Characterization of Phosphopantetheinyl Hydrolase from Mycobacterium tuberculosis.

Phosphopantetheinyl hydrolase, PptH (Rv2795c), is a recently discovered enzyme from Mycobacterium tuberculosis that removes 4'-phosphopantetheine (Ppt) from holo-carrier proteins (CPs) and thereby opposes the action of phosphopantetheinyl transferases (PPTases). PptH is the first structurally characterized enzyme of the phosphopantetheinyl hydrolase family. However, conditions for optimal activity of PptH have not been defined, and only one substrate has been identified. Here, we provide biochemical characterization of PptH and demonstrate that the enzyme hydrolyzes Ppt in vitro from more than one M. tuberculosis holo-CP as well as holo-CPs from other organisms. PptH provided the only detectable activity in mycobacterial lysates that dephosphopantetheinylated acyl carrier protein M (AcpM), suggesting that PptH is the main Ppt hydrolase in M. tuberculosis. We could not detect a role for PptH in coenzyme A (CoA) salvage, and PptH was not required for virulence of M. tuberculosis during infection of mice. It remains to be determined why mycobacteria conserve a broadly acting phosphohydrolase that removes the Ppt prosthetic group from essential CPs. We speculate that the enzyme is critical for aspects of the life cycle of M. tuberculosis that are not routinely modeled. IMPORTANCE Tuberculosis (TB), caused by Mycobacterium tuberculosis, was the leading cause of death from an infectious disease before COVID, yet the in vivo essentiality and function of many of the protein-encoding genes expressed by M. tuberculosis are not known. We biochemically characterize M. tuberculosis's phosphopantetheinyl hydrolase, PptH, a protein unique to mycobacteria that removes an essential posttranslational modification on proteins involved in synthesis of lipids important for the bacterium's cell wall and virulence. We demonstrate that the enzyme has broad substrate specificity, but it does not appear to have a role in coenzyme A (CoA) salvage or virulence in a mouse model of TB.

Macrocyclic Peptides that Selectively Inhibit the Mycobacterium tuberculosis Proteasome.

Treatment of tuberculosis (TB) currently takes at least 6 months. Latent Mycobacterium tuberculosis (Mtb) is phenotypically tolerant to most anti-TB drugs. A key hypothesis is that drugs that kill nonreplicating (NR) Mtb may shorten treatment when used in combination with conventional drugs. The Mtb proteasome (Mtb20S) could be such a target because its pharmacological inhibition kills NR Mtb and its genetic deletion renders Mtb unable to persist in mice. Here, we report a series of macrocyclic peptides that potently and selectively target the Mtb20S over human proteasomes, including macrocycle 6. The cocrystal structure of macrocycle 6 with Mtb20S revealed structural bases for the species selectivity. Inhibition of 20S within Mtb by 6 dose dependently led to the accumulation of Pup-tagged GFP that is degradable but resistant to depupylation and death of nonreplicating Mtb under nitrosative stress. These results suggest that compounds of this class have the potential to develop as anti-TB therapeutics.

The DnaK Chaperone System Buffers the Fitness Cost of Antibiotic Resistance Mutations in Mycobacteria.

Chaperones aid in protein folding and maintenance of protein integrity. In doing so, they have the unique ability to directly stabilize resistance-conferring amino acid substitutions in drug targets and to counter the stress imparted by these substitutions, thus supporting heritable antimicrobial resistance (AMR). We asked whether chaperones support AMR in Mycobacterium smegmatis, a saprophytic model of Mycobacterium tuberculosis, the causative agent of tuberculosis (TB). We show that DnaK associates with many drug targets and that DnaK associates more with AMR-conferring mutant RNA polymerase (RNAP) than with wild-type RNAP. In addition, frequency-of-resistance (FOR) and fitness studies reveal that the DnaK system of chaperones supports AMR in antimicrobial targets in mycobacteria, including RNAP and the ribosome. These findings highlight chaperones as potential targets for drugs to overcome AMR in mycobacteria, including M. tuberculosis, as well as in other pathogens.IMPORTANCE AMR is a global problem, especially for TB. Here, we show that mycobacterial chaperones support AMR in M. smegmatis, a nonpathogenic model of M. tuberculosis, the causative agent of TB. In particular, the mycobacterial DnaK system of chaperones supports AMR in the antimicrobial targets RNA polymerase and the ribosome. This is the first report showing a role for protein chaperones in mediating AMR in mycobacteria. Given the widespread role of protein chaperones in enabling genomic diversity, we anticipate that our findings can be extended to other microbes.

Selective Phenylimidazole-Based Inhibitors of the Mycobacterium tuberculosis Proteasome.

Proteasomes of pathogenic microbes have become attractive targets for anti-infectives. Coevolving with its human host, Mycobacterium tuberculosis (Mtb) has developed mechanisms to resist host-imposed nitrosative and oxidative stresses. Genetic deletion or pharmacological inhibition of the Mtb proteasome (Mtb20S) renders nonreplicating Mtb susceptible to reactive nitrogen species in vitro and unable to survive in the lungs of mice, validating the Mtb proteasome as a promising target for anti-Mtb agents. Using a structure-guided and flow chemistry-enabled study of structure-activity relationships, we developed phenylimidazole-based peptidomimetics that are highly potent for Mtb20S. X-ray structures of selected compounds with Mtb20S shed light on their selectivity for mycobacterial over human proteasomes.

Mycobacterium tuberculosis cysteine biosynthesis genes mec+-cysO-cysM confer resistance to clofazimine.

The Mycobacterium tuberculosis mec+-cysO-cysM gene cluster was shown to be part of a novel cysteine biosynthesis pathway in vitro, but little is known about its essentiality or role in M. tuberculosis physiology. In this study, we generate a knock out of the mec+-cysO-cysM gene cluster in M. tuberculosis and show that the gene cluster is not essential under a variety of conditions, suggesting redundancy in pathways for cysteine biosynthesis in M. tuberculosis. The cysteine biosynthesis gene cluster is essential for resistance for clofazimine, a peroxide-producing anti-leprosy drug. Therefore, although under most conditions the pathway is not essential, it likely has an important role in defense against oxidative stress in M. tuberculosis.

Opposing reactions in coenzyme A metabolism sensitize Mycobacterium tuberculosis to enzyme inhibition.

Mycobacterium tuberculosis (Mtb) is the leading infectious cause of death in humans. Synthesis of lipids critical for Mtb's cell wall and virulence depends on phosphopantetheinyl transferase (PptT), an enzyme that transfers 4'-phosphopantetheine (Ppt) from coenzyme A (CoA) to diverse acyl carrier proteins. We identified a compound that kills Mtb by binding and partially inhibiting PptT. Killing of Mtb by the compound is potentiated by another enzyme encoded in the same operon, Ppt hydrolase (PptH), that undoes the PptT reaction. Thus, loss-of-function mutants of PptH displayed antimicrobial resistance. Our PptT-inhibitor cocrystal structure may aid further development of antimycobacterial agents against this long-sought target. The opposing reactions of PptT and PptH uncover a regulatory pathway in CoA physiology.

Targeting the Proteostasis Network for Mycobacterial Drug Discovery.

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), remains one of the world's deadliest infectious diseases and urgently requires new antibiotics to treat drug-resistant strains and to decrease the duration of therapy. During infection, Mtb encounters numerous stresses associated with host immunity, including hypoxia, reactive oxygen and nitrogen species, mild acidity, nutrient starvation, and metal sequestration and intoxication. The Mtb proteostasis network, composed of chaperones, proteases, and a eukaryotic-like proteasome, provides protection from stresses and chemistries of host immunity by maintaining the integrity of the mycobacterial proteome. In this Review, we explore the proteostasis network as a noncanonical target for antibacterial drug discovery.

Identification of a Mycothiol-Dependent Nitroreductase from Mycobacterium tuberculosis.

The success of Mycobacterium tuberculosis (Mtb) as a pathogen depends on the redundant and complex mechanisms it has evolved for resisting nitrosative and oxidative stresses inflicted by host immunity. Improving our understanding of these defense pathways can reveal vulnerable points in Mtb pathogenesis. In this study, we combined genetic, structural, computational, biochemical, and biophysical approaches to identify a novel enzyme class represented by Rv2466c. We show that Rv2466c is a mycothiol-dependent nitroreductase of Mtb and can reduce the nitro group of a novel mycobactericidal compound using mycothiol as a cofactor. In addition to its function as a nitroreductase, Rv2466c confers partial protection to menadione stress.

Mycobacterium tuberculosis Proteasome Accessory Factor A (PafA) Can Transfer Prokaryotic Ubiquitin-Like Protein (Pup) between Substrates.

The protein degradation machinery of Mycobacterium tuberculosis includes a proteasome and a ubiquitin-like protein (Pup). Proteasome accessory factor A (PafA) attaches Pup to proteins to target them for degradation by the proteasome. Free Pup is unstable and never observed in extracts of M. tuberculosis, an observation that led us to hypothesize that PafA may need alternative sources of Pup. Here, we show that PafA can move Pup from one proteasome substrate, inositol 1-phosphate synthetase (Ino1), to two different proteins, malonyl coenzyme A (CoA)-acyl carrier protein transacylase (FabD) and lonely guy (Log). This apparent "transpupylation" reaction required a previously unrecognized depupylase activity in PafA, and, surprisingly, this depupylase activity was much more efficient than the activity of the dedicated depupylase Dop (deamidase of Pup). Thus, PafA can potentially use both newly synthesized Pup and recycled Pup to doom proteins for degradation.IMPORTANCE Unlike eukaryotes, which contain hundreds of ubiquitin ligases, Pup-containing bacteria appear to have a single ligase to pupylate dozens if not hundreds of different proteins. The observation that PafA can depupylate and transpupylate in vitro offers new insight into how protein stability is regulated in proteasome-bearing bacteria. Importantly, PafA and the dedicated depupylase Dop are each required for the full virulence of Mycobacterium tuberculosis Thus, inhibition of both enzymes may be extremely attractive for the development of therapeutics against tuberculosis.

N-methylation of a bactericidal compound as a resistance mechanism in Mycobacterium tuberculosis.

The rising incidence of antimicrobial resistance (AMR) makes it imperative to understand the underlying mechanisms. Mycobacterium tuberculosis (Mtb) is the single leading cause of death from a bacterial pathogen and estimated to be the leading cause of death from AMR. A pyrido-benzimidazole, 14, was reported to have potent bactericidal activity against Mtb. Here, we isolated multiple Mtb clones resistant to 14. Each had mutations in the putative DNA-binding and dimerization domains of rv2887, a gene encoding a transcriptional repressor of the MarR family. The mutations in Rv2887 led to markedly increased expression of rv0560c. We characterized Rv0560c as an S-adenosyl-L-methionine-dependent methyltransferase that N-methylates 14, abolishing its mycobactericidal activity. An Mtb strain lacking rv0560c became resistant to 14 by mutating decaprenylphosphoryl-ß-d-ribose 2-oxidase (DprE1), an essential enzyme in arabinogalactan synthesis; 14 proved to be a nanomolar inhibitor of DprE1, and methylation of 14 by Rv0560c abrogated this activity. Thus, 14 joins a growing list of DprE1 inhibitors that are potently mycobactericidal. Bacterial methylation of an antibacterial agent, 14, catalyzed by Rv0560c of Mtb, is a previously unreported mechanism of AMR.

Identification of Rv3852 as an Agrimophol-Binding Protein in Mycobacterium tuberculosis.

Mycobacterial tuberculosis (Mtb) is able to preserve its intrabacterial pH (pHIB) near neutrality in the acidic phagosomes of immunologically activated macrophages and to cause lethal pathology in immunocompetent mice. In contrast, when its ability to maintain pHIB homeostasis is genetically compromised, Mtb dies in acidic phagosomes and is attenuated in the mouse. Compounds that phenocopy the genetic disruption of Mtb's pHIB homeostasis could serve as starting points for drug development in their own right or through identification of their targets. A previously reported screen of a natural product library identified a phloroglucinol, agrimophol, that lowered Mtb's pHIB and killed Mtb at an acidic extrabacterial pH. Inability to identify agrimophol-resistant mutants of Mtb suggested that the compound may have more than one target. Given that polyphenolic compounds may undergo covalent reactions, we attempted an affinity-based method for target identification. The structure-activity relationship of synthetically tractable polyhydroxy diphenylmethane analogs with equivalent bioactivity informed the design of a bioactive agrimophol alkyne. After click-chemistry reaction with azido-biotin and capture on streptavidin, the biotinylated agrimophol analog pulled down the Mtb protein Rv3852, a predicted membrane protein that binds DNA in vitro. A ligand-protein interaction between agrimophol and recombinant Rv3852 was confirmed by isothermal calorimetry (ITC) and led to disruption of Rv3852's DNA binding function. However, genetic deletion of rv3852 in Mtb did not phenocopy the effect of agrimophol on Mtb, perhaps because of redundancy of its function.

Target-based screen against a periplasmic serine protease that regulates intrabacterial pH homeostasis in Mycobacterium tuberculosis.

Mycobacterium tuberculosis (Mtb) maintains its intrabacterial pH (pHIB) near neutrality in the acidic environment of phagosomes within activated macrophages. A previously reported genetic screen revealed that Mtb loses this ability when the mycobacterial acid resistance protease (marP) gene is disrupted. In the present study, a high throughput screen (HTS) of compounds against the protease domain of MarP identified benzoxazinones as inhibitors of MarP. A potent benzoxazinone, BO43 (6-chloro-2-(2'-methylphenyl)-4H-1,3-benzoxazin-4-one), acylated MarP and lowered Mtb's pHIB and survival during incubation at pH 4.5. BO43 had similar effects on MarP-deficient Mtb, suggesting the existence of additional target(s). Reaction of an alkynyl-benzoxazinone, BO43T, with Mycobacterium bovis variant bacille Calmette-Guérin (BCG) followed by click chemistry with azido-biotin identified both the MarP homologue and the high temperature requirement A1 (HtrA1) homologue, an essential protein. Thus, the chemical probe identified through a target-based screen not only reacted with its intended target in the intact cells but also implicated an additional enzyme that had eluded a genetic screen biased against essential genes.