Antibiotic resistance in Mycobacterium tuberculosis alters tolerance to cell wall-targeting inhibitors.

Background: A limited ability to eliminate drug-resistant strains of Mycobacterium tuberculosis is a major contributor to the morbidity of TB. Complicating this problem, little is known about how drug resistance-conferring mutations alter the ability of M. tuberculosis to tolerate antibiotic killing. Here, we investigated if drug-resistant strains of M. tuberculosis have an altered ability to tolerate killing by cell wall-targeting inhibitors. Methods: Bacterial killing and MIC assays were used to test for antibiotic tolerance and synergy against a panel of drug-resistant M. tuberculosis strains. Results: Our results demonstrate that vancomycin and thioacetazone exhibit increased killing of diverse drug-resistant strains. Mutations in mmaA4 and mmpL3 increased vancomycin killing, which was consistent with vancomycin synergizing with thioacetazone and MmpL3-targeting inhibitors. In contrast, mutations in the mce1 operon conferred tolerance to vancomycin. Conclusions: Overall, this work demonstrates how drug-resistant strains experience perturbations in cell-wall production that alters their tolerance to killing by cell wall-targeting inhibitors.

A dual-targeting succinate dehydrogenase and F1Fo-ATP synthase inhibitor rapidly sterilizes replicating and non-replicating Mycobacterium tuberculosis.

Mycobacterial bioenergetics is a validated target space for antitubercular drug development. Here, we identify BB2-50F, a 6-substituted 5-(N,N-hexamethylene)amiloride derivative as a potent, multi-targeting bioenergetic inhibitor of Mycobacterium tuberculosis. We show that BB2-50F rapidly sterilizes both replicating and non-replicating cultures of M. tuberculosis and synergizes with several tuberculosis drugs. Target identification experiments, supported by docking studies, showed that BB2-50F targets the membrane-embedded c-ring of the F1Fo-ATP synthase and the catalytic subunit (substrate-binding site) of succinate dehydrogenase. Biochemical assays and metabolomic profiling showed that BB2-50F inhibits succinate oxidation, decreases the activity of the tricarboxylic acid (TCA) cycle, and results in succinate secretion from M. tuberculosis. Moreover, we show that the lethality of BB2-50F under aerobic conditions involves the accumulation of reactive oxygen species. Overall, this study identifies BB2-50F as an effective inhibitor of M. tuberculosis and highlights that targeting multiple components of the mycobacterial respiratory chain can produce fast-acting antimicrobials.

The evolution of antibiotic resistance is associated with collateral drug phenotypes in Mycobacterium tuberculosis.

The increasing incidence of drug resistance in Mycobacterium tuberculosis has diminished the efficacy of almost all available antibiotics, complicating efforts to combat the spread of this global health burden. Alongside the development of new drugs, optimised drug combinations are needed to improve treatment success and prevent the further spread of antibiotic resistance. Typically, antibiotic resistance leads to reduced sensitivity, yet in some cases the evolution of drug resistance can lead to enhanced sensitivity to unrelated drugs. This phenomenon of collateral sensitivity is largely unexplored in M. tuberculosis but has the potential to identify alternative therapeutic strategies to combat drug-resistant strains that are unresponsive to current treatments. Here, by using drug susceptibility profiling, genomics and evolutionary studies we provide evidence for the existence of collateral drug sensitivities in an isogenic collection M. tuberculosis drug-resistant strains. Furthermore, in proof-of-concept studies, we demonstrate how collateral drug phenotypes can be exploited to select against and prevent the emergence of drug-resistant strains. This study highlights that the evolution of drug resistance in M. tuberculosis leads to collateral drug responses that can be exploited to design improved drug regimens.

Deletion of Rv2571c confers resistance to arylamide compounds in Mycobacterium tuberculosis.

Tuberculosis, caused by Mycobacterium tuberculosis, is an urgent global health problem requiring new drugs, new drug targets and an increased understanding of antibiotic resistance. We have determined the mode of resistance to a series of arylamide compounds in M. tuberculosis We isolated M. tuberculosis resistant mutants to two arylamide compounds which are inhibitory to growth under host-relevant conditions (butyrate as a sole carbon source). Thirteen mutants were characterized, and all had mutations in Rv2571c; mutations included a premature stop codon and frameshifts as well as non-synonymous polymorphisms. We isolated a further ten strains with mutations in Rv2571c with resistance. Complementation with a wild-type copy of Rv2571c restored arylamide sensitivity. Over-expression of Rv2571c was toxic in both wild-type and mutant backgrounds. We constructed M. tuberculosis strains with an unmarked deletion of the entire Rv2571c gene by homologous recombination and confirmed that these were resistant to the arylamide series. Rv2571c is a member of the aromatic amino acid transport family and has a fusaric acid resistance domain which is associated with compound transport. Since loss or inactivation of Rv2571c leads to resistance, we propose that Rv2571c is involved in the import of arylamide compounds.

Characterizing in vivo loss of virulence of an HN878 Mycobacterium tuberculosis isolate from a genetic duplication event.

The increase of global cases of drug resistant (DR) Mycobacterium tuberculosis (M.tb) is a serious problem for the tuberculosis research community and the goals to END TB by 2030. Due to the need for advancing and screening next generation therapeutics and vaccines, we aimed to design preclinical DR models of Beijing lineage M.tb HN878 strain in different mouse backgrounds. We found escalating sensitivities of morbidity due to low dose aerosol challenge (50-100 bacilli) in CB6F1, C57BL/6 and SWR mice, respectively. We also observed that pulmonary bacterial burden at morbidity endpoints correlated inversely with survival over time between mouse strains. Interestingly, with in vitro passaging and in the process of selecting individual DR mutant colonies, we observed a significant decrease in in vivo HN878 strain virulence, which correlated with the acquisition of a large genetic duplication. We confirmed that low passage infection stocks with no or low prevalence of the duplication, including stocks directly acquired from the BEI resources biorepository, retained virulence, measured by morbidity over time. These data help confirm previous reports and emphasize the importance of monitoring virulence and stock fidelity.

Uncovering interactions between mycobacterial respiratory complexes to target drug-resistant Mycobacterium tuberculosis.

Mycobacterium tuberculosis remains a leading cause of infectious disease morbidity and mortality for which new drug combination therapies are needed. Mycobacterial bioenergetics has emerged as a promising space for the development of novel therapeutics. Further to this, unique combinations of respiratory inhibitors have been shown to have synergistic or synthetic lethal interactions, suggesting that combinations of bioenergetic inhibitors could drastically shorten treatment times. Realizing the full potential of this unique target space requires an understanding of which combinations of respiratory complexes, when inhibited, have the strongest interactions and potential in a clinical setting. In this review, we discuss (i) chemical-interaction, (ii) genetic-interaction and (iii) chemical-genetic interaction studies to explore the consequences of inhibiting multiple mycobacterial respiratory components. We provide potential mechanisms to describe the basis for the strongest interactions. Finally, whilst we place an emphasis on interactions that occur with existing bioenergetic inhibitors, by highlighting interactions that occur with alternative respiratory components we envision that this information will provide a rational to further explore alternative proteins as potential drug targets and as part of unique drug combinations.

Impaired Succinate Oxidation Prevents Growth and Influences Drug Susceptibility in Mycobacterium tuberculosis.

Succinate is a major focal point in mycobacterial metabolism and respiration, serving as both an intermediate of the tricarboxylic acid (TCA) cycle and a direct electron donor for the respiratory chain. Mycobacterium tuberculosis encodes multiple enzymes predicted to be capable of catalyzing the oxidation of succinate to fumarate, including two different succinate dehydrogenases (Sdh1 and Sdh2) and a separate fumarate reductase (Frd) with possible bidirectional behavior. Previous attempts to investigate the essentiality of succinate oxidation in M. tuberculosis have relied on the use of single-gene deletion mutants, raising the possibility that the remaining enzymes could catalyze succinate oxidation in the absence of the other. To address this, we report on the use of mycobacterial CRISPR interference (CRISPRi) to construct single, double, and triple transcriptional knockdowns of sdhA1, sdhA2, and frdA in M. tuberculosis. We show that the simultaneous knockdown of sdhA1 and sdhA2 is required to prevent succinate oxidation and overcome the functional redundancy within these enzymes. Succinate oxidation was demonstrated to be essential for the optimal growth of M. tuberculosis, with the combined knockdown of sdhA1 and sdhA2 significantly impairing the activity of the respiratory chain and preventing growth on a range of carbon sources. Moreover, impaired succinate oxidation was shown to influence the activity of cell wall-targeting antibiotics and bioenergetic inhibitors against M. tuberculosis. Together, these data provide fundamental insights into mycobacterial physiology, energy metabolism, and antimicrobial susceptibility. IMPORTANCE New drugs are urgently required to combat the tuberculosis epidemic that claims 1.5 million lives annually. Inhibitors of mycobacterial energy metabolism have shown significant promise clinically; however, further advancing this nascent target space requires a more fundamental understanding of the respiratory enzymes and pathways used by Mycobacterium tuberculosis. Succinate is a major focal point in mycobacterial metabolism and respiration; yet, the essentiality of succinate oxidation and the consequences of inhibiting this process are poorly defined. In this study, we demonstrate that impaired succinate oxidation prevents the optimal growth of M. tuberculosis on a range of carbon sources and significantly reduces the activity of the electron transport chain. Moreover, we show that impaired succinate oxidation both positively and negatively influences the activity of a variety of antituberculosis drugs. Combined, these findings provide fundamental insights into mycobacterial physiology and drug susceptibility that will be useful in the continued development of bioenergetic inhibitors.

Deciphering functional redundancy and energetics of malate oxidation in mycobacteria.

Oxidation of malate to oxaloacetate, catalyzed by either malate dehydrogenase (Mdh) or malate quinone oxidoreductase (Mqo), is a critical step of the tricarboxylic acid cycle. Both Mqo and Mdh are found in most bacterial genomes, but the level of functional redundancy between these enzymes remains unclear. A bioinformatic survey revealed that Mqo was not as widespread as Mdh in bacteria but that it was highly conserved in mycobacteria. We therefore used mycobacteria as a model genera to study the functional role(s) of Mqo and its redundancy with Mdh. We deleted mqo from the environmental saprophyte Mycobacterium smegmatis, which lacks Mdh, and found that Mqo was essential for growth on nonfermentable carbon sources. On fermentable carbon sources, the Δmqo mutant exhibited delayed growth and lowered oxygen consumption and secreted malate and fumarate as terminal end products. Furthermore, heterologous expression of Mdh from the pathogenic species Mycobacterium tuberculosis shortened the delayed growth on fermentable carbon sources and restored growth on nonfermentable carbon sources at a reduced growth rate. In M. tuberculosis, CRISPR interference of either mdh or mqo expression resulted in a slower growth rate compared to controls, which was further inhibited when both genes were knocked down simultaneously. These data reveal that exergonic Mqo activity powers mycobacterial growth under nonenergy limiting conditions and that endergonic Mdh activity complements Mqo activity, but at an energetic cost for mycobacterial growth. We propose Mdh is maintained in slow-growing mycobacterial pathogens for use under conditions such as hypoxia that require reductive tricarboxylic acid cycle activity.

An amiloride derivative is active against the F1Fo-ATP synthase and cytochrome bd oxidase of Mycobacterium tuberculosis.

Increasing antimicrobial resistance compels the search for next-generation inhibitors with differing or multiple molecular targets. In this regard, energy conservation in Mycobacterium tuberculosis has been clinically validated as a promising new drug target for combatting drug-resistant strains of M. tuberculosis. Here, we show that HM2-16F, a 6-substituted derivative of the FDA-approved drug amiloride, is an anti-tubercular inhibitor with bactericidal properties comparable to the FDA-approved drug bedaquiline (BDQ; Sirturo®) and inhibits the growth of bedaquiline-resistant mutants. We show that HM2-16F weakly inhibits the F1Fo-ATP synthase, depletes ATP, and affects the entry of acetyl-CoA into the Krebs cycle. HM2-16F synergizes with the cytochrome bcc-aa3 oxidase inhibitor Q203 (Telacebec) and co-administration with Q203 sterilizes in vitro cultures in 14 days. Synergy with Q203 occurs via direct inhibition of the cytochrome bd oxidase by HM2-16F. This study shows that amiloride derivatives represent a promising discovery platform for targeting energy generation in drug-resistant tuberculosis.

Multiplexed transcriptional repression identifies a network of bactericidal interactions between mycobacterial respiratory complexes.

Mycobacterium tuberculosis remains a leading cause of infectious disease morbidity and mortality for which new drug combination therapies are needed. Combinations of respiratory inhibitors can have synergistic or synthetic lethal interactions with sterilizing activity, suggesting that regimens with multiple bioenergetic inhibitors could shorten treatment times. However, realizing this potential requires an understanding of which combinations of respiratory complexes, when inhibited, have the strongest consequences on bacterial growth and viability. Here we have used multiplex CRISPR interference (CRISPRi) and Mycobacterium smegmatis as a physiological and molecular model for mycobacterial respiration to identify interactions between respiratory complexes. In this study, we identified synthetic lethal and synergistic interactions between respiratory complexes and demonstrated how the engineering of CRISPRi-guide sequences can be used to further explore networks of interacting gene pairs. These results provide fundamental insights into the functions of and interactions between bioenergetic complexes and the utility of CRISPRi in designing drug combinations.

Utilization of CRISPR interference to investigate the contribution of genes to pathogenesis in a macrophage model of Mycobacterium tuberculosis infection.

OBJECTIVES: There is an urgent need for novel drugs that target unique cellular pathways to combat infections caused by Mycobacterium tuberculosis. CRISPR interference (CRISPRi)-mediated transcriptional repression has recently been developed for use in mycobacteria as a genetic tool for identifying and validating essential genes as novel drug targets. Whilst CRISPRi has been applied to extracellular bacteria, no studies to date have determined whether CRISPRi can be used in M. tuberculosis infection models. METHODS: Using the human monocytic macrophage-like THP-1 cell line as a model for M. tuberculosis infection we investigated if CRISPRi can be activated within intracellular M. tuberculosis. RESULTS: The transcriptional repression of two candidate M. tuberculosis genes, i.e. mmpL3 and qcrB, leads to a reduction in viable M. tuberculosis within infected THP-1 cells. The reduction in viable colonies is dependent on both the level of CRISPRi-mediated repression and the duration of repression. CONCLUSIONS: These results highlight the utility of CRISPRi in exploring mycobacterial gene function and essentiality under a variety of conditions pertinent to host infection.

CRISPR interference identifies vulnerable cellular pathways with bactericidal phenotypes in Mycobacterium tuberculosis.

Mycobacterium tuberculosis remains a leading cause of death for which new drugs are needed. The identification of drug targets has been advanced by high-throughput and targeted genetic deletion strategies. Each though has limitations including the inability to distinguish between levels of vulnerability, lethality, and scalability as a molecular tool. Using mycobacterial CRISPR interference in combination with phenotypic screening, we have overcome these individual issues to investigate essentiality, vulnerability and lethality for 94 target genes from a diverse array of cellular pathways, many of which are potential antibiotic targets. Essential genes involved in cell wall synthesis and central cellular functions were equally vulnerable and often had bactericidal consequences. Conversely, essential genes involved in metabolism, oxidative phosphorylation, or amino acid synthesis were less vulnerable to inhibition and frequently bacteriostatic. In conclusion, this study provides novel insights into mycobacterial genetics and biology that will help to prioritize potential drug targets.

Novel Trifluoromethyl Pyrimidinone Compounds With Activity Against Mycobacterium tuberculosis.

The identification and development of new anti-tubercular agents are a priority research area. We identified the trifluoromethyl pyrimidinone series of compounds in a whole-cell screen against Mycobacterium tuberculosis. Fifteen primary hits had minimum inhibitory concentrations (MICs) with good potency IC90 is the concentration at which M. tuberculosis growth is inhibited by 90% (IC90 < 5 µM). We conducted a structure-activity relationship investigation for this series. We designed and synthesized an additional 44 molecules and tested all analogs for activity against M. tuberculosis and cytotoxicity against the HepG2 cell line. Substitution at the 5-position of the pyrimidinone with a wide range of groups, including branched and straight chain alkyl and benzyl groups, resulted in active molecules. Trifluoromethyl was the preferred group at the 6-position, but phenyl and benzyl groups were tolerated. The 2-pyridyl group was required for activity; substitution on the 5-position of the pyridyl ring was tolerated but not on the 6-position. Active molecules from the series demonstrated low selectivity, with cytotoxicity against eukaryotic cells being an issue. However, there were active and non-cytotoxic molecules; the most promising molecule had an MIC (IC90) of 4.9 µM with no cytotoxicity (IC50 > 100 µM). The series was inactive against Gram-negative bacteria but showed good activity against Gram-positive bacteria and yeast. A representative molecule from this series showed rapid concentration-dependent bactericidal activity against replicating M. tuberculosis bacilli with ~4 log kill in <7 days. Overall the biological properties were promising, if cytotoxicity could be reduced. There is scope for further medicinal chemistry optimization to improve the properties without major change in structural features.

Multiple Mutations in Mycobacterium tuberculosis MmpL3 Increase Resistance to MmpL3 Inhibitors.

The Mycobacterium tuberculosis protein MmpL3 performs an essential role in cell wall synthesis, since it effects the transport of trehalose monomycolates across the inner membrane. Numerous structurally diverse pharmacophores have been identified as inhibitors of MmpL3 largely based on the identification of resistant isolates with mutations in MmpL3. For some compounds, it is possible there are different primary or secondary targets. Here, we have investigated resistance to the spiral amine class of compounds. Isolation and sequencing of resistant mutants demonstrated that all had mutations in MmpL3. We hypothesized that if additional targets of this pharmacophore existed, then successive rounds to generate resistant isolates might reveal mutations in other loci. Since compounds were still active against resistant isolates, albeit with reduced potency, we isolated resistant mutants in this background at higher concentrations. After a second round of isolation with the spiral amine, we found additional mutations in MmpL3. To increase our chance of finding alternative targets, we ran a third round of isolation using a different molecule scaffold (AU1235, an adamantyl urea). Surprisingly, we obtained further mutations in MmpL3. Multiple mutations in MmpL3 increased the level and spectrum of resistance to different pharmacophores but did not incur a fitness cost in vitro These results support the hypothesis that MmpL3 is the primary mechanism of resistance and likely target for these pharmacophores.IMPORTANCEMycobacterium tuberculosis is a major global human pathogen, and new drugs and new drug targets are urgently required. Cell wall biosynthesis is a major target of current tuberculosis drugs and of new agents under development. Several new classes of molecules appear to have the same target, MmpL3, which is involved in the export and synthesis of the mycobacterial cell wall. However, there is still debate over whether MmpL3 is the primary or only target for these classes. We wanted to confirm the mechanism of resistance for one series. We identified mutations in MmpL3 which led to resistance to the spiral amine series. High-level resistance to these compounds and two other series was conferred by multiple mutations in the same protein (MmpL3). These mutations did not reduce growth rate in culture. These results support the hypothesis that MmpL3 is the primary mechanism of resistance and likely target for these pharmacophores.

Transcriptional Inhibition of the F1F0-Type ATP Synthase Has Bactericidal Consequences on the Viability of Mycobacteria.

Bedaquiline, an inhibitor of the mycobacterial ATP synthase, has revolutionized the treatment of Mycobacterium tuberculosis infection. Although a potent inhibitor, it is characterized by poorly understood delayed time-dependent bactericidal activity. Here, we demonstrate that in contrast to bedaquiline, the transcriptional inhibition of the ATP synthase in M. tuberculosis and Mycobacterium smegmatis has rapid bactericidal activity. These results validate the mycobacterial ATP synthase as a drug target with the potential for rapid bactericidal activity.

Two for the price of one: Attacking the energetic-metabolic hub of mycobacteria to produce new chemotherapeutic agents.

Cellular bioenergetics is an area showing promise for the development of new antimicrobials, antimalarials and cancer therapy. Enzymes involved in central carbon metabolism and energy generation are essential mediators of bacterial physiology, persistence and pathogenicity, lending themselves natural interest for drug discovery. In particular, succinate and malate are two major focal points in both the central carbon metabolism and the respiratory chain of Mycobacterium tuberculosis. Both serve as direct links between the citric acid cycle and the respiratory chain due to the quinone-linked reactions of succinate dehydrogenase, fumarate reductase and malate:quinone oxidoreductase. Inhibitors against these enzymes therefore hold the promise of disrupting two distinct, but essential, cellular processes at the same time. In this review, we discuss the roles and unique adaptations of these enzymes and critically evaluate the role that future inhibitors of these complexes could play in the bioenergetics target space.

Utilization of CRISPR Interference To Validate MmpL3 as a Drug Target in Mycobacterium tuberculosis.

There is an urgent need for novel therapeutics to treat Mycobacterium tuberculosis infections. Genetic strategies for validating novel targets are available, yet their time-consuming nature limits their utility. Here, using MmpL3 as a model target, we report on the application of mycobacterial CRISPR interference for the rapid validation of target essentiality and compound mode of action. This strategy has the potential to rapidly accelerate tuberculosis drug discovery.

Efficacy and Improved Resistance Potential of a Cofactor-Independent InhA Inhibitor of Mycobacterium tuberculosis in the C3HeB/FeJ Mouse Model.

AN12855 is a direct, cofactor-independent inhibitor of InhA in Mycobacterium tuberculosis In the C3HeB/FeJ mouse model with caseous necrotic lung lesions, AN12855 proved efficacious with a significantly lower resistance frequency than isoniazid. AN12855 drug levels were better retained in necrotic lesions and caseum where the majority of hard to treat, extracellular bacilli reside. Owing to these combined attributes, AN12855 represents a promising alternative to the frontline antituberculosis agent isoniazid.