Clp-targeting BacPROTACs impair mycobacterial proteostasis and survival.

The ClpC1:ClpP1P2 protease is a core component of the proteostasis system in mycobacteria. To improve the efficacy of antitubercular agents targeting the Clp protease, we characterized the mechanism of the antibiotics cyclomarin A and ecumicin. Quantitative proteomics revealed that the antibiotics cause massive proteome imbalances, including upregulation of two unannotated yet conserved stress response factors, ClpC2 and ClpC3. These proteins likely protect the Clp protease from excessive amounts of misfolded proteins or from cyclomarin A, which we show to mimic damaged proteins. To overcome the Clp security system, we developed a BacPROTAC that induces degradation of ClpC1 together with its ClpC2 caretaker. The dual Clp degrader, built from linked cyclomarin A heads, was highly efficient in killing pathogenic Mycobacterium tuberculosis, with >100-fold increased potency over the parent antibiotic. Together, our data reveal Clp scavenger proteins as important proteostasis safeguards and highlight the potential of BacPROTACs as future antibiotics.

Metabolites from Streptomyces aureus (VTCC43181) and Their Inhibition of Mycobacterium tuberculosis ClpC1 Protein.

Tuberculosis is one of the most common infectious diseases in the world, caused by Mycobacterium tuberculosis. The outbreak of multiple drug-resistant tuberculosis has become a major challenge to prevent this disease worldwide. ClpC1 is a Clp ATPase protein of Mycobacterium tuberculosis, functioning as a chaperon when combined with the Clp complex. ClpC1 has emerged as a new target to discover anti-tuberculosis drugs. This study aimed to explore the ClpC1 inhibitors from actinomycetes, which have been known to provide abundant sources of antibiotics. Two cyclic peptides, including nocardamin (1), halolitoralin A (3), and a lactone pleurone (2), were isolated from the culture of Streptomyces aureus (VTCC43181). The structures of these compounds were determined based on the detailed analysis of their spectral data and comparison with references. This is the first time these compounds have been isolated from S. aureus. Compounds 1-3 were evaluated for their affection of ATPase activity of the recombinant ClpC1 protein. Of these compounds, halolitoralin A (1), a macrocyclic peptide, was effective for the ATPase hydrolysis of the ClpC1 protein.

Antibacterial peptide CyclomarinA creates toxicity by deregulating the Mycobacterium tuberculosis ClpC1-ClpP1P2 protease.

The ring-forming AAA+ hexamer ClpC1 associates with the peptidase ClpP1P2 to form a central ATP-driven protease in Mycobacterium tuberculosis (Mtb). ClpC1 is essential for Mtb viability and has been identified as the target of antibacterial peptides like CyclomarinA (CymA) that exhibit strong toxicity toward Mtb. The mechanistic actions of these drugs are poorly understood. Here, we dissected how ClpC1 activity is controlled and how this control is deregulated by CymA. We show that ClpC1 exists in diverse activity states correlating with its assembly. The basal activity of ClpC1 is low, as it predominantly exists in an inactive nonhexameric resting state. We show that CymA stimulates ClpC1 activity by promoting formation of supercomplexes composed of multiple ClpC1 hexameric rings, enhancing ClpC1-ClpP1P2 degradation activity toward various substrates. Both the ClpC1 resting state and the CymA-induced alternative assembly state rely on interactions between the ClpC1 coiled-coil middle domains (MDs). Accordingly, we found that mutation of the conserved aromatic F444 residue located at the MD tip blocks MD interactions and prevents assembly into higher order complexes, thereby leading to constitutive ClpC1 hexamer formation. We demonstrate that this assembly state exhibits the highest ATPase and proteolytic activities, yet its heterologous expression in Escherichia coli is toxic, indicating that the formation of such a state must be tightly controlled. Taken together, these findings define the basis of control of ClpC1 activity and show how ClpC1 overactivation by an antibacterial drug generates toxicity.

Pneumococcal proteins ClpC and UvrC as novel host plasminogen binding factors.

Two plasminogen binding proteins were identified from a mouse infected with Streptococcus pneumoniae. The pneumococcal proteins were annotated as ATP-dependent Clp protease ATP-binding subunit (ClpC) and excinuclease ABC subunit C (UvrC) using the isobaric tags for relative and absolute quantification (iTRAQ) method. Recombinants of both proteins showed significant binding to plasminogen and were found to promote plasminogen activation by tissue-type plasminogen activator. In addition, ClpC and UvrC were LytA-dependently released into the culture supernatant and bound to the bacterial surface. These results suggest that S. pneumoniae releases ClpC and UvrC by autolysis and recruits them to the bacterial surface, where they bind to plasminogen and promote its activation, contributing to extracellular matrix degradation and tissue invasion.

Total Synthesis and Biological Evaluation of Modified Ilamycin Derivatives.

Ilamycins/rufomycins are marine cycloheptapeptides containing unusual amino acids. Produced by Streptomyces sp., these compounds show potent activity against a range of mycobacteria, including multidrug-resistant strains of Mycobacterium tuberculosis. The cyclic peptides target the AAA+ protein ClpC1 that, together with the peptidases ClpP1/ClpP2, forms an essential ATP-driven protease. Derivatives of the ilamycins with a simplified tryptophane unit are synthesized in a straightforward manner. The ilamycin derivative 26 with a cyclic hemiaminal structure is active in the nM-range against several mycobacterial strains and shows no significant cytotoxicity. In contrast, derivative 27, with a glutamic acid at this position, is significantly less active, with MICs in the mid µM-range. Detailed investigations of the mode of action of 26 indicate that 26 deregulates ClpC1 activity and strongly enhances ClpC1-WT ATPase activity. The consequences of 26 on ClpC1 proteolytic activities were substrate-specific, suggesting dual effects of 26 on ClpC1-WT function. The positive effect relates to ClpC1-WT ATPase activation, and the negative to competition with substrates for binding to the ClpC1 NTD.

Isolation and molecular characterization of Polychromophilus spp. (Haemosporida: Plasmodiidae) from the Asian long-fingered bat (Miniopterus fuliginosus) and Japanese large-footed bat (Myotis macrodactylus) in Japan.

Bats (order, Chiroptera) account for more than one-fifth of all mammalian species in the world and are infected by various intra-erythrocytic parasites of the family Plasmodiidae (Apicomplexa: Haemosporida), including Polychromophilus Dionisi, 1899. Recent advance in the molecular characterization of haemosporidian isolates has enabled their accurate identification, particularly in the last decade. Studies are actively conducted in tropical regions, Europe, and Australia; however, data on haemosporidian infection in bats in Asian temperate areas, including Japan, remain limited. In this study, 75 bats of 4 species (Miniopterus fuliginosus, Myotis macrodactylus, Rhinolophus nippon, and Rhinolophus cornutus) were captured at three sites in western Japan (Yamaguchi Prefecture), and haemosporidian parasites were screened microscopically and molecularly via nested polymerase chain reaction (PCR) targeting the cytochrome b (cytb), cytochrome c oxidase subunit I (cox-1), apicoplast caseinolytic protease C (clpc), and nuclear elongation factor 2 (EF2) genes. The survey detected Polychromophilus melanipherus in 15 (40.5%) miniopterid bats (M. fuliginosus) and Polychromophilus murinus in 6 (46.2%) vespertilionid bats (M. macrodactylus), whereas none of the 25 rhinolophid bats (R. nippon and R. cornutus) was infected, indicating the robust host specificity for miniopterid (P. melanipherus) and vespertilionid (P. murinus) bats regardless of orthotopic nesting. The 15 Polychromophilus cytb sequences obtained from 11 miniopterid and 4 vespertilionid bats were classified into six cytb haplotypes (three for each species), showing no region-specific variation in a phylogenetic tree of Polychromophilus isolates in the Old World. Similarly, multiple haplotypes (seven for cox-1 and nine for clpc) and genotypes (three for EF2) were characterized for the Japanese isolates of Polychromophilus, and the results were consistent with those based on a haemosporidian cytb analysis. Bat flies (Nycteribia allotopa and another undetermined Nycteribia sp.) collected from the body surface of bats harbored Polychromophilus oocysts on the external surface of the midgut. This is the first study to report the isolation and molecular characterization of Polychromophilus spp. in miniopterid and vespertilionid bats in the temperate area of Asia (western Japan). Future studies should evaluate the global prevalence of haemosporidian infections in bats.

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

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.

Overcoming the Challenges of Pyrazinamide Susceptibility Testing in Clinical Mycobacterium tuberculosis Isolates.

Pyrazinamide (PZA) is one of the first-line agents used for the treatment of tuberculosis. However, current phenotypic PZA susceptibility testing in the Bactec MGIT 960 system is unreliable, and false resistance is well documented. Rapid identification of resistance-associated mutations can confirm the phenotypic result. This study aimed to investigate the use of genotypic methods in combination with phenotypic susceptibility testing for confirmation of PZA-resistant Mycobacterium tuberculosis isolates. Sanger sequencing and/or whole-genome sequencing were performed to detect mutations in pncA, rpsA, panD, and clpC1. Isolates were screened for heteroresistance, and PZA susceptibility testing was performed using the Bactec MGIT 960 system using a reduced inoculum to investigate false resistance. Overall, 40 phenotypically PZA-resistant isolates were identified. Of these, PZA resistance was confirmed in 22/40 (55%) isolates by detecting mutations in the pncA, rpsA, and panD genes. Of the 40 isolates, 16 (40%) were found to be susceptible using the reduced inoculum method (i.e., false resistance). No mutations were detected in two PZA-resistant isolates. False resistance was observed in isolates with MICs close to the critical concentration. In particular, East African Indian strains (lineage 1) appeared to have an elevated MIC that is close to the critical concentration. While this study illustrates the complexity and challenges associated with PZA susceptibility testing of M. tuberculosis, we conclude that a combination of genotypic and phenotypic drug susceptibility testing methods is required for accurate detection of PZA resistance.

Atypical Genetic Basis of Pyrazinamide Resistance in Monoresistant Mycobacterium tuberculosis.

Pyrazinamide (PZA) is a widely used antitubercular chemotherapeutic. Typically, PZA resistance (PZA-R) emerges in Mycobacterium tuberculosis strains with existing resistance to isoniazid and rifampin (i.e., multidrug resistance [MDR]) and is conferred by loss-of-function pncA mutations that inhibit conversion to its active form, pyrazinoic acid (POA). PZA-R departing from this canonical scenario is poorly understood. Here, we genotyped pncA and purported alternative PZA-R genes (panD, rpsA, and clpC1) with long-read sequencing of 19 phenotypically PZA-monoresistant isolates collected in Sweden and compared their phylogenetic and genomic characteristics to a large set of MDR PZA-R (MDRPZA-R) isolates. We report the first association of ClpC1 mutations with PZA-R in clinical isolates, in the ClpC1 promoter (clpC1p-138) and the N terminus of ClpC1 (ClpC1Val63Ala). Mutations have emerged in both these regions under POA selection in vitro, and the N-terminal region of ClpC1 has been implicated further, through its POA-dependent efficacy in PanD proteolysis. ClpC1Val63Ala mutants spanned 4 Indo-Oceanic sublineages. Indo-Oceanic isolates invariably harbored ClpC1Val63Ala and were starkly overrepresented (odds ratio [OR] = 22.2, P < 0.00001) among PZA-monoresistant isolates (11/19) compared to MDRPZA-R isolates (5/80). The genetic basis of Indo-Oceanic isolates' overrepresentation in PZA-monoresistant tuberculosis (TB) remains undetermined, but substantial circumstantial evidence suggests that ClpC1Val63Ala confers low-level PZA resistance. Our findings highlight ClpC1 as potentially clinically relevant for PZA-R and reinforce the importance of genetic background in the trajectory of resistance development.

The apicoplast of Haemoproteus columbae: A comparative study of this organelle genome in Haemosporida.

Apicomplexa is a phylum of parasitic protozoa; among them are the order Haemosporida, vector-borne parasites that include those that cause malaria (genus Plasmodium). Most Apicomplexa species have a non-photosynthetic plastid or apicoplast. Given its unique metabolic pathways, this organelle is considered a target for malaria therapeutics. Regardless of its importance, there is a paucity of complete apicoplast genome data hindering comparative studies. Here, the Haemoproteus (Haemoproteus) columbae apicoplast genome (lineage HAECOL1) was obtained using next-generation sequencing. This genome was included in a comparative analysis with other plastids. This 29.8 kb circular genome shares the same structure found in Plasmodium parasites. It is A + T rich (87.7%), comparable but at the higher end of A + T content observed in Plasmodium species (85.5-87.2%). As expected, considering its high A + T content, the synonymous codon usage (RSCU) and the effective number of codons (ENc) showed a moderate codon bias. Several apicoplast genes have a phylogenetic signal. However, unlike mitochondrial genes, single-gene phylogenies have low support in haemosporidian clades that diverged recently. The H. columbae apicoplast genome suggests that the apicoplast function may be conserved across Haemosporida. This parasite could be a model to study this organelle in a non-mammalian system.

ClpC-Mediated Sporulation Regulation at Engulfment Stage in Bacillus anthracis.

Bacterial sporulation is a conserved process utilized by members of Bacillus genus and Clostridium in response to stress such as nutrient or temperature. Sporulation initiation is triggered by stress signals perceived by bacterial cell that leads to shutdown of metabolic pathways of bacterial cells. The mechanism of sporulation involves a complex network that is regulated at various checkpoints to form the viable bacterial spore. Engulfment is one such check point that drives the required cellular rearrangement necessary for the spore assembly and is mediated by bacterial proteolytic machinery that involves association of various Clp ATPases and ClpP protease. The present study highlights the importance of degradation of an anti-sigma factor F, SpoIIAB by ClpCP proteolytic machinery playing a crucial role in culmination of engulfment process during the sporulation in Bacillus anthracis.

The ClpCP Complex Modulates Respiratory Metabolism in Staphylococcus aureus and Is Regulated in a SrrAB-Dependent Manner.

The staphylococcal respiratory regulator (SrrAB) modulates energy metabolism in Staphylococcus aureus Studies have suggested that regulated protein catabolism facilitates energy homeostasis. Regulated proteolysis in S. aureus is achieved through protein complexes composed of a peptidase (ClpQ or ClpP) in association with an AAA+ family ATPase (typically, ClpC or ClpX). In the present report, we tested the hypothesis that SrrAB regulates a Clp complex to facilitate energy homeostasis in S. aureus Strains deficient in one or more Clp complexes were attenuated for growth in the presence of puromycin, which causes enrichment of misfolded proteins. A ΔsrrAB strain had increased sensitivity to puromycin. Epistasis experiments suggested that the puromycin sensitivity phenotype of the ΔsrrAB strain was a result of decreased ClpC activity. Consistent with this, transcriptional activity of clpC was decreased in the ΔsrrAB mutant, and overexpression of clpC suppressed the puromycin sensitivity of the ΔsrrAB strain. We also found that ClpC positively influenced respiration and that it did so upon association with ClpP. In contrast, ClpC limited fermentative growth, while ClpP was required for optimal fermentative growth. Metabolomics studies demonstrated that intracellular metabolic profiles of the ΔclpC and ΔsrrAB mutants were distinct from those of the wild-type strain, supporting the notion that both ClpC and SrrAB affect central metabolism. We propose a model wherein SrrAB regulates energy homeostasis, in part, via modulation of regulated proteolysis.IMPORTANCE Oxygen is used as a substrate to derive energy by the bacterial pathogen Staphylococcus aureus during infection; however, S. aureus can also grow fermentatively in the absence of oxygen. To successfully cause infection, S. aureus must tailor its metabolism to take advantage of respiratory activity. Different proteins are required for growth in the presence or absence of oxygen; therefore, when cells transition between these conditions, several proteins would be expected to become unnecessary. In this report, we show that regulated proteolysis is used to modulate energy metabolism in S. aureus We report that the ClpCP protein complex is involved in specifically modulating aerobic respiratory growth but is dispensable for fermentative growth.

Rufomycin Targets ClpC1 Proteolysis in Mycobacterium tuberculosis and M. abscessus.

ClpC1 is an emerging new target for the treatment of Mycobacterium tuberculosis infections, and several cyclic peptides (ecumicin, cyclomarin A, and lassomycin) are known to act on this target. This study identified another group of peptides, the rufomycins (RUFs), as bactericidal to M. tuberculosis through the inhibition of ClpC1 and subsequent modulation of protein degradation of intracellular proteins. Rufomycin I (RUFI) was found to be a potent and selective lead compound for both M. tuberculosis (MIC, 0.02 µM) and Mycobacterium abscessus (MIC, 0.4 µM). Spontaneously generated mutants resistant to RUFI involved seven unique single nucleotide polymorphism (SNP) mutations at three distinct codons within the N-terminal domain of clpC1 (V13, H77, and F80). RUFI also significantly decreased the proteolytic capabilities of the ClpC1/P1/P2 complex to degrade casein, while having no significant effect on the ATPase activity of ClpC1. This represents a marked difference from ecumicin, which inhibits ClpC1 proteolysis but stimulates the ATPase activity, thereby providing evidence that although these peptides share ClpC1 as a macromolecular target, their downstream effects are distinct, likely due to differences in binding.

Development of high throughput screening methods for inhibitors of ClpC1P1P2 from Mycobacteria tuberculosis.

Tuberculosis affects about 100 million people worldwide and causes nearly 2 million deaths annually. It has been estimated that one third of all humans is infected with latent Mycobacterium tuberculosis (Mtb). Moreover, Mtb has become increasingly resistant to available antibiotics. Consequently, it is important to identify and characterize new therapeutic targets in Mtb and to synthesize selective inhibitors. ClpP1, ClpP2 and their associated regulatory ATPases, ClpX and ClpC1 are required for the growth of Mtb and for its virulence during murine infection and are highly attractive drug targets, especially since they are not present in the cytosol of mammalian cells, and they differ markedly from the mitochondrial ClpP complex. The importance of these proteins in Mtb is emphasized by the existence of several natural antibiotics targeting this system. In order to find new inhibitors of ClpC1P1P2 system, we developed an assay based on the ATP-dependent degradation of a fluorescent protein substrate. The hits obtained were further characterized with a set of secondary assays to identify precise targets within a complex. A large library of compounds was screened and led to the identification of a ClpC1 ATPase inhibitor demonstrating that this approach can be used in future searches for anti-TB agents.

Co-suppression of NbClpC1 and NbClpC2 alters plant morphology with changed hormone levels in Nicotiana benthamiana.

KEY MESSAGE: Co-suppression of chaperonic ClpC1 and ClpC2 in Nicotiana benthamiana significantly affect the development and exogenous application of gibberellin partially rescue the developmental defects. Over the past decade, the Clp protease complex has been identified as being implicated in plastid protein quality control in plant cells. CLPC1 and CLPC2 proteins form the chaperone subunits of the Clp protease complex and unfold protein substrates to thread them into the ClpP complex. Here, using the technique of virus-induced gene silencing (VIGS), we suppressed both Nicotiana benthamiana ClpC1 and ClpC2 (NbClpC1/C2) functioning as chaperone subunits in the protease complex. Co-suppression of NbClpC1/C2 caused chlorosis and retarded-growth phenotype with no seed formation and significantly reduced root length. We found that co-suppression of NbClpC1/C2 also affected stomata and trichome formation and vascular bundle differentiation and patterning. Analysis of phytohormones revealed significant alteration and imbalance of major hormones in the leaves of NbClpC1/C2 co-suppressed plant. We also found that application of gibberellin (GA3) partially rescued the developmental defects. Co-suppression of NbClpC1/C2 significantly affected the development of N. benthamiana and exogenous application of GA3 partially rescued the developmental defects. Overall, our findings demonstrate that CLPC1 and CLPC2 proteins have a pivotal role in plant growth and development.

Phylogenetic analysis predicts structural divergence for proteobacterial ClpC proteins.

Regulated proteolysis is required in all organisms for the removal of misfolded or degradation-tagged protein substrates in cellular quality control pathways. The molecular machines that catalyze this process are known as ATP-dependent proteases with examples that include ClpAP and ClpCP. Clp/Hsp100 subunits form ring-structures that couple the energy of ATP binding and hydrolysis to protein unfolding and subsequent translocation of denatured protein into the compartmentalized ClpP protease for degradation. Copies of the clpA, clpC, clpE, clpK, and clpL genes are present in all characterized bacteria and their gene products are highly conserved in structure and function. However, the evolutionary relationship between these proteins remains unclear. Here we report a comprehensive phylogenetic analysis that suggests divergent evolution yielded ClpA from an ancestral ClpC protein and that ClpE/ClpL represent intermediates between ClpA/ClpC. This analysis also identifies a group of proteobacterial ClpC proteins that are likely not functional in regulated proteolysis. Our results strongly suggest that bacterial ClpC proteins should not be assumed to all function identically due to the structural differences identified here.

The caseinolytic protease complex component CLPC1 in Arabidopsis maintains proteome and RNA homeostasis in chloroplasts.

BACKGROUND: Homeostasis of the proteome is critical to the development of chloroplasts and also affects the expression of certain nuclear genes. CLPC1 facilitates the translocation of chloroplast pre-proteins and mediates protein degradation. RESULTS: We found that proteins involved in photosynthesis are dramatically decreased in their abundance in the clpc1 mutant, whereas many proteins involved in chloroplast transcription and translation were increased in the mutant. Expression of the full-length CLPC1 protein, but not of the N-terminus-deleted CLPC1 (ΔN), in the clpc1 mutant background restored the normal levels of most of these proteins. Interestingly, the ΔN complementation line could also restore some proteins affected by the mutation to normal levels. We also found that that the clpc1 mutation profoundly affects transcript levels of chloroplast genes. Sense transcripts of many chloroplast genes are up-regulated in the clpc1 mutant. The level of SVR7, a PPR protein, was affected by the clpc1 mutation. We showed that SVR7 might be a target of CLPC1 as CLPC1-SVR7 interaction was detected through co-immunoprecipitation. CONCLUSION: Our study indicates that in addition to its role in maintaining proteome homeostasis, CLPC1 and likely the CLP proteasome complex also play a role in transcriptome homeostasis through its functions in maintaining proteome homeostasis.

Mycobacterium tuberculosis ClpC1 N-Terminal Domain Is Dispensable for Adaptor Protein-Dependent Allosteric Regulation.

ClpC1 hexamers couple the energy of ATP hydrolysis to unfold and, subsequently, translocate specific protein substrates into the associated ClpP protease. Substrate recognition by ATPases associated with various cellular activities (AAA+) proteases is driven by the ATPase component, which selectively determines protein substrates to be degraded. The specificity of these unfoldases for protein substrates is often controlled by an adaptor protein with examples that include MecA regulation of Bacillus subtilis ClpC or ClpS-mediated control of Escherichia coli ClpA. No adaptor protein-mediated control has been reported for mycobacterial ClpC1. Using pulldown and stopped-flow fluorescence methods, we report data demonstrating that Mycobacterium tuberculosis ClpC1 catalyzed unfolding of an SsrA-tagged protein is negatively impacted by association with the ClpS adaptor protein. Our data indicate that ClpS-dependent inhibition of ClpC1 catalyzed SsrA-dependent protein unfolding does not require the ClpC1 N-terminal domain but instead requires the presence of an interaction surface located in the ClpC1 Middle Domain. Taken together, our results demonstrate for the first time that mycobacterial ClpC1 is subject to adaptor protein-mediated regulation in vitro.

Missense Mutations in the Unfoldase ClpC1 of the Caseinolytic Protease Complex Are Associated with Pyrazinamide Resistance in Mycobacterium tuberculosis.

Previously, we showed that mutations in Mycobacterium tuberculosis panD, involved in coenzyme A biosynthesis, cause resistance against pyrazinoic acid, the bioactive component of the prodrug pyrazinamide. To identify additional resistance mechanisms, we isolated mutants resistant against pyrazinoic acid and subjected panD wild-type strains to whole-genome sequencing. Eight of the nine resistant strains harbored missense mutations in the unfoldase ClpC1 associated with the caseinolytic protease complex.

Anti-tuberculosis lead molecules from natural products targeting Mycobacterium tuberculosis ClpC1.

Tuberculosis (TB) is a serious and potentially fatal disease caused by Mycobacterium tuberculosis (M. tb). The occurrence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) M. tb is a significant public health concern because most of the anti-TB drugs that have been in use for over 40 years are no longer effective for the treatment of these infections. Recently, new anti-TB lead compounds such as cyclomarin A, lassomycin, and ecumicin, which are cyclic peptides from actinomycetes, have shown potent anti-TB activity against MDR and XDR M. tb as well as drug-susceptible M. tb in vitro. The target molecule of these antibiotics is ClpC1, a protein that is essential for the growth of M. tb. In this review, we introduce the three anti-TB lead compounds as potential anti-TB therapeutic agents targeting ClpC1 and compare them with the existing anti-TB drugs approved by the US Food and Drug Administration.