ClpP Activity Research Articles

Mitochondrial Protease ClpP is a Target for the Anticancer Compounds ONC201 and Related Analogues.

ONC201 is a first-in-class imipridone molecule currently in clinical trials for the treatment of multiple cancers. Despite enormous clinical potential, the mechanism of action is controversial. To investigate the mechanism of ONC201 and identify compounds with improved potency, we tested a series of novel ONC201 analogues (TR compounds) for effects on cell viability and stress responses in breast and other cancer models. The TR compounds were found to be ∼50–100 times more potent at inhibiting cell proliferation and inducing the integrated stress response protein ATF4 than ONC201. Using immobilized TR compounds, we identified the human mitochondrial caseinolytic protease P (ClpP) as a specific binding protein by mass spectrometry. Affinity chromatography/drug competition assays showed that the TR compounds bound ClpP with ∼10-fold higher affinity compared to ONC201. Importantly, we found that the peptidase activity of recombinant ClpP was strongly activated by ONC201 and the TR compounds in a dose- and time-dependent manner with the TR compounds displaying a ∼10–100 fold increase in potency over ONC201. Finally, siRNA knockdown of ClpP in SUM159 cells reduced the response to ONC201 and the TR compounds, including induction of CHOP, loss of the mitochondrial proteins (TFAM, TUFM), and the cytostatic effects of these compounds. Thus, we report that ClpP directly binds ONC201 and the related TR compounds and is an important biological target for this class of molecules. Moreover, these studies provide, for the first time, a biochemical basis for the difference in efficacy between ONC201 and the TR compounds.

Molecular Basis of ClpP Protease Activation by Small Molecules

Bacterial ClpP is a highly conserved, self‐compartmentalizing, cylindrical serine protease required for maintaining cellular proteostasis. Acyldepsipeptides (ADEPs) and activators of self‐compartmentalized proteases (ACPs) are small molecules that cause the dysregulation and activation of ClpP, which leads to bacterial cell death, highlighting their potential use as novel antibiotics. However, the molecular basis of ClpP activation by small molecules remains undetermined. Structural changes in ClpP from Neisseria meningitidis and Escherichia coli upon binding to novel ACP and ADEP analogs were probed by X‐ray crystallography, methyl‐TROSY NMR, hydrogen‐deuterium exchange, and small angle X‐ray scattering. ACP and ADEP were found to induce distinct conformational changes in the tertiary structure of ClpP. However, ordered N‐terminal axial loops, reorganization of the hydrogen bonding network at the entrance pore of ClpP, and reduced structural heterogeneity of the ClpP cylinder are common features of activator‐bound complexes. Activated ClpP mutants were obtained that recapitulate these effects. Based on our data, we propose a unified mechanism for compound‐based ClpP activation.Support or Funding InformationCanadian Institutes of Health ResearchThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Perrault syndrome type 3 caused by diverse molecular defects in CLPP

The maintenance of mitochondrial protein homeostasis (proteostasis) is crucial for correct cellular function. Recently, several mutations in the mitochondrial protease CLPP have been identified in patients with Perrault syndrome 3 (PRLTS3). These mutations can be arranged into two groups, those that cluster near the docking site (hydrophobic pocket, Hp) for the cognate unfoldase CLPX (i.e. T145P and C147S) and those that are adjacent to the active site of the peptidase (i.e. Y229D). Here we report the biochemical consequence of mutations in both regions. The Y229D mutant not only inhibited CLPP-peptidase activity, but unexpectedly also prevented CLPX-docking, thereby blocking the turnover of both peptide and protein substrates. In contrast, Hp mutations cause a range of biochemical defects in CLPP, from no observable change to CLPP activity for the C147S mutant, to dramatic disruption of most activities for the “gain-of-function” mutant T145P - including loss of oligomeric assembly and enhanced peptidase activity.

The Role of ClpP Protease in Bacterial Pathogenesis and Human Diseases.

In prokaryotic cells and eukaryotic organelles, the ClpP protease plays an important role in proteostasis. The disruption of the ClpP function has been shown to influence the infectivity and virulence of a number of bacterial pathogens. More recently, ClpP has been found to be involved in various forms of carcinomas and in Perrault syndrome, which is an inherited condition characterized by hearing loss in males and females and by ovarian abnormalities in females. Hence, targeting ClpP is a potentially viable, attractive option for the treatment of different ailments. Herein, the biochemical and cellular activities of ClpP are discussed along with the mechanisms by which ClpP affects bacterial pathogenesis and various human diseases. In addition, a comprehensive overview is given of the new classes of compounds in development that target ClpP. Many of these compounds are currently primarily aimed at treating bacterial infections. Some of these compounds inhibit ClpP activity, while others activate the protease and lead to its dysregulation. The ClpP activators are remarkable examples of small molecules that inhibit protein-protein interactions but also result in a gain of function.

Consequences of Depsipeptide Substitution on the ClpP Activation Activity of Antibacterial Acyldepsipeptides.

The acyldepsipeptide (ADEP) antibiotics operate through a clinically unexploited mechanism of action and thus have attracted attention from several antibacterial development groups. The ADEP scaffold is synthetically tractable, and deep-seated modifications have produced extremely potent antibacterial leads against Gram-positive pathogens. Although newly identified ADEP analogs demonstrate remarkable antibacterial activity against bacterial isolates and in mouse models of bacterial infections, stability issues pertaining to the depsipeptide core remain. To date, no study has been reported on the natural ADEP scaffold that evaluates the sole importance of the macrocyclic linkage on target engagement, molecular conformation, and bioactivity. To address this gap in ADEP structure-activity relationships, we synthesized three ADEP analogs that only differ in the linkage motif (i.e., ester, amide, and N-methyl amide) and provide a side-by-side comparison of conformational behavior and biological activity. We demonstrate that while replacement of the naturally occurring ester linkage with a secondary amide maintains in vitro biochemical activity, this simple substitution results in a significant drop in whole-cell activity. This study provides direct evidence that ester to amide linkage substitution is unlikely to provide a reasonable solution for ADEP instability.

RelA Mutant Enterococcus faecium with Multiantibiotic Tolerance Arising in an Immunocompromised Host.

ABSTRACTSerious bacterial infections in immunocompromised patients require highly effective antibacterial therapy for cure, and thus, this setting may reveal novel mechanisms by which bacteria circumvent antibiotics in the absence of immune pressure. Here, an infant with leukemia developed vancomycin-resistant Enterococcus faecium (VRE) bacteremia that persisted for 26 days despite appropriate antibiotic therapy. Sequencing of 22 consecutive VRE isolates identified the emergence of a single missense mutation (L152F) in relA, which constitutively activated the stringent response, resulting in elevated baseline levels of the alarmone guanosine tetraphosphate (ppGpp). Although the mutant remained susceptible to both linezolid and daptomycin in clinical MIC testing and during planktonic growth, it demonstrated tolerance to high doses of both antibiotics when growing in a biofilm. This biofilm-specific gain in resistance was reflected in the broad shift in transcript levels caused by the mutation. Only an experimental biofilm-targeting ClpP-activating antibiotic was able to kill the mutant strain in an established biofilm. The relA mutation was associated with a fitness trade-off, forming smaller and less-well-populated biofilms on biological surfaces. We conclude that clinically relevant relA mutations can emerge during prolonged VRE infection, causing baseline activation of the stringent response, subsequent antibiotic tolerance, and delayed eradication in an immunocompromised state.

The development of small-molecule modulators for ClpP protease activity.

The global spread of antibiotic resistance among important human pathogens emphasizes the need to find new antibacterial drugs with a novel mode of action. The ClpP protease has been shown to demonstrate its pivotal importance to both the survival and the virulence of pathogenic bacteria during host infection. Deregulating ClpP activity either through overactivation or inhibition could lead to antibacterial activity, declaiming the dual molecular mechanism for small-molecule modulation. Recently, natural products acyldepsipeptides (ADEPs) have been identified as a new class of antibiotics that activate ClpP to a dysfunctional state in the absence of cognate ATPases. ADEPs in combination with rifampicin eradicate deep-seated mouse biofilm infections. In addition, several non-ADEP compounds have been identified as activators of the ClpP proteolytic core without the involvement of ATPases. These findings indicate a general principle for killing dormant cells, the activation and corruption of the ClpP protease, rather than through conventional inhibition. Deletion of the clpP gene reduced the virulence of Staphylococcus aureus, thus making it an ideal antivirulence target. Multiple inhibitors have been developed in order to attenuate the production of extracellular virulence factors of bacteria through covalent modifications on serine in the active site or disruption of oligomerization of ClpP. Interestingly, due to the unusual composition and activation mechanism of ClpP in Mycobacterium tuberculosis, mycobacteria are killed by ADEPs through inhibition of ClpP activity rather than overactivation. In this short review, we will summarize recent progress in the development of small molecules modulating ClpP protease activity for both antibiotics and antivirulence.

Characterization of Gain-of-Function Mutant Provides New Insights into ClpP Structure.

ATP-dependent Clp protease (ClpP), a highly conserved serine protease in vast bacteria, could be converted into a noncontrollable enzyme capable of degrading mature proteins in the presence of acyldepsipeptides (ADEPs). Here, we design such a gain-of-function mutant of Staphylococcus aureus ClpP (SaClpP) capable of triggering the same level of dysfunctional activity that occurs upon ADEPs treatment. The SaClpPY63A mutant degrades FtsZ in vivo and inhibits staphylococcal growth. The crystal structure of SaClpPY63A indicates that Asn42 would be an important domino to fall for further activation of ClpP. Indeed, the SaClpPN42AY63A mutant demonstrates promoted self-activated proteolysis, which is a result of an enlarged entrance pore as observed in cryo-electron microscopy images. In addition, the expression of the engineered clpP allele phenocopies treatment with ADEPs; inhibition of cell division occurs as does showing sterilizing with rifampicin antibiotics. Collectively, we show that the gain-of-function SaClpPN42AY63A mutant becomes a fairly nonspecific protease and kills persisters by degrading over 500 proteins, thus providing new insights into the structure of the ClpP protease.

Sclerotiamide: The First Non-Peptide-Based Natural Product Activator of Bacterial Caseinolytic Protease P.

Caseinolytic protease P (ClpP) maintains essential roles in bacterial homeostasis. As such, both the inhibition and activation of this enzyme result in bactericidal activity, making ClpP a promising target for antibacterial drug development. Herein, we report the results of a fluorescence-based screen of ∼450 structurally diverse fungal and bacterial secondary metabolites. Sclerotiamide (1), a paraherquamide-related indolinone, was identified as the first non-peptide-based natural product activator of ClpP. Structure-activity relationships arising from the initial screen, preliminary biochemical evaluation of 1, and rationale for the exploitation of this chemotype to develop novel ClpP activators are presented.

Mitochondrial ClpP activity is required for cisplatin resistance in human cells.

In human cells ClpP and ClpX are imported into the mitochondrial matrix, where they interact to form the ATP-dependent protease ClpXP and play a role in the mitochondrial unfolded protein response. We find that reducing the levels of mitochondrial ClpP or ClpX renders human cancer cells more sensitive to cisplatin, a widely used anti-cancer drug. Conversely, overexpression of HClpP desensitizes cells to cisplatin. Overexpression of inactive HClpP–S97A had no effect. Cisplatin resistance correlated with decreased cellular accumulation of cisplatin and decreased levels of diguanosine–cisplatin adducts in both mitochondrial and genomic DNA. In contrast, higher levels of cisplatin–DNA adducts were found in cells in which HClpP had been depleted. Changes in the levels of ClpP had no effect on the levels of CTR1, a copper transporter that contributes to cisplatin uptake. However, the levels of ATP7A and ATP7B, copper efflux pumps that help eliminate cisplatin from cells, were increased when HClpP was overexpressed. HClpP levels were elevated in cervical carcinoma cells (KB‐CP20) and hepatoma cells (BEL-7404-CP20) independently selected for cisplatin resistance. The data indicate that robust HClpXP activity positively affects the ability of cells to efflux cisplatin and suggest that targeting HClpP or HClpX would offer a novel mechanism for sensitizing cancer cells to cisplatin.

Examination of a Structural Model of Peptidomimicry by Cyclic Acyldepsipeptide Antibiotics in Their Interaction with the ClpP Peptidase.

The cyclic acyldepsipeptide (ADEP) antibiotics act by binding the ClpP peptidase and dysregulating its activity. Their exocyclic N-acylphenylalanine is thought to structurally mimic the ClpP-binding, (I/L)GF tripeptide loop of the peptidase's accessory ATPases. We found that ADEP analogues with exocyclic N-acyl tripeptides or dipeptides resembling the (I/L)GF motif were weak ClpP activators and had no bioactivity. In contrast, ADEP analogues possessing difluorophenylalanine N-capped with methyl-branched acyl groups-like the side chains of residues in the (I/L)GF motifs-were superior to the parent ADEP with respect to both ClpP activation and bioactivity. We contend that the ADEP's N-acylphenylalanine moiety is not simply a stand-in for the ATPases' (I/L)GF motif; it likely has physicochemical properties that are better suited for ClpP binding. Further, our finding that the methyl-branching on the acyl group of the ADEPs improves activity opens new avenues for optimization.

AAA+ chaperones and acyldepsipeptides activate the ClpP protease via conformational control.

The Clp protease complex degrades a multitude of substrates, which are engaged by a AAA+ chaperone such as ClpX and subsequently digested by the dynamic, barrel-shaped ClpP protease. Acyldepsipeptides (ADEPs) are natural product-derived antibiotics that activate ClpP for chaperone-independent protein digestion. Here we show that both protein and small-molecule activators of ClpP allosterically control the ClpP barrel conformation. We dissect the catalytic mechanism with chemical probes and show that ADEP in addition to opening the axial pore directly stimulates ClpP activity through cooperative binding. ClpP activation thus reaches beyond active site accessibility and also involves conformational control of the catalytic residues. Moreover, we demonstrate that substoichiometric amounts of ADEP potently prevent binding of ClpX to ClpP and, at the same time, partially inhibit ClpP through conformational perturbance. Collectively, our results establish the hydrophobic binding pocket as a major conformational regulatory site with implications for both ClpXP proteolysis and ADEP-based anti-bacterial activity.

Structure and activation of a heteromeric protease complex.

New drug therapies are needed for treating Mycobacteria tuberculosis infections because strains with multiple drug resistances have arisen that tolerate almost all existing antibiotics (1). Regulated protein degradation is a universal process found in all kingdoms of life and is crucial for development, virulence, and pathogenicity in many bacteria, including M. tuberculosis (2⇓–4). Regulated degradation is principally carried out by energy-dependent proteases, such as the caseinolytic peptidase P (ClpP)-dependent family of proteases (5). These proteases consist of two oligomeric components: an ATP-consuming complex that is selective for substrate engagement, unfolding, and translocation (e.g., ClpX, ClpA, ClpC), and a chambered peptidase (ClpP) with sequestered active site serines that catalyze peptide bond hydrolysis. In many bacteria, a single clpP gene encodes a single protein that assembles into a homomeric tetradecamer; however, M. tuberculosis has two ClpP orthologs that are active only in a heteromeric ClpP1P2 complex (6). Other cases of multiple ClpP paralogs have been found in the genomes of both prokaryotic and eukaryotic organisms (7), but there has been limited characterization of these heteromeric complexes at the atomic scale. In PNAS, Schmitz et al. report the crystal structure of the M. tuberculosis ClpP1P2 complex, revealing the first high-resolution structure of a heteromeric ClpP complex and demonstrating how binding partners can dramatically affect ClpP activity through allosteric changes (8). The ClpP peptidase chamber can be envisioned as a static box that simply waits for delivery of unfolded substrates by the attendant ATPase and will digest whatever is passed to it (Fig. 1). However, recent biochemical and structural studies reveal a richer communication … [↵][1]1To whom correspondence should be addressed. Email: pchien{at}biochem.umass.edu. [1]: #xref-corresp-1-1

A simple fragment of cyclic acyldepsipeptides is necessary and sufficient for ClpP activation and antibacterial activity.

The development of new antibacterial agents, particularly those with unique biological targets, is essential to keep pace with the inevitable emergence of drug resistance in pathogenic bacteria. We identified the minimal structural component of the cyclic acyldepsipeptide (ADEP) antibiotics that exhibits antibacterial activity. We found that N-acyldifluorophenylalanine fragments function via the same mechanism of action as ADEPs, as evidenced by the requirement of ClpP for the fragments' antibacterial activity, the ability of fragments to activate Bacillus subtilis ClpP in vitro, and the capacity of an N-acyldifluorophenylalanine affinity matrix to capture ClpP from B. subtilis cell lysates. N-acyldifluorophenylalanine fragments are much simpler in structure than the full ADEPs and are also highly amenable to structural diversification. Thus, the stage has been set for the development of non-peptide activators of ClpP that can be used as antibacterial agents.

Targeting The Mitochondrial ClpP As a Novel Therapeutic Strategy For Acute Myeloid Leukemia

Caseinolytic protease (ClpP) is a mitochondrial enzyme complex with structural similarity to the cytoplasmic proteasome, but little is known about its function in the mitochondria. We identified ClpP as a potential therapeutic target for acute myeloid leukemia (AML) through an shRNA screen to identify mitochondrial proteins that are necessary for the viability of AML cells.We measured ClpP expression in 511 AML samples and 21 samples of normal CD34+ hematopoietic cells using a reverse phase protein array. ClpP was over-expressed in 45% of primary AML samples and expression occurred across FAB subtypes, cytogenetic risk groups, molecular mutations, and CD34+ expression subsets.Next, we evaluated the effects of ClpP knockdown on the growth and viability of AML cells using 3 independent shRNA constructs in lentiviral vectors. In leukemic cell lines that express high levels of ClpP, (OCI-AML2 and K562), knockdown of ClpP reduced growth and viability by > 80%. Importantly, no changes in growth or viability were observed following knockdown of ClpP in HL60 cells, which have lower basal levels of ClpP expression.As a chemical approach to evaluate the effects of ClpP inhibition on AML and normal hematopoietic cells, we synthesized a beta-lactone bacterial ClpP inhibitor, (3RS,4RS)-3-(non-8-en-1-yl)-4-(2-(pyridin-3-yl)ethyl)oxetan-2-one (also known as A2-32-01). Demonstrating specificity for the target, A2-32-01 inhibited the enzymatic activity of human recombinant ClpP, but not chymotrypsin-, trypsin-, or capsase-like enzymatic activity. A2-32-01 induced cell death in TEX, OCI-AML2, and K562 leukemia cells at concentrations that matched its ability to inhibit ClpP activity. Similar to the genetic studies, A2-32-01 did not kill HL60 cells. A2-32-01 was selectively cytotoxic to primary AML cells expressing ClpP over normal hematopoietic cells or AML cells with low ClpP expression. In addition, A2-32-01 reduced the clonogenic growth and bone marrow engraftment of primary AML cells, demonstrating the ability of ClpP inhibition to target AML progenitor/stem cells. Mechanistically, we demonstrated that A2-32-01 disrupted mitochondrial membrane potential in TEX cells and primary AML samples sensitive to A2-32-01, but not in normal hematopoietic cells.To date, the substrates of the mitochondrial ClpP are unknown. Therefore, we defined the interactome map of ClpP in HEK293 cells using mass spectrometry and the BirA tagging method whereby near-neighbors of ClpP are marked with biotin. Fifty-eight mitochondrial proteins preferentially interacted with ClpP over controls and the proteins were primarily components of the respiratory chain and mitochondrial translation apparatus. Thus, ClpP appears to be important to maintain the integrity of mitochondrial respiration. In support of this hypothesis, genetic or chemical ClpP inhibition was cytotoxic to 143B rhabdomyosarcoma cells, but not their rho-zero counterparts that lack mitochondrial DNA and oxidative phosphorylation.Next, we evaluated whether ClpP was required for the growth of AML cells in vivo. We knocked down ClpP in TEX cells with shRNA and injected the cells into the femur of NSGF mice. Compared to cells infected with control shRNA, knockdown of ClpP significantly reduced the engraftment of the cells (control shRNA 15.12 ± 4.576 % vs. ClpP knockdown 0.6180 ± 0.1976 % engraftment).Finally, to evaluate the toxicity of ClpP inhibition, we generated ClpP -/- mice. ClpP -/- mice were viable with normal peripheral blood counts and hematopoietic progenitor cells isolated from their bone marrow showed no significant reduction in clonogenic growth compared to those from wild type mice. Moreover, the abundance of Lin-, Sca-1+, c-kit+ hematopoietic progenitor cells in the bone marrow of ClpP -/- mice was equivalent to that in ClpP +/+ controls.Thus, these data suggest that ClpP inhibition can effectively target a subset of AML, while sparing normal hematopoietic cells. Disclosures:No relevant conflicts of interest to declare.

Structural determinants stabilizing the axial channel of ClpP for substrate translocation

Acyldepsipeptides (ADEPs) antibiotics bind to Escherichia coli ClpP mimicking the interactions that the IGL/F loops in ClpA or ClpX ATPases establish with the hydrophobic pockets surrounding the axial pore of the tetradecamer that the protease forms. ADEP binding induces opening of the gates blocking the axial channel of ClpP and allowing protein substrates to be translocated and hydrolysed in the degradation chamber. To identify the structural determinants stabilizing the open conformation of the axial channel for efficient substrate translocation, we constructed ClpP variants with amino acid substitutions in the N-terminal region that forms the axial gates. We found that adoption of a β-hairpin loop by this region and the integrity of the hydrophobic cluster at the base of this loop are necessary elements for the axial gate to efficiently translocate protein substrates. Analysis of ClpP variants from Bacillus subtilis suggested that the identified structural requirements of the axial channel for efficient translocation are conserved between Gram-positive and Gram-negative bacteria. These findings provide mechanistic insights into the activation of ClpP by ADEPs as well as the gating mechanism of the protease in the context of the ClpAP and ClpXP complexes.

The ClpP peptidase of Wolbachia endobacteria is a novel target for drug development against filarial infections

Filarial infections causing lymphatic filariasis or onchocerciasis (river blindness) can be treated with antibiotics (e.g. doxycycline) targeting the essential endosymbiotic Wolbachia bacteria. The depletion of Wolbachia inhibits worm development and causes worm death. Available antibiotics have restrictions for use in children and pregnant or breastfeeding women. Therefore, alternative antibiotics are needed that can be given to all members of the population and that are active with a shorter therapy time. Antibiotics of the acyldepsipeptide class have been shown to inhibit the growth of bacteria by overactivating the peptidase ClpP. The novel mode of action of this class of antibiotics could lead to faster killing of intracellular bacteria. To characterize acyldepsipeptide activity against the Wolbachia ClpP. The activity of acyldepsipeptides was investigated against Wolbachia in vitro in insect cells and also against worms in culture. In addition, structural effects were investigated by fluorescence microscopy and electron microscopy. The activity of ClpP was also investigated in vitro. We show that acyldepsipeptides are active against recombinant Wolbachia ClpP and endobacteria resident within insect cells in vitro, and some derivatives were also active against filarial worms in culture. As a consequence of treatment, the worms became immotile and died, the latter confirmed by a viability assay. The mode of action of the acyldepsipeptides in Wolbachia is the dysregulation of ClpP, causing the uncontrolled degradation of proteins, including the cell division protein FtsZ. Our results demonstrate that wolbachial ClpP is a target for further antifilarial antibiotic discovery.

Insights into Structural Network Responsible for Oligomerization and Activity of Bacterial Virulence Regulator Caseinolytic Protease P (ClpP) Protein

The barrel-shaped caseinolytic protease P (ClpP) is a main virulence regulator in the bacterial pathogen Staphylococcus aureus (SaClpP). It consists of two heptameric rings forming a homotetradecamer with an inner chamber that houses the 14 active sites. We recently showed that SaClpP is able to adopt a compressed, inactive conformation. We present here the 2.3 Å resolution structure of SaClpP in its closed, active conformation as well as the structure of the S98A mutant. Comprehensive mutational analysis aiming at destabilizing one or the other or both conformations was able to pinpoint key residues involved in this catalytic switch and in the heptamer-heptamer interaction. By probing the active site serine with a covalently modifying β-lactone probe, we could show that the tetradecameric organization is essential for a proper formation of the active site. Structural data suggest that a highly conserved hydrogen-bonding network links oligomerization to activity. A comparison of ClpP structures from different organisms provides suggestive evidence for the presence of a universal mechanism regulating ClpP activity in which binding of one subunit to the corresponding subunit on the other ring interface is necessary for the functional assembly of the catalytic triad and thus for protease function. This mechanism ensures controlled access to the active sites of a highly unspecific protease.

Validation of the Essential ClpP Protease in Mycobacterium tuberculosis as a Novel Drug Target

Mycobacterium tuberculosis is a pathogen of major global importance. Validated drug targets are required in order to develop novel therapeutics for drug-resistant strains and to shorten therapy. The Clp protease complexes provide a means for quality control of cellular proteins; the proteolytic activity of ClpP in concert with the ATPase activity of the ClpX/ClpC subunits results in degradation of misfolded or damaged proteins. Thus, the Clp system plays a major role in basic metabolism, as well as in stress responses and pathogenic mechanisms. M. tuberculosis has two ClpP proteolytic subunits. Here we demonstrate that ClpP1 is essential for viability in this organism in culture, since the gene could only be deleted from the chromosome when a second functional copy was provided. Overexpression of clpP1 had no effect on growth in aerobic culture or viability under anaerobic conditions or during nutrient starvation. In contrast, clpP2 overexpression was toxic, suggesting different roles for the two homologs. We synthesized known activators of ClpP protease activity; these acyldepsipeptides (ADEPs) were active against M. tuberculosis. ADEP activity was enhanced by the addition of efflux pump inhibitors, demonstrating that ADEPs gain access to the cell but that export occurs. Taken together, the genetic and chemical validation of ClpP as a drug target leads to new avenues for drug discovery.

Acyl depsipeptide (ADEP) resistance in Streptomyces

ADEP, a molecule of the acyl depsipeptide family, has an antibiotic activity with a unique mode of action. ADEP binding to the ubiquitous protease ClpP alters the structure of the enzyme. Access of protein to the ClpP proteolytic chamber is therefore facilitated and its cohort regulatory ATPases (ClpA, ClpC, ClpX) are not required. The consequent uncontrolled protein degradation in the cell appears to kill the ADEP-treated bacteria. ADEP is produced by Streptomyces hawaiiensis. Most sequenced genomes of Streptomyces have five clpP genes, organized as two distinct bicistronic operons, clpP1clpP2 and clpP3clpP4, and a single clpP5 gene. We investigated whether the different Clp proteases are all sensitive to ADEP. We report that ClpP1 is a target of ADEP whereas ClpP3 is largely insensitive. In wild-type Streptomyces lividans, clpP3clpP4 expression is constitutively repressed and the reason for the maintenance of this operon in Streptomyces has been elusive. ClpP activity is indispensable for survival of actinomycetes; we therefore tested whether the clpP3clpP4 operon, encoding an ADEP-insensitive Clp protease, contributes to a mechanism of ADEP resistance by target substitution. We report that in S. lividans, inactivation of ClpP1ClpP2 production or protease activity is indeed a mode of resistance to ADEP although it is neither the only nor the most frequent mode of resistance. The ABC transporter SclAB (orthologous to the Streptomyces coelicolor multidrug resistance pump SCO4959-SCO4960) is also able to confer ADEP resistance, and analysis of strains with sclAB deletions indicates that there are also other mechanisms of ADEP resistance.