The human pathogen Mycobacterium tuberculosis (Mtb) harbors a well-orchestrated Clp (caseinolytic protease) proteolytic machinery consisting of two oligomeric segments, a barrel-shaped heterotetradecameric protease core comprising the ClpP1 and ClpP2 subunits, and hexameric ring-like ATP-dependent unfoldases composed of ClpX or ClpC1. The roles of the ClpP1P2 protease subunits are well-established in Mtb, but the potential roles of the associated unfoldases, such as ClpC1, remain elusive. Using a CRISPR interference–mediated gene silencing approach, here we demonstrate that clpC1 is indispensable for the extracellular growth of Mtb and for its survival in macrophages. The results from isobaric tags for relative and absolute quantitation–based quantitative proteomic experiments with clpC1- and clpP2-depleted Mtb cells suggested that the ClpC1P1P2 complex critically maintains the homeostasis of various growth-essential proteins in Mtb, several of which contain intrinsically disordered regions at their termini. We show that the Clp machinery regulates dosage-sensitive proteins such as the small heat shock protein Hsp20, which exists in a dodecameric conformation. Further, we observed that Hsp20 is poorly expressed in WT Mtb and that its expression is greatly induced upon depletion of clpC1 or clpP2. Remarkably, high Hsp20 protein levels were detected in the clpC1(−) or clpP2(−) knockdown strains but not in the parental bacteria, despite significant induction of hsp20 transcripts. In summary, the cellular levels of oligomeric proteins such as Hsp20 are maintained post-translationally through their recognition, disassembly, and degradation by ClpC1, which requires disordered ends in its protein substrates.

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Persisters are a subpopulation of bacteria that resist killing by antibiotics, even though they are genetically similar to their drug-susceptible counterpart. Like in several other bacteria, persisters are also reported in the human pathogen Mycobacterium tuberculosis (Mtb). Stochastic formation of Mtb persisters with a high level of antimicrobial tolerance set the stage for subsequent multidrug-resistant mutations. Despite significant advancement in our understanding, much remains to be learnt about the biology of this drug-recalcitrant bacterial subpopulation. Most of the information pertaining to the metabolic evolution required for emergence of drug tolerance in tuberculosis (TB) pathogens has come from transcriptional, metabolomic, and mutagenesis studies. Since proteins are the key functional molecules regulating the majority of metabolic activities in the cell, investigation of the whole-cell protein expression profile will further provide valuable insights into the physiology of Mtb persisters. We performed a quantitative proteomic analysis of Mtb H37Rv cultured under an in vitro persistence model to identify the proteomic profile of the phenotypic drug-tolerant bacterial population. Our study reveals that proteins related to intermediary metabolism and respiration, cell-wall and cell processes, lipid metabolism, information pathways, and virulence, detoxification and adaptation functional categories are primarily modulated in the persister subpopulation. Further, we demonstrate that various surface-localized mycobacterial membrane protein large (MmpL) proteins, which exhibit a high level of expression in Mtb persisters, are crucial for the mycobacterial survival during persistent growth state. A drug-induced persister subpopulation of Mtb exhibit various differentially regulated proteins that might be critical in mitigating the antimicrobial effect of drugs and can be further explored to develop novel anti-TB agents. The peptide identifications and tandem mass spectra (MS/MS) have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD013621.