality of its composite members was studied, novel substrates and degrons were characterized, and pathways directly or indirectly regulated by the protease complex were identified. By better defining the broad roles of the Clp system in mycobacterial physiology, we hope these studies contribute to a better understanding of its function in the proteostasis network and aid in the development of improved treatments against this recalcitrant pathogen.
Recommended Citation
d'Andrea, Felipe Badke, "Inquiries into the Mycobacterial Caseinolytic Protease System" (2024). Student Theses and Dissertations. 798.
https://digitalcommons.rockefeller.edu/student_theses_and_dissertations/798
Comments
A Thesis Presented to the Faculty of The Rockefeller University in Partial Fulfillment of the Requirements for the degree of Doctor of Philosophy