The unfoldase ClpC1 of Mycobacterium tuberculosis regulates the expression of a distinct subset of proteins having intrinsically disordered termini

Abstract

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.

Keywords: ATPase; ClpC1 unfoldase; Hsp20; Mycobacterium tuberculosis; caseinolytic protease; heat shock protein (HSP); intrinsically disordered region; protein degradation; proteolysis; tuberculosis.

Figure 1.

ClpC1 is essential for mycobacterial growth. A and B, validation of CRISPRi-mediated silencing of genes by quantitative RT-PCR. Quantitative RT-PCR analysis showing levels of clpC1 (A) and clpP2 (B) transcripts in the respective knockdown strains of Mtb H₃₇Rv. C–E, in vitro growth analysis of different strains of Mtb H₃₇Rv. Growth of clpC1(−) and clpP2(−) strains was monitored relative to control in the presence of ATc (50 ng/ml) by measuring A₆₀₀ of bacterial cultures (C) or bacterial CFUs (D and E) at regular intervals. Inset in C shows visual representation of growth defect after depletion of clpC1 and clpP2 respectively, compared with control. Mean values from three repeat experiments ± S.D. are shown. The p values were determined by Student's t test.

Figure 2.

Intracellular survival analysis in THP1-derived human macrophages. Intracellular survival of Mtb H₃₇Rv depleted for clpC1 (A) or clpP2 (B) in THP1-derived macrophages was analyzed by estimation of CFU at successive time points after infection. Infection was performed with ATc-pretreated bacteria at 1:2 MOI for 4 h followed by washing to remove extracellular bacteria. Infected cells were maintained in the presence of 200 ng/ml ATc to suppress the expression of respective genes. Mean values from three repeat experiments ± S.D. are shown. The p values were determined by Student's t test.

Figure 3.

Comparative analysis of differentially accumulated proteins in Mtb H₃₇Rv depleted with clpC1 and clpP2. A, schematic of the work-flow for iTRAQ analysis. Lysates were prepared from respective knockdown strains after 4 days of depletion with 50 ng/ml ATc, which was subsequently labeled with four-plex iTRAQ labels followed by LC–MS analysis, as described in the text. B and C, approximately 50% of total Mtb proteins (n = 1827) were common in the two different iTRAQ experiments (B), which comprise of 219 proteins showing differential abundance (by ≥1.5-fold) after depletion of both clpC1 and clpP2 (C). D, functional categorization of ClpC1P1P2-regulatory proteins in Mtb. Shown is the percentage of distribution of various accumulated substrates under different functional categories. Functional classification was performed using the Mycobrowser database (RRID:SCR_018242) of proteins accumulated in clpC1(−) and clpP2(−) strains.

Figure 4.

Analysis of DPRs in ClpC1P1P2-regulated proteins. A, shown is the frequency of DPRs in proteins accumulated in clpC1(−) and clpP2(−) strains. The 219 proteins differentially accumulated in clpC1(−) and clpP2(−) strains were further categorized into 186 proteins enriched with ≥50% DPRs at the terminal 15-aa region. Moreover, 127 of these contain ≤20% ORs, indicating the possibility of unstructured region at the termini. B, list of proteins accumulated in clpC1(−) and clp2(−) strains, that exhibit ≥50% intrinsic disordered region and ≤20% OR at both the N and the C termini.

Figure 5.

Dosage sensitivity of Hsp20 is mediated by ClpC1P1P2 via recognition of its C terminus. A, expression analysis of hsp20. Comparative analysis of hsp20 transcripts and its protein levels in the individual knockdown strains of Mtb H₃₇Rv reveals relatively significant accumulation of Hsp20 protein in the clpC1(−) and clpP2(−) compared with its expression in the control strain. Transcripts were quantitated by real-time PCR, whereas protein levels were obtained from iTRAQ-based LC–MS studies. B, expression analysis of Hsp20 by immunoblotting. Assessment of Hsp20 expression levels in control and different knockdown strains of Mtb H₃₇Rv by anti-Hsp20 immunoblotting further corroborates its post-transcriptional regulation by ClpC1 and ClpP2 (middle). The upper portion of the blot was cut and probed with anti-YidC, which served as control (top); levels of YidC remain constant, indicating equal loading of samples. Comparable loading of samples was also confirmed by Ponceau S staining of the blot (bottom). C, conformational analysis of purified Hsp20. Evaluation of purified Hsp20 by Coomassie Brilliant Blue–stained denatured polyacrylamide gel shows multiple protein bands migrating at higher molecular masses, indicating polydisperse conformation, which was also confirmed by size-exclusion chromatography, as well as by anti-Hsp20 immunoblotting of different fractions. D, homology modeling of Mtb Hsp20. Homology modeling by SWISS-MODEL reveals dodecameric conformation with a distinct solvent-exposed floppy region at the C terminus (see inset). E and F, in vivo expression analysis of Hsp20. Despite showing significant overexpression of mRNA transcripts following ATc treatment, N-ter cMyc-tagged Hsp20 (cMyc-Hsp20) with a free C terminus fails to show expression by anti-cMyc immunoblotting in WT M. smegmatis (E). Contrary to this, its derivative with C-ter cMyc tag (Hsp20-cMyc) exhibits remarkable overexpression upon incubation with ATc (F). The upper portions of the same blots in E and F were probed with anti-PknB antibody as control for validating equal loading of samples. The values in graphs represent the means ± S.D. from multiple experiments. The p value was determined by Student's t test.

Figure 6.

Role of C-terminal region of Hsp20 on its regulation by ClpC1. A, expression analysis of full-length and truncated Hsp20. Although full-length protein is expressed only in the clpP2(−) strain and not in the WT bacteria, deletion of the terminal 10 aa from the C terminus restores expression of cMyc-Hsp20 in the WT cells similar to its level in the clpP2(−) strain. B–D, association of Hsp20 with ClpC1 requires C-terminal sequence. Kinetics of ClpC1 association with full-length dodecamer (B) and C-terminal truncated dimer (C) or dodecamer (D) Hsp20 by BLI–Octet indicates the requirement of C-terminal sequence in the recognition of Hsp20 by ClpC1. The results are representative of three independent experiments.

Figure 7.

Schematic representation of a model depicting critical requirement of terminal disordered region for recognition of substrate by ClpC1. A and B, terminal disordered region serves as recognition signal for effective engagement, unfolding, and degradation of the prospective ClpC1 substrate. A, the solvent-exposed C-terminal residues in Hsp20, a model substrate of ClpC1, provide a unique conformation that facilitates engagement of Hsp20 with the N-terminal region of ClpC1 followed by unfolding and introduction to the protease chamber. B, in the absence of the C-ter tail, the Hsp20 is unable to interact with ClpC1, which prevents its proteolysis by Clp machinery.