Loss-of-function mutations in ndh do not confer delamanid, ethionamide, isoniazid, or pretomanid resistance in Mycobacterium tuberculosis.

Results from clinical strains and knockouts of the H37Rv and CDC1551 laboratory strains demonstrated that ndh (Rv1854c) is not a resistance-conferring gene for isoniazid, ethionamide, delamanid, or pretomanid in Mycobacterium tuberculosis. This difference in the susceptibility to NAD-adduct-forming drugs compared with other mycobacteria may be driven by differences in the absolute intrabacterial NADH concentration.

The promises and limitations of N-acetylcysteine as a potentiator of first-line and second-line tuberculosis drugs.

N-acetylcysteine (NAC) is most commonly used for the treatment of acetaminophen overdose and acetaminophen-induced liver injury. In patients infected with Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), NAC is given to treat hepatotoxicity induced by TB drugs. We had previously shown that cysteine, a derivative of NAC, potentiated the activity of isoniazid, a first-line TB drug, by preventing the emergence of INH resistance and persistence in M. tuberculosis in vitro. Herein, we demonstrate that in vitro, NAC has the same boosting activity with various combinations of first- and second-line TB drugs against drug-susceptible and multidrug-resistant M. tuberculosis strains. Similar to cysteine, NAC increased M. tuberculosis respiration. However, in M. tuberculosis-infected mice, the addition of NAC did not augment the activity of first- or second-line TB drugs. A comparison of the activity of NAC combined with TB drugs in murine and human macrophage cell lines revealed that studies in mice might not be recapitulated during host infection in vivo.

Plasticity of Mycobacterium tuberculosis NADH dehydrogenases and their role in virulence.

Worldwide control of the tuberculosis (TB) epidemic has not been achieved, and the latest statistics show that the TB problem might be more endemic than previously thought. Although drugs and a TB vaccine are available, TB eradication faces the challenges of increasing occurrences of multidrug-resistant and extensively drug-resistant Mycobacterium tuberculosis (Mtb) strains. To forestall this trend, the development of drugs targeting novel pathways is actively pursued. Recently, enzymes of the electron transport chain (ETC) have been determined to be the targets of potent antimycobacterial drugs such as bedaquiline. We focused on the three NADH dehydrogenases (Ndh, NdhA, and Nuo) of the Mtb ETC with the purpose of defining their role and essentiality in Mtb Each NADH dehydrogenase was deleted in both virulent and BSL2-approved Mtb strains, from which the double knockouts ΔndhΔnuoAN and ΔndhAΔnuoAN were constructed. The ΔndhΔndhA double knockout could not be obtained, suggesting that at least one type II NADH dehydrogenase is required for Mtb growth. Δndh and ΔndhΔnuoAN showed growth defects in vitro and in vivo, susceptibility to oxidative stress, and redox alterations, while the phenotypes of ΔndhA, ΔnuoAN, and ΔndhAΔnuoAN were similar to the parental strain. Interestingly, although ΔnuoAN had no phenotype in vivo, ΔndhΔnuoAN was the most severely attenuated strain in mice, suggesting a key role for Nuo in vivo when Ndh is absent. We conclude that Ndh is the main NADH dehydrogenase of Mtb and that compounds that could target both Ndh and Nuo would be good candidates for TB drug development.

Arginine-deprivation-induced oxidative damage sterilizes Mycobacterium tuberculosis.

Reactive oxygen species (ROS)-mediated oxidative stress and DNA damage have recently been recognized as contributing to the efficacy of most bactericidal antibiotics, irrespective of their primary macromolecular targets. Inhibitors of targets involved in both combating oxidative stress as well as being required for in vivo survival may exhibit powerful synergistic action. This study demonstrates that the de novo arginine biosynthetic pathway in Mycobacterium tuberculosis (Mtb) is up-regulated in the early response to the oxidative stress-elevating agent isoniazid or vitamin C. Arginine deprivation rapidly sterilizes the Mtb de novo arginine biosynthesis pathway mutants ΔargB and ΔargF without the emergence of suppressor mutants in vitro as well as in vivo. Transcriptomic and flow cytometry studies of arginine-deprived Mtb have indicated accumulation of ROS and extensive DNA damage. Metabolomics studies following arginine deprivation have revealed that these cells experienced depletion of antioxidant thiols and accumulation of the upstream metabolite substrate of ArgB or ArgF enzymes. ΔargB and ΔargF were unable to scavenge host arginine and were quickly cleared from both immunocompetent and immunocompromised mice. In summary, our investigation revealed in vivo essentiality of the de novo arginine biosynthesis pathway for Mtb and a promising drug target space for combating tuberculosis.

Characterization of Large Deletion Mutants of Mycobacterium tuberculosis Selected for Isoniazid Resistance.

Large genomic deletions (LGDs) (6 to 63 kbp) were observed in isoniazid-resistant Mycobacterium tuberculosis mutants derived from four M. tuberculosis strains. These LGDs had no growth defect in vitro but could be defective in intracellular growth and showed various sensitivities toward oxidative stress despite lacking katG The LGD regions comprise 74 genes, mostly of unknown function, that may be important for M. tuberculosis intracellular growth and protection against oxidative stress.

3-(Phenethylamino)demethyl(oxy)aaptamine as an anti-dormant mycobacterial substance: Isolation, evaluation and total synthesis.

3-(Phenethylamino)demethyl(oxy)aaptamine (1) was re-discovered from the marine sponge of Aaptos sp. as an anti-dormant mycobacterial substance through the bioassay-guided separation. Compound 1 showed potent anti-microbial activity against Mycobacterium bovis BCG with a minimum inhibitory concentration of 0.75 µg/mL under both aerobic conditions and hypoxic conditions inducing dormant state. Compound 1 was also effective against pathogenic M. tuberculosis strains including clinical multidrug-resistant strains. Furthermore, the successful total syntheses of 1 and its analog 3-aminodemethyl(oxy)aaptamine (2) afford sufficient quantities for further biological studies.

Enhanced respiration prevents drug tolerance and drug resistance in Mycobacterium tuberculosis.

Persistence, manifested as drug tolerance, represents a significant obstacle to global tuberculosis control. The bactericidal drugs isoniazid and rifampicin kill greater than 99% of exponentially growing Mycobacterium tuberculosis (Mtb) cells, but the remaining cells are persisters, cells with decreased metabolic rate, refractory to killing by these drugs, and able to generate drug-resistant mutants. We discovered that the combination of cysteine or other small thiols with either isoniazid or rifampicin prevents the formation of drug-tolerant and drug-resistant cells in Mtb cultures. This effect was concentration- and time-dependent, relying on increased oxygen consumption that triggered enhanced production of reactive oxygen species. In infected murine macrophages, the addition of N-acetylcysteine to isoniazid treatment potentiated the killing of Mtb Furthermore, we demonstrate that the addition of small thiols to Mtb drug treatment shifted the menaquinol/menaquinone balance toward a reduced state that stimulates Mtb respiration and converts persister cells to metabolically active cells. This prevention of both persister cell formation and drug resistance leads ultimately to mycobacterial cell death. Strategies to enhance respiration and initiate oxidative damage should improve tuberculosis chemotherapies.

Mycobacterium tuberculosis universal stress protein Rv2623 interacts with the putative ATP binding cassette (ABC) transporter Rv1747 to regulate mycobacterial growth.

We have previously shown that the Mycobacterium tuberculosis universal stress protein Rv2623 regulates mycobacterial growth and may be required for the establishment of tuberculous persistence. Here, yeast two-hybrid and affinity chromatography experiments have demonstrated that Rv2623 interacts with one of the two forkhead-associated domains (FHA I) of Rv1747, a putative ATP-binding cassette transporter annotated to export lipooligosaccharides. FHA domains are signaling protein modules that mediate protein-protein interactions to modulate a wide variety of biological processes via binding to conserved phosphorylated threonine (pT)-containing oligopeptides of the interactors. Biochemical, immunochemical and mass spectrometric studies have shown that Rv2623 harbors pT and specifically identified threonine 237 as a phosphorylated residue. Relative to wild-type Rv2623 (Rv2623WT), a mutant protein in which T237 has been replaced with a non-phosphorylatable alanine (Rv2623T237A) exhibits decreased interaction with the Rv1747 FHA I domain and diminished growth-regulatory capacity. Interestingly, compared to WT bacilli, an M. tuberculosis Rv2623 null mutant (ΔRv2623) displays enhanced expression of phosphatidyl-myo-inositol mannosides (PIMs), while the ΔRv1747 mutant expresses decreased levels of PIMs. Animal studies have previously shown that ΔRv2623 is hypervirulent, while ΔRv1747 is growth-attenuated. Collectively, these data have provided evidence that Rv2623 interacts with Rv1747 to regulate mycobacterial growth; and this interaction is mediated via the recognition of the conserved Rv2623 pT237-containing FHA-binding motif by the Rv1747 FHA I domain. The divergent aberrant PIM profiles and the opposing in vivo growth phenotypes of ΔRv2623 and ΔRv1747, together with the annotated lipooligosaccharide exporter function of Rv1747, suggest that Rv2623 interacts with Rv1747 to modulate mycobacterial growth by negatively regulating the activity of Rv1747; and that Rv1747 might function as a transporter of PIMs. Because these glycolipids are major mycobacterial cell envelope components that can impact on the immune response, our findings raise the possibility that Rv2623 may regulate bacterial growth, virulence, and entry into persistence, at least in part, by modulating the levels of bacillary PIM expression, perhaps through negatively regulating the Rv1747-dependent export of the immunomodulatory PIMs to alter host-pathogen interaction, thereby influencing the fate of M. tuberculosis in vivo.

Deletion of a dehydratase important for intracellular growth and cording renders rough Mycobacterium abscessus avirulent.

Mycobacterium abscessus (Mabs) is a rapidly growing Mycobacterium and an emerging pathogen in humans. Transitioning from a smooth (S) high-glycopeptidolipid (GPL) producer to a rough (R) low-GPL producer is associated with increased virulence in zebrafish, which involves the formation of massive serpentine cords, abscesses, and rapid larval death. Generating a cord-deficient Mabs mutant would allow us to address the contribution of cording in the physiopathological signs of the R variant. Herein, a deletion mutant of MAB_4780, encoding a dehydratase, distinct from the ß-hydroxyacyl-ACP dehydratase HadABC complex, was constructed in the R morphotype. This mutant exhibited an alteration of the mycolic acid composition and a pronounced defect in cording. This correlated with an extremely attenuated phenotype not only in wild-type but also in immunocompromised zebrafish embryos lacking either macrophages or neutrophils. The abolition of granuloma formation in embryos infected with the dehydratase mutant was associated with a failure to replicate in macrophages, presumably due to limited inhibition of the phagolysosomal fusion. Overall, these results indicate that MAB_4780 is required for Mabs to successfully establish acute and lethal infections. Therefore, targeting MAB_4780 may represent an attractive antivirulence strategy to control Mabs infections, refractory to most standard chemotherapeutic interventions. The combination of a dehydratase assay with a high-resolution crystal structure of MAB_4780 opens the way to identify such specific inhibitors.

Separable roles for Mycobacterium tuberculosis ESX-3 effectors in iron acquisition and virulence.

Mycobacterium tuberculosis (Mtb) encodes five type VII secretion systems (T7SS), designated ESX-1-ESX-5, that are critical for growth and pathogenesis. The best characterized is ESX-1, which profoundly impacts host cell interactions. In contrast, the ESX-3 T7SS is implicated in metal homeostasis, but efforts to define its function have been limited by an inability to recover deletion mutants. We overcame this impediment using medium supplemented with various iron complexes to recover mutants with deletions encompassing select genes within esx-3 or the entire operon. The esx-3 mutants were defective in uptake of siderophore-bound iron and dramatically accumulated cell-associated mycobactin siderophores. Proteomic analyses of culture filtrate revealed that secretion of EsxG and EsxH was codependent and that EsxG-EsxH also facilitated secretion of several members of the proline-glutamic acid (PE) and proline-proline-glutamic acid (PPE) protein families (named for conserved PE and PPE N-terminal motifs). Substrates that depended on EsxG-EsxH for secretion included PE5, encoded within the esx-3 locus, and the evolutionarily related PE15-PPE20 encoded outside the esx-3 locus. In vivo characterization of the mutants unexpectedly showed that the ESX-3 secretion system plays both iron-dependent and -independent roles in Mtb pathogenesis. PE5-PPE4 was found to be critical for the siderophore-mediated iron-acquisition functions of ESX-3. The importance of this iron-acquisition function was dependent upon host genotype, suggesting a role for ESX-3 secretion in counteracting host defense mechanisms that restrict iron availability. Further, we demonstrate that the ESX-3 T7SS secretes certain effectors that are important for iron uptake while additional secreted effectors modulate virulence in an iron-independent fashion.

Defining a temporal order of genetic requirements for development of mycobacterial biofilms.

Most mycobacterial species spontaneously form biofilms, inducing unique growth physiologies and reducing drug sensitivity. Biofilm growth progresses through three genetically programmed stages: substratum attachment, intercellular aggregation and architecture maturation. Growth of Mycobacterium smegmatis biofilms requires multiple factors including a chaperonin (GroEL1) and a nucleoid-associated protein (Lsr2), although how their activities are linked remains unclear. Here it is shown that Lsr2 participates in intercellular aggregation, but substratum attachment of Lsr2 mutants is unaffected, thereby genetically distinguishing these developmental stages. Further, a suppressor mutation in a glycopeptidolipid synthesis gene (mps) that results in hyperaggregation of cells and fully restores the form and functions of Δlsr2 mutant biofilms was identified. Suppression by the mps mutation is specific to Δlsr2; it does not rescue the maturation-deficient biofilms of a ΔgroEL1 mutant, thereby differentiating the process of aggregation from maturation. Gene expression analysis supports a stepwise process of maturation, highlighted by temporally separated, transient inductions of iron and nitrogen import genes. Furthermore, GroEL1 activity is required for induction of nitrogen, but not iron, import genes. Together, the findings begin to define molecular checkpoints during development of mycobacterial biofilms.

Vitamin C Potentiates the Killing of Mycobacterium tuberculosis by the First-Line Tuberculosis Drugs Isoniazid and Rifampin in Mice.

The treatment of drug-susceptible tuberculosis (TB) is long and cumbersome. Mismanagement of TB treatment can lead to the emergence of drug resistance in patients, so shortening the treatment duration could significantly improve TB chemotherapy and prevent the development of drug resistance. We previously discovered that high concentrations of vitamin C sterilize cultures of drug-susceptible and drug-resistant Mycobacterium tuberculosis Here, we tested subinhibitory concentration of vitamin C in combination with TB drugs against M. tuberculosisin vitro and in a mouse model of M. tuberculosis infection. In vivo, we showed that the vitamin C level in mouse serum can be increased by intraperitoneal injection of vitamin C to reach vitamin C levels close to the concentrations required for activity in vitro Although vitamin C had no activity by itself in M. tuberculosis-infected mice, the combination of vitamin C with the first-line TB drugs isoniazid and rifampin reduced the bacterial burden in the lungs of M. tuberculosis-infected mice faster than isoniazid and rifampin combined in two independent experiments. These experiments suggest that the addition of vitamin C to first-line TB drugs could shorten TB treatment. Vitamin C, an inexpensive and nontoxic compound, could easily be added to the TB pharmacopeia to substantially improve chemotherapy outcome, which would have a significant impact on the worldwide TB community.

Auranofin exerts broad-spectrum bactericidal activities by targeting thiol-redox homeostasis.

Infections caused by antibiotic-resistant bacteria are a rising public health threat and make the identification of new antibiotics a priority. From a cell-based screen for bactericidal compounds against Mycobacterium tuberculosis under nutrient-deprivation conditions we identified auranofin, an orally bioavailable FDA-approved antirheumatic drug, as having potent bactericidal activities against both replicating and nonreplicating M. tuberculosis. We also found that auranofin is active against other Gram-positive bacteria, including Bacillus subtilis and Enterococcus faecalis, and drug-sensitive and drug-resistant strains of Enterococcus faecium and Staphylococcus aureus. Our biochemical studies showed that auranofin inhibits the bacterial thioredoxin reductase, a protein essential in many Gram-positive bacteria for maintaining the thiol-redox balance and protecting against reactive oxidative species. Auranofin decreases the reducing capacity of target bacteria, thereby sensitizing them to oxidative stress. Finally, auranofin was efficacious in a murine model of methicillin-resistant S. aureus infection. These results suggest that the thioredoxin-mediated redox cascade of Gram-positive pathogens is a valid target for the development of antibacterial drugs, and that the existing clinical agent auranofin may be repurposed to aid in the treatment of several important antibiotic-resistant pathogens.

Small Molecules Targeting Mycobacterium tuberculosis Type II NADH Dehydrogenase Exhibit Antimycobacterial Activity.

The generation of ATP through oxidative phosphorylation is an essential metabolic function for Mycobaterium tuberculosis (Mtb), regardless of the growth environment. The type II NADH dehydrogenase (Ndh-2) is the conduit for electrons into the pathway, and is absent in the mammalian genome, thus making it a potential drug target. Herein, we report the identification of two types of small molecules as selective inhibitors for Ndh-2 through a multicomponent high-throughput screen. Both compounds block ATP synthesis, lead to effects consistent with loss of NADH turnover, and importantly, exert bactericidal activity against Mtb. Extensive medicinal chemistry optimization afforded the best analogue with an MIC of 90 nm against Mtb. Moreover, the two scaffolds have differential inhibitory activities against the two homologous Ndh-2 enzymes in Mtb, which will allow precise control over Ndh-2 function in Mtb to facilitate the assessment of this anti-TB drug target.

Determinants of the Inhibition of DprE1 and CYP2C9 by Antitubercular Thiophenes.

Mycobacterium tuberculosis (Mtb) DprE1, an essential isomerase for the biosynthesis of the mycobacterial cell wall, is a validated target for tuberculosis (TB) drug development. Here we report the X-ray crystal structures of DprE1 and the DprE1 resistant mutant (Y314C) in complexes with TCA1 derivatives to elucidate the molecular basis of their inhibitory activities and an unconventional resistance mechanism, which enabled us to optimize the potency of the analogs. The selected lead compound showed excellent in vitro and in vivo activities, and low risk of toxicity profile except for the inhibition of CYP2C9. A crystal structure of CYP2C9 in complex with a TCA1 analog revealed the similar interaction patterns to the DprE1-TCA1 complex. Guided by the structures, an optimized molecule was generated with differential inhibitory activities against DprE1 and CYP2C9, which provides insights for development of a clinical candidate to treat TB.

Succinate dehydrogenase is the regulator of respiration in Mycobacterium tuberculosis.

In chronic infection, Mycobacterium tuberculosis bacilli are thought to enter a metabolic program that provides sufficient energy for maintenance of the protonmotive force, but is insufficient to meet the demands of cellular growth. We sought to understand this metabolic downshift genetically by targeting succinate dehydrogenase, the enzyme which couples the growth processes controlled by the TCA cycle with the energy production resulting from the electron transport chain. M. tuberculosis contains two operons which are predicted to encode succinate dehydrogenase enzymes (sdh-1 and sdh-2); we found that deletion of Sdh1 contributes to an inability to survive long term stationary phase. Stable isotope labeling and mass spectrometry revealed that Sdh1 functions as a succinate dehydrogenase during aerobic growth, and that Sdh2 is dispensable for this catalysis, but partially overlapping activities ensure that the loss of one enzyme can incompletely compensate for loss of the other. Deletion of Sdh1 disturbs the rate of respiration via the mycobacterial electron transport chain, resulting in an increased proportion of reduced electron carrier (menaquinol) which leads to increased oxygen consumption. The loss of respiratory control leads to an inability to recover from stationary phase. We propose a model in which succinate dehydrogenase is a governor of cellular respiration in the adaptation to low oxygen environments.

Phosphorylation of KasB regulates virulence and acid-fastness in Mycobacterium tuberculosis.

Mycobacterium tuberculosis bacilli display two signature features: acid-fast staining and the capacity to induce long-term latent infections in humans. However, the mechanisms governing these two important processes remain largely unknown. Ser/Thr phosphorylation has recently emerged as an important regulatory mechanism allowing mycobacteria to adapt their cell wall structure/composition in response to their environment. Herein, we evaluated whether phosphorylation of KasB, a crucial mycolic acid biosynthetic enzyme, could modulate acid-fast staining and virulence. Tandem mass spectrometry and site-directed mutagenesis revealed that phosphorylation of KasB occurred at Thr334 and Thr336 both in vitro and in mycobacteria. Isogenic strains of M. tuberculosis with either a deletion of the kasB gene or a kasB_T334D/T336D allele, mimicking constitutive phosphorylation of KasB, were constructed by specialized linkage transduction. Biochemical and structural analyses comparing these mutants to the parental strain revealed that both mutant strains had mycolic acids that were shortened by 4-6 carbon atoms and lacked trans-cyclopropanation. Together, these results suggested that in M. tuberculosis, phosphorylation profoundly decreases the condensing activity of KasB. Structural/modeling analyses reveal that Thr334 and Thr336 are located in the vicinity of the catalytic triad, which indicates that phosphorylation of these amino acids would result in loss of enzyme activity. Importantly, the kasB_T334D/T336D phosphomimetic and deletion alleles, in contrast to the kasB_T334A/T336A phosphoablative allele, completely lost acid-fast staining. Moreover, assessing the virulence of these strains indicated that the KasB phosphomimetic mutant was attenuated in both immunodeficient and immunocompetent mice following aerosol infection. This attenuation was characterized by the absence of lung pathology. Overall, these results highlight for the first time the role of Ser/Thr kinase-dependent KasB phosphorylation in regulating the later stages of mycolic acid elongation, with important consequences in terms of acid-fast staining and pathogenicity.

Antituberculosis thiophenes define a requirement for Pks13 in mycolic acid biosynthesis.

We report a new class of thiophene (TP) compounds that kill Mycobacterium tuberculosis by the previously uncharacterized mechanism of Pks13 inhibition. An F79S mutation near the catalytic Ser55 site in Pks13 conferred TP resistance in M. tuberculosis. Overexpression of wild-type Pks13 resulted in TP resistance, and overexpression of the Pks13(F79S) mutant conferred high resistance. In vitro, TP inhibited fatty acyl-AMP loading onto Pks13. TP inhibited mycolic acid biosynthesis in wild-type M. tuberculosis, but it did so to a much lesser extent in TP-resistant M. tuberculosis. TP treatment was bactericidal and equivalent to treatment with the first-line drug isoniazid, but it was less likely to permit emergent resistance. Combined isoniazid and TP treatment resulted in sterilizing activity. Computational docking identified a possible TP-binding groove within the Pks13 acyl carrier protein domain. This study confirms that M. tuberculosis Pks13 is required for mycolic acid biosynthesis, validates it as a druggable target and demonstrates the therapeutic potential of simultaneously inhibiting multiple targets in the same biosynthetic pathway.

Evolution of a thienopyrimidine antitubercular relying on medicinal chemistry and metabolomics insights.

The metabolic instability of an antitubercular small molecule CD117 was addressed through iterative alteration of a key sulfide substituent and interrogation of the effect on growth inhibition of cultured Mycobacterium tuberculosis. This process was informed by studies of the intramycobacterial metabolism of CD117 and its inactive carboxylic acid derivative. Isoxazole 4e and thiazole 4m demonstrated significant gains in mouse liver microsomal stability with slight losses in whole-cell activity. This work illustrates the challenges of antitubercular hit evolution, requiring a balance of chemical and biological insights.

Propionate prevents loss of the PDIM virulence lipid in Mycobacterium tuberculosis.

Phthiocerol dimycocerosate (PDIM) is an essential virulence lipid of Mycobacterium tuberculosis. In vitro culturing rapidly selects for spontaneous PDIM-negative mutants that have attenuated virulence and increased cell wall permeability, thus impacting the relevance of experimental findings. PDIM loss can also reduce the efficacy of the BCG Pasteur vaccine. Here we show that vancomycin susceptibility can rapidly screen for M. tuberculosis PDIM production. We find that metabolic deficiency of methylmalonyl-CoA impedes the growth of PDIM-producing bacilli, selecting for PDIM-negative variants. Supplementation with odd-chain fatty acids, cholesterol or vitamin B12 restores PDIM-positive bacterial growth. Specifically, we show that propionate supplementation enhances PDIM-producing bacterial growth and selects against PDIM-negative mutants, analogous to in vivo conditions. Our study provides a simple approach to screen for and maintain PDIM production, and reveals how discrepancies between the host and in vitro nutrient environments can attenuate bacterial pathogenicity.