Mycobacterium tuberculosis (Mtb) contains two clpP genes, both of which are essential for viability. We expressed and purified Mtb ClpP1 and ClpP2 separately. Although each formed a tetradecameric structure and was processed, they lacked proteolytic activity. We could, however, reconstitute an active, mixed ClpP1P2 complex after identifying N‐blocked dipeptides that stimulate dramatically (>1000‐fold) ClpP1P2 activity against certain peptides and proteins. These activators function cooperatively to induce the dissociation of ClpP1 and ClpP2 tetradecamers into heptameric rings, which then re‐associate to form the active ClpP1P2 2‐ring mixed complex. No analogous small molecule‐induced enzyme activation mechanism involving dissociation and re‐association of multimeric rings has been described. ClpP1P2 possesses chymotrypsin and caspase‐like activities, and ClpP1 and ClpP2 differ in cleavage preferences. The regulatory ATPase ClpC1 was purified and shown to increase hydrolysis of proteins by ClpP1P2, but not peptides. ClpC1 did not activate ClpP1 or ClpP2 homotetradecamers and stimulated ClpP1P2 only when both ATP and a dipeptide activator were present. ClpP1P2 activity, its unusual activation mechanism and ClpC1 ATPase represent attractive drug targets to combat tuberculosis.

Granulomas are the pathological hallmark of tuberculosis (TB). However, their function and mechanisms of formation remain poorly understood. To understand the role of granulomas in TB, we analyzed the proteomes of granulomas from subjects with tuberculosis in an unbiased fashion. Using laser capture microdissection, mass spectrometry and confocal microscopy, we generated detailed molecular maps of human granulomas. We found that the centers of granulomas possess a pro-inflammatory environment characterized by anti-microbial peptides, ROS and pro-inflammatory eicosanoids. Conversely, the tissue surrounding the caseum possesses a comparatively anti-inflammatory signature. These findings are consistent across a set of six subjects and in rabbits. While the balance between systemic pro- and anti-inflammatory signals is crucial to TB disease outcome, here we find that these signals are physically segregated within each granuloma. The protein and lipid snapshots of human and rabbit lesions analysed here suggest that the pathologic response to TB is shaped by the precise anatomical localization of these inflammatory pathways during the development of the granuloma.

In most bacteria, Clp protease is a conserved, non-essential serine protease that regulates the response to various stresses. Mycobacteria, including Mycobacterium tuberculosis (Mtb) and Mycobacterium smegmatis, unlike most well studied prokaryotes, encode two ClpP homologs, ClpP1 and ClpP2, in a single operon. Here we demonstrate that the two proteins form a mixed complex (ClpP1P2) in mycobacteria. Using two different approaches, promoter replacement, and a novel system of inducible protein degradation, leading to inducible expression of clpP1 and clpP2, we demonstrate that both genes are essential for growth and that a marked depletion of either one results in rapid bacterial death. ClpP1P2 protease appears important in degrading missense and prematurely terminated peptides, as partial depletion of ClpP2 reduced growth specifically in the presence of antibiotics that increase errors in translation. We further show that the ClpP1P2 protease is required for the degradation of proteins tagged with the SsrA motif, a tag co-translationally added to incomplete protein products. Using active site mutants of ClpP1 and ClpP2, we show that the activity of each subunit is required for proteolysis, for normal growth of Mtb in vitro and during infection of mice. These observations suggest that the Clp protease plays an unusual and essential role in Mtb and may serve as an ideal target for antimycobacterial therapy.

The 1,5-diarylpyrrole derivative BM212 was previously shown to be active against multidrug-resistant clinical isolates and Mycobacterium tuberculosis residing within macrophages as well as against Mycobacterium avium and other atypical mycobacteria. To determine its mechanism of action, we identified the cellular target. Spontaneous Mycobacterium smegmatis, Mycobacterium bovis BCG, and M. tuberculosis H37Rv mutants that were resistant to BM212 were isolated. By the screening of genomic libraries and by whole-genome sequencing, we found that all the characterized mutants showed mutations in the mmpL3 gene, allowing us to conclude that resistance to BM212 maps to the MmpL3 protein, a member of the MmpL (mycobacterial membrane protein, large) family. Susceptibility was unaffected by the efflux pump inhibitors reserpine, carbonylcyanide m-chlorophenylhydrazone, and verapamil. Uptake/efflux experiments with [ 14 C]BM212 demonstrated that resistance is not driven by the efflux of BM212. Together, these data strongly suggest that the MmpL3 protein is the cellular target of BM212.T he rise of multidrug-resistant (MDR) and extensively drugresistant (XDR) Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), makes the validation of new antitubercular agents a major global priority. Since tubercular drug resistance is chromosomally encoded (17), chemotherapeutic agents directed against new cellular targets are likely to be effective against both drug-sensitive and drug-resistant M. tuberculosis strains (5,12,13,18). Target identification and validation are usually achieved by either genetic or chemical approaches. The former has the advantage of identifying a likely cellular target a priori but yields no information with regard to the druggability of the target and the access of the drug to the target (a particular problem in mycobacteria [23]). It is therefore not surprising that no current antitubercular agents have been identified through rational drug design (23). Alternatively, the identification of a cellular target candidate through chemical screening has the advantage of knowing that the compound can bind and affect the cellular target in vivo. The identification of the target for an active compound allows the rational modification of lead candidates through medicinal chemistry while ensuring that the compound retains activity against its primary target. However, finding which proteins are inhibited by a compound can be quite challenging.We randomly screened a library of compounds to identify structures of interest for further development. Several azole compounds containing imidazole, pyrrole, toluidine, or methanamine groups were tested for antimycobacterial activity. Among them, 1-{[1,5-bis(4-chlorophenyl)-2-methyl-1H-pyrrol-3-yl]methyl}-4-methylpiperazine (BM212) (Fig. 1) proved to be active against multidrug-resistant clinical isolates, against M. tuberculosis residing within macrophages, and against Mycobacterium avium as well as other nontuberculous mycobacteria (7). The identification of BM21...

Proteases have been successfully targeted for the treatment of several diseases, including hypertension, type 2 diabetes, multiple myeloma, HIV and hepatitis C virus infections. Given the demonstrated pharmacological tractability of this enzyme family and the pressing need for novel drugs to combat antibiotic resistance, proteases have also attracted interest as antibacterial targets--particularly the widely conserved intracellular bacterial degradative proteases, which are often indispensable for normal bacterial growth or virulence. This Review summarizes the roles of the key prokaryotic degradative proteases, with a focus on the initial efforts and associated challenges in developing specific therapeutic modulators of these enzymes as novel classes of antibacterial drugs.

The epigenome and three-dimensional (3D) –genomic architecture are emerging as key factors in the dynamic regulation of different transcriptional programs required for neuronal functions. Here we utilize an activity-dependent tagging system in mice to determine the epigenetic state, 3D-genome architecture, and transcriptional landscape of engram cells over the lifespan of memory formation and recall. Our findings reveal that memory encoding leads to an epigenetic priming event, marked by increased accessibility of enhancers without corresponding transcriptional changes. Memory consolidation subsequently results in spatial reorganization of large chromatin segments and promoter-enhancer interactions. Finally, with reactivation, engram neurons utilize a subset of de novo long-range interactions, where primed enhancers were brought in contact with their respective promoters to up-regulate genes involved in local protein translation in synaptic compartments. Collectively, our work elucidates the comprehensive transcriptional and epigenomic landscape across the lifespan of memory formation and recall in the hippocampal engram ensemble. The formation and preservation of long-term memories depends on coordinated gene expression and synthesis of synaptic proteins 1 . These molecular processes act within a specific population of neurons, referred to as engram cells 2 – 4 . Recent approaches using activity-dependent expression of reporters, provided a framework for exploring the engram ensemble 5 – 8 , but the molecular mechanisms that govern memory storage and retrieval remain poorly understood. Specifically, epigenetic modifications and 3D -genomic architecture are emerging as a key factors in dynamic regulation of gene expression 9 – 17 , and there is an increasing appreciation of their importance in neuronal function, development and disease 14 , 16 , 18 Here, we utilized the Targeted Recombination in Active Populations (TRAP) mouse model 5 , 6 , in which activated neurons expressing the Activity Regulated Cytoskeleton Associated Protein, ( Arc ) gene, are permanently tagged in an inducible manner. Activated neurons during memory encoding, consolidation and recall were sorted and subjected to nuclear RNA sequencing (nRNA-seq), Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) and chromosome conformation capture (Hi-C). Our data demonstrates that memory encoding leads to a genome-wide increase in chromatin accessibility, without expected changes in gene expression. Furthermore, we demonstrate that late phase of memory consolidation was associated with re-localization of large chromatin segments (sub-compartments) from...

Unlike most bacterial species, Mycobacterium tuberculosis depends on the Clp proteolysis system for survival even in in vitro conditions. We hypothesized that Clp is required for the physiologic turnover of mycobacterial proteins whose accumulation is deleterious to bacterial growth and survival. To identify cellular substrates, we employed quantitative proteomics and transcriptomics to identify the set of proteins that accumulated upon the loss of functional Clp protease. Among the set of potential Clp substrates uncovered, we were able to unambiguously identify WhiB1, an essential transcriptional repressor capable of auto-repression, as a substrate of the mycobacterial Clp protease. Dysregulation of WhiB1 turnover had a toxic effect that was not rescued by repression of whiB1 transcription. Thus, under normal growth conditions, Clp protease is the predominant regulatory check on the levels of potentially toxic cellular proteins. Our findings add to the growing evidence of how post-translational regulation plays a critical role in the regulation of bacterial physiology.

A novel type of antibacterial screening method, a target mechanism-based whole-cell screening method, was developed to combine the advantages of target mechanism- and whole-cell-based approaches. A mycobacterial reporter strain with a synthetic phenotype for caseinolytic protease (ClpP1P2) activity was engineered, allowing the detection of inhibitors of this enzyme inside intact bacilli. A high-throughput screening method identified bortezomib, a human 26S proteasome drug, as a potent inhibitor of ClpP1P2 activity and bacterial growth. A battery of secondary assays was employed to demonstrate that bortezomib indeed exerts its antimicrobial activity via inhibition of ClpP1P2: Down- or upmodulation of the intracellular protease level resulted in hyper- or hyposensitivity of the bacteria, the drug showed specific potentiation of translation error-inducing aminoglycosides, ClpP1P2-specific substrate WhiB1 accumulated upon exposure, and growth inhibition potencies of bortezomib derivatives correlated with ClpP1P2 inhibition potencies. Furthermore, molecular modeling showed that the drug can bind to the catalytic sites of ClpP1P2. This work demonstrates the feasibility of target mechanism-based whole-cell screening, provides chemical validation of ClpP1P2 as a target, and identifies a drug in clinical use as a new lead compound for tuberculosis therapy.