Targeting Mycobacterium tuberculosis pH-driven adaptation.

Mycobacterium tuberculosis (Mtb) senses and adapts to host environmental cues as part of its pathogenesis. One important cue sensed by Mtb is the acidic pH of its host niche - the macrophage. Acidic pH induces widespread transcriptional and metabolic remodelling in Mtb. These adaptations to acidic pH can lead Mtb to slow its growth and promote pathogenesis and antibiotic tolerance. Mutants defective in pH-dependent adaptations exhibit reduced virulence in macrophages and animal infection models, suggesting that chemically targeting these pH-dependent pathways may have therapeutic potential. In this review, we discuss mechanisms by which Mtb regulates its growth and metabolism at acidic pH. Additionally, we consider the therapeutic potential of disrupting pH-driven adaptations in Mtb and review the growing class of compounds that exhibit pH-dependent activity or target pathways important for adaptation to acidic pH.

Discovery and characterization of antimycobacterial nitro-containing compounds with distinct mechanisms of action and in vivo efficacy.

Nitro-containing compounds have emerged as important agents in the control of tuberculosis (TB). From a whole-cell high-throughput screen for Mycobacterium tuberculosis (Mtb) growth inhibitors, 10 nitro-containing compounds were prioritized for characterization and mechanism of action studies. HC2209, HC2210, and HC2211 are nitrofuran-based prodrugs that need the cofactor F420 machinery for activation. Unlike pretomanid which depends only on deazaflavin-dependent nitroreductase (Ddn), these nitrofurans depend on Ddn and possibly another F420-dependent reductase for activation. These nitrofurans also differ from pretomanid in their potent activity against Mycobacterium abscessus. Four dinitrobenzamides (HC2217, HC2226, HC2238, and HC2239) and a nitrofuran (HC2250) are proposed to be inhibitors of decaprenyl-phosphoryl-ribose 2'-epimerase 1 (DprE1), based on isolation of resistant mutations in dprE1. Unlike other DprE1 inhibitors, HC2250 was found to be potent against non-replicating persistent bacteria, suggesting additional targets. Two of the compounds, HC2233 and HC2234, were found to have potent, sterilizing activity against replicating and non-replicating Mtb in vitro, but a proposed mechanism of action could not be defined. In a pilot in vivo efficacy study, HC2210 was orally bioavailable and efficacious in reducing bacterial load by ~1 log in a chronic murine TB infection model.

Response to Hypoxia and the Ensuing Dysregulation of Inflammation Impacts Mycobacterium tuberculosis Pathogenicity.

Rationale: Different Mycobacterium tuberculosis (Mtb) strains exhibit variable degrees of virulence in humans and animal models. Differing stress response strategies used by different strains of Mtb could influence virulence. Objectives: We compared the virulence of two strains of Mtb with use in animal model research: CDC1551 and Erdman. Methods: Rhesus macaques, which develop human-like tuberculosis attributes and pathology, were infected with a high dose of either strain via aerosol, and virulence was compared by bacterial burden and pathology. Measurements and Main Results: Infection with Erdman resulted in significantly shorter times to euthanasia and higher bacterial burdens and greater systemic inflammation and lung pathology relative to those infected with CDC1551. Macaques infected with Erdman also exhibited significantly higher early inflammatory myeloid cell influx to the lung, greater macrophage and T cell activity, and higher expression of lung remodeling (extracellular matrix) genes, consistent with greater pathology. Expression of NOTCH4 (neurogenic locus notch homolog 4) signaling, which is induced in response to hypoxia and promotes undifferentiated cellular state, was also higher in Erdman-infected lungs. The granulomas generated by Erdman, and not CDC1551, infection appeared to have larger regions of necrosis, which is strongly associated with hypoxia. To better understand the mechanisms of differential hypoxia induction by these strains, we subjected both to hypoxia in vitro. Erdman induced higher concentrations of DosR regulon relative to CDC1551. The DosR regulon is the global regulator of response to hypoxia in Mtb and critical for its persistence in granulomas. Conclusions: Our results show that the response to hypoxia is a critical mediator of virulence determination in Mtb, with potential impacts on bacillary persistence, reactivation, and efficiency of therapeutics.

ppe51 Variants Enable Growth of Mycobacterium tuberculosis at Acidic pH by Selectively Promoting Glycerol Uptake.

In defined media supplemented with single carbon sources, Mycobacterium tuberculosis (Mtb) exhibits carbon source specific growth restriction. When supplied with glycerol as the sole carbon source at pH 5.7, Mtb establishes a metabolically active state of nonreplicating persistence known as acid growth arrest. We hypothesized that acid growth arrest on glycerol is not a metabolic restriction, but rather an adaptive response. To test this hypothesis, we selected for and identified several Mtb mutants that could grow under these restrictive conditions. All mutations were mapped to the ppe51 gene and resulted in variants with 3 different amino acid substitutions- S211R, E215K, and A228D. Expression of the ppe51 variants in Mtb promoted growth at acidic pH showing that the mutant alleles are sufficient to cause the dominant gain-of-function, Enhanced Acid Growth (EAG) phenotype. Testing growth on other single carbon sources showed the PPE51 variants specifically enhanced growth on glycerol, suggesting PPE51 plays a role in glycerol uptake. Using radiolabeled glycerol, enhanced glycerol uptake was observed in Mtb expressing the PPE51 (S211R) variant, with glycerol overaccumulation in triacylglycerol. Notably, the EAG phenotype is deleterious for growth in macrophages, where the mutants have selectively faster replication and reduced survival in activated macrophages compared to resting macrophages. Recombinant PPE51 protein exhibited differential thermostability in the wild type (WT) or S211R variants in the presence of glycerol, supporting the model that EAG substitutions alter PPE51-glycerol interactions. Together, these findings support that PPE51 variants selectively promote glycerol uptake and that slowed growth at acidic pH is an important adaptive mechanism required for macrophage pathogenesis. IMPORTANCE It is puzzling why Mycobacterium tuberculosis (Mtb) cannot grow on glycerol at acidic pH, as it has a carbon source and oxygen, everything it needs to grow. In this study, we found that Mtb limits uptake of glycerol at acidic pH to restrict its growth and that mutations in ppe51 promote uptake of glycerol at acidic pH and enable growth. That is, Mtb can grow well at acidic pH on glycerol, but has adapted instead to stop growth. Notably, ppe51 variants exhibit enhanced replication and reduced survival in activated macrophages, supporting a role for pH-dependent slowed growth during macrophage pathogenesis.

WhiB6 regulation of ESX-1 gene expression is controlled by a negative feedback loop in Mycobacterium marinum.

ESX (ESAT-6 system) export systems play diverse roles across mycobacterial species. Interestingly, genetic disruption of ESX systems in different species does not result in an accumulation of protein substrates in the mycobacterial cell. However, the mechanisms underlying this observation are elusive. We hypothesized that the levels of ESX substrates were regulated by a feedback-control mechanism, linking the levels of substrates to the secretory status of ESX systems. To test this hypothesis, we used a combination of genetic, transcriptomic, and proteomic approaches to define export-dependent mechanisms regulating the levels of ESX-1 substrates in Mycobacterium marinum WhiB6 is a transcription factor that regulates expression of genes encoding ESX-1 substrates. We found that, in the absence of the genes encoding conserved membrane components of the ESX-1 system, the expression of the whiB6 gene and genes encoding ESX-1 substrates were reduced. Accordingly, the levels of ESX-1 substrates were decreased, and WhiB6 was not detected in M. marinum strains lacking genes encoding ESX-1 components. We demonstrated that, in the absence of EccCb1, a conserved ESX-1 component, substrate gene expression was restored by constitutive, but not native, expression of the whiB6 gene. Finally, we found that the loss of WhiB6 resulted in a virulent M. marinum strain with reduced ESX-1 secretion. Together, our findings demonstrate that the levels of ESX-1 substrates in M. marinum are fine-tuned by negative feedback control, linking the expression of the whiB6 gene to the presence, not the functionality, of the ESX-1 membrane complex.

Identification of New MmpL3 Inhibitors by Untargeted and Targeted Mutant Screens Defines MmpL3 Domains with Differential Resistance.

The Mycobacterium tuberculosis mycolate flippase MmpL3 has been the proposed target for multiple inhibitors with diverse chemical scaffolds. This diversity in chemical scaffolds has made it difficult to predict compounds that inhibit MmpL3 without whole-genome sequencing of isolated resistant mutants. Here, we describe the identification of four new inhibitors that select for resistance mutations in mmpL3. Using these resistant mutants, we conducted a targeted whole-cell phenotypic screen of 163 novel M. tuberculosis growth inhibitors for differential growth inhibition of wild-type M. tuberculosis compared to the growth of a pool of 24 unique mmpL3 mutants. The screen successfully identified six additional putative MmpL3 inhibitors. The compounds were bactericidal both in vitro and against intracellular M. tuberculosisM. tuberculosis cells treated with these compounds were shown to accumulate trehalose monomycolates, have reduced levels of trehalose dimycolate, and displace an MmpL3-specific probe, supporting MmpL3 as the target. The inhibitors were mycobacterium specific, with several also showing activity against the nontuberculous mycobacterial species M. abscessus Cluster analysis of cross-resistance profiles generated by dose-response experiments for each combination of 13 MmpL3 inhibitors against each of the 24 mmpL3 mutants defined two clades of inhibitors and two clades of mmpL3 mutants. Pairwise combination studies of the inhibitors revealed interactions that were specific to the clades identified in the cross-resistance profiling. Additionally, modeling of resistance-conferring substitutions to the MmpL3 crystal structure revealed clade-specific localization of the residues to specific domains of MmpL3, with the clades showing differential resistance. Several compounds exhibited high solubility and stability in microsomes and low cytotoxicity in macrophages, supporting their further development. The combined study of multiple mutants and novel compounds provides new insights into structure-function interactions of MmpL3 and small-molecule inhibitors.

Inhibitors of Mycobacterium tuberculosis DosRST signaling and persistence.

The Mycobacterium tuberculosis (Mtb) DosRST two-component regulatory system promotes the survival of Mtb during non-replicating persistence (NRP). NRP bacteria help drive the long course of tuberculosis therapy; therefore, chemical inhibition of DosRST may inhibit the ability of Mtb to establish persistence and thus shorten treatment. Using a DosRST-dependent fluorescent Mtb reporter strain, a whole-cell phenotypic high-throughput screen of a ∼540,000 compound small-molecule library was conducted. The screen discovered novel inhibitors of the DosRST regulon, including three compounds that were subject to follow-up studies: artemisinin, HC102A and HC103A. Under hypoxia, all three compounds inhibit Mtb-persistence-associated physiological processes, including triacylglycerol synthesis, survival and antibiotic tolerance. Artemisinin functions by disabling the heme-based DosS and DosT sensor kinases by oxidizing ferrous heme and generating heme-artemisinin adducts. In contrast, HC103A inhibits DosS and DosT autophosphorylation activity without targeting the sensor kinase heme.

HC2091 Kills Mycobacterium tuberculosis by Targeting the MmpL3 Mycolic Acid Transporter.

Tuberculosis, caused by the intracellular pathogen Mycobacterium tuberculosis, is a deadly disease that requires a long course of treatment. The emergence of drug-resistant strains has driven efforts to discover new small molecules that can kill the bacterium. Here, we report characterizations of the compound HC2091, which kills M. tuberculosis in a time- and dose-dependent manner in vitro and inhibits M. tuberculosis growth in macrophages. Whole-genome sequencing of spontaneous HC2091-resistant mutants identified single-nucleotide variants in the mmpL3 mycolic acid transporter gene. HC2091-resistant mutants do not exhibit cross-resistance with the well-characterized Mycobacterium membrane protein large 3 (MmpL3) inhibitor SQ109, suggesting a distinct mechanism of interaction with MmpL3. Additionally, HC2091 does not modulate bacterial membrane potential or kill nonreplicating M. tuberculosis, thus acting differently from other known MmpL3 inhibitors. RNA sequencing (RNA-seq) transcriptional profiling and lipid profiling of M. tuberculosis treated with HC2091 or SQ109 show that the two compounds target a similar pathway. HC2091 has a chemical structure dissimilar to those of previously described MmpL3 inhibitors, supporting the notion that HC2091 is a new class of MmpL3 inhibitor.

Mycobacterium tuberculosis Reporter Strains as Tools for Drug Discovery and Development.

Reporter strains have proven to be powerful tools to study Mycobacterium tuberculosis (Mtb) physiology. Transcriptional and translational reporter strains are engineered by fusing a readout gene, encoding a fluorescent, luminescent or enzymatic protein, downstream of a promoter or in-frame with a gene of interest. When the reporter is expressed, it generates a signal that acts as a synthetic phenotype, enabling the study of physiologies that might have otherwise been hidden. This review will discuss approaches for generating reporter strains in Mtb and how they can be used as tools for high-throughput genetic and small molecule screening and as biomarkers for examining Mtb responses to drug or immune stresses during animal infections. Fluorescent reporter strains have an added benefit in that they can be used for single-cell studies both in vitro and in vivo, thus enabling the study of mechanisms underlying phenotypic heterogeneity. Recent examples of the use of Mtb reporter strains will be presented with a focus on how they can be used as tools for drug discovery and development. © 2018 IUBMB Life, 70(9):818-825, 2018.

A Nonsense Mutation in Mycobacterium marinum That Is Suppressible by a Novel Mechanism.

Mycobacterial pathogens use the ESAT-6 system 1 (Esx-1) exporter to promote virulence. Previously, we used gene disruption and complementation to conclude that the MMAR_0039 gene in Mycobacterium marinum is required to promote Esx-1 export. Here we applied molecular genetics, proteomics, and whole-genome sequencing to demonstrate that the MMAR_0039 gene is not required for Esx-1 secretion or virulence. These findings suggest that we initially observed an indirect mechanism of genetic complementation. We identified a spontaneous nonsense mutation in a known Esx-1-associated gene which causes a loss of Esx-1 activity. We show that the Esx-1 function was restored by nonsense suppression. Moreover, we identified a polar mutation in the ppsC gene which reduced cellular impermeability but did not impact cytotoxicity in macrophages. Our studies reveal insight into Esx-1 export, nonsense suppression, and cell envelope lipid biogenesis.

Novel inhibitors of cholesterol degradation in Mycobacterium tuberculosis reveal how the bacterium's metabolism is constrained by the intracellular environment.

Mycobacterium tuberculosis (Mtb) relies on a specialized set of metabolic pathways to support growth in macrophages. By conducting an extensive, unbiased chemical screen to identify small molecules that inhibit Mtb metabolism within macrophages, we identified a significant number of novel compounds that limit Mtb growth in macrophages and in medium containing cholesterol as the principle carbon source. Based on this observation, we developed a chemical-rescue strategy to identify compounds that target metabolic enzymes involved in cholesterol metabolism. This approach identified two compounds that inhibit the HsaAB enzyme complex, which is required for complete degradation of the cholesterol A/B rings. The strategy also identified an inhibitor of PrpC, the 2-methylcitrate synthase, which is required for assimilation of cholesterol-derived propionyl-CoA into the TCA cycle. These chemical probes represent new classes of inhibitors with novel modes of action, and target metabolic pathways required to support growth of Mtb in its host cell. The screen also revealed a structurally-diverse set of compounds that target additional stage(s) of cholesterol utilization. Mutants resistant to this class of compounds are defective in the bacterial adenylate cyclase Rv1625/Cya. These data implicate cyclic-AMP (cAMP) in regulating cholesterol utilization in Mtb, and are consistent with published reports indicating that propionate metabolism is regulated by cAMP levels. Intriguingly, reversal of the cholesterol-dependent growth inhibition caused by this subset of compounds could be achieved by supplementing the media with acetate, but not with glucose, indicating that Mtb is subject to a unique form of metabolic constraint induced by the presence of cholesterol.

SPARTA: Simple Program for Automated reference-based bacterial RNA-seq Transcriptome Analysis.

BACKGROUND: Many tools exist in the analysis of bacterial RNA sequencing (RNA-seq) transcriptional profiling experiments to identify differentially expressed genes between experimental conditions. Generally, the workflow includes quality control of reads, mapping to a reference, counting transcript abundance, and statistical tests for differentially expressed genes. In spite of the numerous tools developed for each component of an RNA-seq analysis workflow, easy-to-use bacterially oriented workflow applications to combine multiple tools and automate the process are lacking. With many tools to choose from for each step, the task of identifying a specific tool, adapting the input/output options to the specific use-case, and integrating the tools into a coherent analysis pipeline is not a trivial endeavor, particularly for microbiologists with limited bioinformatics experience. RESULTS: To make bacterial RNA-seq data analysis more accessible, we developed a Simple Program for Automated reference-based bacterial RNA-seq Transcriptome Analysis (SPARTA). SPARTA is a reference-based bacterial RNA-seq analysis workflow application for single-end Illumina reads. SPARTA is turnkey software that simplifies the process of analyzing RNA-seq data sets, making bacterial RNA-seq analysis a routine process that can be undertaken on a personal computer or in the classroom. The easy-to-install, complete workflow processes whole transcriptome shotgun sequencing data files by trimming reads and removing adapters, mapping reads to a reference, counting gene features, calculating differential gene expression, and, importantly, checking for potential batch effects within the data set. SPARTA outputs quality analysis reports, gene feature counts and differential gene expression tables and scatterplots. CONCLUSIONS: SPARTA provides an easy-to-use bacterial RNA-seq transcriptional profiling workflow to identify differentially expressed genes between experimental conditions. This software will enable microbiologists with limited bioinformatics experience to analyze their data and integrate next generation sequencing (NGS) technologies into the classroom. The SPARTA software and tutorial are available at sparta.readthedocs.org.

Polysorbates prevent biofilm formation and pathogenesis of Escherichia coli O104:H4.

Escherichia coli biotype O104:H4 recently caused the deadliest E. coli outbreak ever reported. Based on prior results, it was hypothesized that compounds inhibiting biofilm formation by O104:H4 would reduce its pathogenesis. The nonionic surfactants polysorbate 80 (PS80) and polysorbate 20 (PS20) were found to reduce biofilms by ≥ 90% at submicromolar concentrations and elicited nearly complete dispersal of preformed biofilms. PS80 did not significantly impact in vivo colonization in a mouse infection model; however, mice treated with PS80 exhibited almost no intestinal inflammation or tissue damage while untreated mice exhibited robust pathology. As PS20 and PS80 are classified as 'Generally Recognized as Safe' (GRAS) compounds by the Food and Drug Administration (FDA), these compounds have clinical potential to treat future O104:H4 outbreaks.

Slow growth of Mycobacterium tuberculosis at acidic pH is regulated by phoPR and host-associated carbon sources.

During pathogenesis, Mycobacterium tuberculosis (Mtb) colonizes environments, such as the macrophage or necrotic granuloma, that are acidic and rich in cholesterol and fatty acids. The goal of this study was to examine how acidic pH and available carbon sources interact to regulate Mtb physiology. Here we report that Mtb growth at acidic pH requires host-associated carbon sources that function at the intersection of glycolysis and the TCA cycle, such as pyruvate, acetate, oxaloacetate and cholesterol. In contrast, in other tested carbon sources, Mtb fully arrests its growth at acidic pH and establishes a state of non-replicating persistence. Growth-arrested Mtb is resuscitated by the addition of pyruvate suggesting that growth arrest is due to a pH-dependent checkpoint on metabolism. Additionally, we demonstrate that the phoPR two-component regulatory system is required to slow Mtb growth at acidic pH and functions to maintain redox homeostasis. Transcriptional profiling and functional metabolic studies demonstrate that signals from acidic pH and carbon source are integrated to remodel pathways associated with anaplerotic central metabolism, lipid anabolism and the regeneration of oxidized cofactors. Because phoPR is required for Mtb virulence in animals, we suggest that pH-driven adaptation may be critical to Mtb pathogenesis.

The Carbonic Anhydrase Inhibitor Ethoxzolamide Inhibits the Mycobacterium tuberculosis PhoPR Regulon and Esx-1 Secretion and Attenuates Virulence.

Mycobacterium tuberculosis must sense and adapt to host environmental cues to establish and maintain an infection. The two-component regulatory system PhoPR plays a central role in sensing and responding to acidic pH within the macrophage and is required for M. tuberculosis intracellular replication and growth in vivo. Therefore, the isolation of compounds that inhibit PhoPR-dependent adaptation may identify new antivirulence therapies to treat tuberculosis. Here, we report that the carbonic anhydrase inhibitor ethoxzolamide inhibits the PhoPR regulon and reduces pathogen virulence. We show that treatment of M. tuberculosis with ethoxzolamide recapitulates phoPR mutant phenotypes, including downregulation of the core PhoPR regulon, altered accumulation of virulence-associated lipids, and inhibition of Esx-1 protein secretion. Quantitative single-cell imaging of a PhoPR-dependent fluorescent reporter strain demonstrates that ethoxzolamide inhibits PhoPR-regulated genes in infected macrophages and mouse lungs. Moreover, ethoxzolamide reduces M. tuberculosis growth in both macrophages and infected mice. Ethoxzolamide inhibits M. tuberculosis carbonic anhydrase activity, supporting a previously unrecognized link between carbonic anhydrase activity and PhoPR signaling. We propose that ethoxzolamide may be pursued as a new class of antivirulence therapy that functions by modulating expression of the PhoPR regulon and Esx-1-dependent virulence.

Mycobacterium tuberculosis responds to chloride and pH as synergistic cues to the immune status of its host cell.

The ability of Mycobacterium tuberculosis (Mtb) to thrive in its phagosomal niche is critical for its establishment of a chronic infection. This requires that Mtb senses and responds to intraphagosomal signals such as pH. We hypothesized that Mtb would respond to additional intraphagosomal factors that correlate with maturation. Here, we demonstrate that [Cl⁻] and pH correlate inversely with phagosome maturation, and identify Cl⁻ as a novel environmental cue for Mtb. Mtb responds to Cl⁻ and pH synergistically, in part through the activity of the two-component regulator phoPR. Following identification of promoters responsive to Cl⁻ and pH, we generated a reporter Mtb strain that detected immune-mediated changes in the phagosomal environment during infection in a mouse model. Our study establishes Cl⁻ and pH as linked environmental cues for Mtb, and illustrates the utility of reporter bacterial strains for the study of Mtb-host interactions in vivo.

The Mycobacterium tuberculosis MmpL3 inhibitor MSU-43085 is active in a mouse model of infection.

IMPORTANCE: MmpL3 is a protein that is required for the survival of bacteria that cause tuberculosis (TB) and nontuberculous mycobacterial (NTM) infections. This report describes the discovery and characterization of a new small molecule, MSU-43085, that targets MmpL3 and is a potent inhibitor of Mycobacterium tuberculosis (Mtb) and M. abscessus survival. MSU-43085 is shown to be orally bioavailable and efficacious in an acute model of Mtb infection. However, the analog is inactive against Mtb in chronically infected mice. Pharmacokinetic and metabolite identification studies identified in vivo metabolism of MSU-43085, leading to a short half-life in treated mice. These proof-of-concept studies will guide further development of the MSU-43085 series for the treatment of TB or NTM infections.

Total synthesis of dissectol A, using an enediolate-based Tsuji-Trost reaction.

Dissectol A is a rearranged terpene glycoside isolated from several flowering plants. Starting from glucose, the densely functionalized bicyclic structure has been prepared via site-selective oxidation and an intramolecular allylic alkylation reaction with an enediolate as the nucleophile. Despite earlier reports, dissectol A is not effective at inhibiting DevRS signaling in whole-cell Mycobacterium tuberculosis and does not inhibit growth of the bacterium.

A bacterial E3 ubiquitin ligase targets a host protein kinase to disrupt plant immunity.

Many bacterial pathogens of plants and animals use a type III secretion system to deliver diverse virulence-associated 'effector' proteins into the host cell. The mechanisms by which these effectors act are mostly unknown; however, they often promote disease by suppressing host immunity. One type III effector, AvrPtoB, expressed by the plant pathogen Pseudomonas syringae pv. tomato, has a carboxy-terminal domain that is an E3 ubiquitin ligase. Deletion of this domain allows an amino-terminal region of AvrPtoB (AvrPtoB(1-387)) to be detected by certain tomato varieties leading to immunity-associated programmed cell death. Here we show that a host kinase, Fen, physically interacts with AvrPtoB(1-387 )and is responsible for activating the plant immune response. The AvrPtoB E3 ligase specifically ubiquitinates Fen and promotes its degradation in a proteasome-dependent manner. This degradation leads to disease susceptibility in Fen-expressing tomato lines. Various wild species of tomato were found to exhibit immunity in response to AvrPtoB(1-387 )and not to full-length AvrPtoB. Thus, by acquiring an E3 ligase domain, AvrPtoB has thwarted a highly conserved host resistance mechanism.

Molecular Mechanisms of MmpL3 Function and Inhibition.

Mycobacteria species include a large number of pathogenic organisms such as Mycobacterium tuberculosis, Mycobacterium leprae, and various non-tuberculous mycobacteria. Mycobacterial membrane protein large 3 (MmpL3) is an essential mycolic acid and lipid transporter required for growth and cell viability. In the last decade, numerous studies have characterized MmpL3 with respect to protein function, localization, regulation, and substrate/inhibitor interactions. This review summarizes new findings in the field and seeks to assess future areas of research in our rapidly expanding understanding of MmpL3 as a drug target. An atlas of known MmpL3 mutations that provide resistance to inhibitors is presented, which maps amino acid substitutions to specific structural domains of MmpL3. In addition, chemical features of distinct classes of Mmpl3 inhibitors are compared to provide insights into shared and unique features of varied MmpL3 inhibitors.