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Drug-resistant Mycobacterium tuberculosis is a significant cause of infectious disease morbidity and mortality for which new antimicrobials are urgently needed. Inhibitors of mycobacterial respiratory energy metabolism have emerged as promising next-generation antimicrobials, but a number of targets remain unexplored. Succinate dehydrogenase (SDH), a focal point in mycobacterial central carbon metabolism and respiratory energy production, is required for growth and survival in M. tuberculosis under a number of conditions, highlighting the potential of inhibitors targeting mycobacterial SDH enzymes. To advance SDH as a novel drug target in M. tuberculosis , we utilized a combination of biochemical screening and in-silico deep learning technologies to identify multiple chemical scaffolds capable of inhibiting mycobacterial SDH activity. Antimicrobial susceptibility assays show that lead inhibitors are bacteriostatic agents with activity against wild-type and drug-resistant strains of M. tuberculosis . Mode of action studies on lead compounds demonstrate that the specific inhibition of SDH activity dysregulates mycobacterial metabolism and respiration and results in the secretion of intracellular succinate. Interaction assays demonstrate that the chemical inhibition of SDH activity potentiates the activity of other bioenergetic inhibitors and prevents the emergence of resistance to a variety of drugs. Overall, this study shows that SDH inhibitors are promising next-generation antimicrobials against M. tuberculosis .

Synergistic interactions between chemical inhibitors, whilst informative, can be difficult to interpret, as chemical inhibitors can often have multiple targets, many of which can be unknown. Here, using multiplexed transcriptional repression, we have validated that the simultaneous repression of glutamate racemase and alanine racemase has a synergistic interaction in Mycobacterium tuberculosis . This confirms prior observations from chemical interaction studies and highlights the potential of targeting multiple enzymes involved in mycobacterial cell wall synthesis.

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., (© The Author(s) 2024. Published by Oxford University Press on behalf of British Society for Antimicrobial Chemotherapy.)

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 F 1 F o -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., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2021 Shelton et al.)

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., (© 2023. The Author(s).)

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., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

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.

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 McNeil, Cheung, Waller, Adolph, Chapman, Seeto, Jowsey, Li, Hameed, Zhang and Cook.)

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., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

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 F 1 F o -ATP synthase, depletes ATP, and affects the entry of acetyl-CoA into the Krebs cycle. HM2-16F synergizes with the cytochrome bcc-aa 3 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., (© 2022. The Author(s).)

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., (© The Author(s) 2021. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

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., Competing Interests: We have no conflicts of interest to declare., (© 2021 The Authors.)

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., (© 2021 John Wiley & Sons Ltd.)

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 IC 90 is the concentration at which M. tuberculosis growth is inhibited by 90% (IC 90 < 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 (IC 90 ) of 4.9 μM with no cytotoxicity (IC 50 > 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., Competing Interests: EH, TM, and PH were employed by Eli Lilly and Company. PO was employed by company Apollo Drug Discovery Consulting, LLC. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Hembre, Early, Odingo, Shelton, Anoshchenko, Guzman, Flint, Dennison, McNeil, Korkegian, Ovechkina, Ornstein, Masquelin, Hipskind and Parish.)

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. IMPORTANCE Mycobacterium 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., (Copyright © 2020 McNeil et al.)

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., (Copyright © 2020 American Society for Microbiology.)

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., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2019 American Society for Microbiology.)

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., (Copyright © 2019 Robertson et al.)

There is a need for new combination regimens for tuberculosis. Identifying synergistic drug combinations can avoid toxic side effects and reduce treatment times. Using a fluorescent rifampicin conjugate, we demonstrated that synergy between cell wall inhibitors and rifampicin was associated with increased accumulation of rifampicin. Increased accumulation was also associated with increased cellular permeability., Competing Interests: The authors declare that there are no conflicts of interest., (© 2019 The Authors.)

New antitubercular agents are needed to combat the spread of multidrug- and extensively drug-resistant strains of Mycobacterium tuberculosis . The frontline antitubercular drug isoniazid (INH) targets the mycobacterial enoyl-ACP reductase, InhA. Resistance to INH is predominantly through mutations affecting the prodrug-activating enzyme KatG. Here, we report the identification of the diazaborines as a new class of direct InhA inhibitors. The lead compound, AN12855, exhibited in vitro bactericidal activity against replicating bacteria and was active against several drug-resistant clinical isolates. Biophysical and structural investigations revealed that AN12855 binds to and inhibits the substrate-binding site of InhA in a cofactor-independent manner. AN12855 showed good drug exposure after i.v. and oral delivery, with 53% oral bioavailability. Delivered orally, AN12855 exhibited dose-dependent efficacy in both an acute and chronic murine model of tuberculosis infection that was comparable with INH. Combined, AN12855 is a promising candidate for the development of new antitubercular agents., Competing Interests: The authors declare that they have no conflict of interest.

Isoniazid inhibits Mycobacterium tuberculosis InhA and is a key component of drug regimens that treat tuberculosis. However, the high rate of resistance against isoniazid is a contributing factor to the emergence of multi-drug resistance strains of M. tuberculosis. The 4-hydroxy-2-pyridine NITD-916 is a direct inhibitor of M. tuberculosis InhA that has comparable efficacy to isoniazid in mouse models of TB infection but a lower frequency of resistance. To characterize resistance mechanisms against NITD-916 we isolated resistant mutants in H37Rv (Euro-American lineage) and HN878 (East-Asian lineage) strains of M. tuberculosis. The resistance frequency was similar in both strains. Mutations were identified in residues within or near to the active of InhA or in the fabG1inhA promoter region. All mutants were resistant to NITD-916 but were not cross resistant to isoniazid, despite homology to SNPs identified in isoniazid resistant clinical isolates., (Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Oxazolidinones are promising candidates for the treatment of Mycobacterium tuberculosis infections. We isolated linezolid-resistant strains from H37Rv (Euro-American) and HN878 (East-Asian) strains; resistance frequencies were similar in the two strains. Mutations were identified in ribosomal protein L3 (RplC) and the 23S rRNA ( rrl ). All mutant strains were cross resistant to sutezolid; a subset was cross resistant to chloramphenicol. Mutations in rrl led to growth impairment and decreased fitness that may limit spread in clinical settings., (Copyright © 2017 McNeil et al.)

MmpL3 is a promising target for novel anti-tubercular agents, with numerous compound series identified as MmpL3 inhibitors. Despite this, there is an incomplete understanding of MmpL3 function. Here we show that Mycobacterium smegmatis MmpL3 mutant strains had an altered cell wall hydrophobicity, disrupted membrane potential and growth defects in liquid media. Compensatory mutations that restored normal growth also returned membrane potential to wild-type. M. smegmatis MmpL3 mutant strains were resistant to two anti-tubercular agents, SQ109 and AU1235, but were more sensitive to rifampicin, erythromycin and ampicillin. Exposure of M. smegmatis to AU1235 affected the cell wall composition and increased the potency of rifampicin. However, MmpL3 mutants did not prevent the dissipation of membrane potential following exposure to SQ109. These results demonstrate that in M. smegmatis, MmpL3 contributes to a number of important phenotypes such as membrane potential, cell wall composition, antibiotic susceptibility and fitness.

Objectives Determine trends in incidence and expenditure for perinatal drug exposure and neonatal abstinence syndrome (NAS) among Louisiana's Medicaid population. We also describe the maternal characteristics of NAS affected infants. Methods Retrospective cohort analysis using linked Medicaid and vital records data from 2003 to 2013. Conducted incidence and cost trends for drug exposed infants with and without NAS. Also performed comparison statistics among drug exposed infants with and without NAS and those not drug exposed. Results As rate of perinatal drug exposure increased, NAS rate per 1000 live Medicaid births also increased, from 2.1 (2003) to 3.6 (2007) to 8.0 (2013) (P for trend <0.0001). Total medical cost paid by Medicaid also increased from $1.3 million to $3.6 million to $8.7 million (P for trend <0.0001). Compared with drug exposed infants without NAS and those not drug exposed, infants with NAS were more likely to be white, have feeding difficulties, respiratory distress syndrome, sepsis, and seizures, all of which had an association at P < 0.0001. Over one-third (33.2%) of the mothers of infants with NAS had an opioid dependency in combination with a mental illness; with depression being most common. Conclusions for Practice Over an 11-year period, NAS rate among Louisiana's Medicaid infants quadrupled and the cost for caring for the affected infants increased six-fold. Medicaid, as the predominant payer for pregnant women and children affected by substance use disorders, must play a more active role in expanding access to comprehensive substance abuse treatment programs.

SdhE is required for the flavinylation and activation of succinate dehydrogenase and fumarate reductase (FRD). In addition, SdhE is conserved in proteobacteria (α, β and γ) and eukaryotes. Although the function of this recently characterized family of proteins has been determined, almost nothing is known about how their genes are regulated. Here, the RsmA (CsrA) and RsmC (HexY) post-transcriptional and post-translational regulators have been identified and shown to repress sdhEygfX expression in Serratia sp. ATCC 39006. Conversely, the flagella master regulator complex, FlhDC, activated sdhEygfX transcription. To investigate the hierarchy of control, we developed a novel approach that utilized endogenous CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR associated) genome-editing by a type I-F system to generate a chromosomal point mutation in flhC. Mutation of flhC alleviated the ability of RsmC to repress sdhEygfX expression, whereas RsmA acted in both an FlhDC-dependent and -independent manner to inhibit sdhEygfX. Mutation of rsmA or rsmC, or overexpression of FlhDC, led to increased prodigiosin, biosurfactant, swimming and swarming. Consistent with the modulation of sdhE by motility regulators, we have demonstrated that SdhE and FRD are required for maximal flagella-dependent swimming. Together, these results demonstrate that regulators of both metabolism and motility (RsmA, RsmC and FlhDC) control the transcription of the sdhEygfX operon.

Clustered regularly interspaced short palindromic repeats (CRISPR), in combination with CRISPR associated (cas) genes, constitute CRISPR-Cas bacterial adaptive immune systems. To generate immunity, these systems acquire short sequences of nucleic acids from foreign invaders and incorporate these into their CRISPR arrays as spacers. This adaptation process is the least characterized step in CRISPR-Cas immunity. Here, we used Pectobacterium atrosepticum to investigate adaptation in Type I-F CRISPR-Cas systems. Pre-existing spacers that matched plasmids stimulated hyperactive primed acquisition and resulted in the incorporation of up to nine new spacers across all three native CRISPR arrays. Endogenous expression of the cas genes was sufficient, yet required, for priming. The new spacers inhibited conjugation and transformation, and interference was enhanced with increasing numbers of new spacers. We analyzed ∼ 350 new spacers acquired in priming events and identified a 5'-protospacer-GG-3' protospacer adjacent motif. In contrast to priming in Type I-E systems, new spacers matched either plasmid strand and a biased distribution, including clustering near the primed protospacer, suggested a bi-directional translocation model for the Cas1:Cas2-3 adaptation machinery. Taken together these results indicate priming adaptation occurs in different CRISPR-Cas systems, that it can be highly active in wild-type strains and that the underlying mechanisms vary., (© The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The activity of the respiratory enzyme fumarate reductase (FRD) is dependent on the covalent attachment of the redox cofactor flavin adenine dinucleotide (FAD). We demonstrate that the FAD assembly factor SdhE, which flavinylates and activates the respiratory enzyme succinate dehydrogenase (SDH), is also required for the complete activation and flavinylation of FRD. SdhE interacted with, and flavinylated, the flavoprotein subunit FrdA, whilst mutations in a conserved RGxxE motif impaired the complete flavinylation and activation of FRD. These results are of widespread relevance because SDH and FRD play an important role in cellular energetics and are required for virulence in many important bacterial pathogens., (Copyright © 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Serratia sp. strain ATCC 39006 is a Gram-negative bacterium and a member of the Enterobacteriaceae that produces various bioactive secondary metabolites, including the tripyrrole red pigment prodigiosin and the β-lactam antibiotic 1-carbapenen-2-em-3-carboxylic acid (a carbapenem). This strain is the only member of the Enterobacteriaceae known to naturally produce gas vesicles, as flotation organelles. Here we present the genome sequence of this strain, which has served as a model for analysis of the biosynthesis and regulation of antibiotic production.

Succinate dehydrogenase (SDH) is an important respiratory enzyme that plays a critical role in the generation of energy in the majority of eukaryotes, bacteria, and archaea. The activity of SDH is dependent on the covalent attachment of the redox cofactor FAD to the flavoprotein subunit SdhA. In the Gram-negative bacteria Escherichia coli and Serratia sp. ATCC 39006, the covalent attachment of FAD to SdhA is dependent on the FAD assembly factor SdhE (YgfY). Although mechanisms have been proposed, experimental evidence that elucidates the molecular details of SdhE-mediated flavinylation are scarce. In this study, truncation and alanine swap mutagenesis of SdhE identified a highly conserved RGxxE motif that was important for SdhE function. Interestingly, RGxxE site-directed variants were not impaired in terms of protein folding or interactions with SdhA. Purification and analysis of SdhA from different mutant backgrounds demonstrated that SdhE interacts with and flavinylates folded SdhA without a requirement for the assembly of the entire SDH complex. SdhA was also partially active in the absence of SdhE, suggesting that SdhA is able to attach FAD through an inefficient autocatalytic mechanism. The results presented are of widespread relevance because SdhE and SDH are required for bacterial pathogenesis and mutations in the eukaryotic homologues of SdhE and SDH are associated with cancer in humans.

Serratia sp. strain ATCC 39006 produces the red-pigmented antibiotic prodigiosin. Prodigiosin biosynthesis is regulated by a complex hierarchy that includes the uncharacterized protein YgfX (DUF1434). The ygfX gene is co-transcribed with sdhE, an FAD assembly factor essential for the flavinylation and activation of the SdhA subunit of succinate dehydrogenase (SDH), a central enzyme in the tricarboxylic acid cycle and electron transport chain. The sdhEygfX operon is highly conserved within the Enterobacteriaceae, suggesting that SdhE and YgfX function together. We performed an extensive mutagenesis to gain molecular insights into the uncharacterized protein YgfX, and have investigated the relationship between YgfX and SdhE. YgfX localized to the membrane, interacted with itself, forming dimers or larger multimers, and interacted with SdhE. The transmembrane helices of YgfX were critical for protein function and the formation of YgfX multimers. Site-directed mutagenesis of residues conserved in DUF1434 proteins revealed a periplasmic tryptophan and a cytoplasmic aspartate that were crucial for YgfX activity. Both of these amino acids were required for the formation of YgfX multimers and interactions with SdhE but not membrane localization. Multiple cell division proteins were identified as putative interaction partners of YgfX and overexpression of YgfX had effects on cell morphology. These findings represent an important step in understanding the function of DUF1434 proteins. In contrast to a recent report, we found no evidence that YgfX and SdhE form a toxin-antitoxin system. In summary, YgfX functions as a multimeric membrane-bound protein that interacts with SdhE, an important FAD assembly factor that controls SDH activity.

Complex II (also known as Succinate dehydrogenase or Succinate-ubiquinone oxidoreductase) is an important respiratory enzyme that participates in both the tricarboxylic acid cycle and electron transport chain. Complex II consists of four subunits including a catalytic flavoprotein (SdhA), an iron-sulphur subunit (SdhB) and two hydrophobic membrane anchors (SdhC and SdhD). Complex II also contains a number of redox cofactors including haem, Fe-S clusters and FAD, which mediate electron transfer from succinate oxidation to the reduction of the mobile electron carrier ubiquinone. The flavin cofactor FAD is an important redox cofactor found in many proteins that participate in oxidation/reduction reactions. FAD is predominantly bound non-covalently to flavoproteins, with only a small percentage of flavoproteins, such as complex II, binding FAD covalently. Aside from a few examples, the mechanisms of flavin attachment have been a relatively unexplored area. This review will discuss the FAD cofactor and the mechanisms used by flavoproteins to covalently bind FAD. Particular focus is placed on the attachment of FAD to complex II with an emphasis on SdhE (a DUF339/SDH5 protein previously termed YgfY), the first protein identified as an assembly factor for FAD attachment to flavoproteins in prokaryotes. The molecular details of SdhE-dependent flavinylation of complex II are discussed and comparisons are made to known cofactor chaperones. Furthermore, an evolutionary hypothesis is proposed to explain the distribution of SdhE homologues in bacterial and eukaryotic species. Mechanisms for regulating SdhE function and how this may be linked to complex II function in different bacterial species are also discussed. This article is part of a Special Issue entitled: Respiratory complex II: Role in cellular physiology and disease., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Conserved uncharacterized genes account for ~30% of genes in both eukaryotic and bacterial genomes and are predicted to encode what are often termed "conserved hypothetical proteins." Many of these proteins have a wide phylogenetic distribution and might play important roles in conserved cellular pathways. Using the bacterium Serratia as a model system, we have investigated two conserved uncharacterized proteins, YgfY (a DUF339 protein, renamed SdhE; succinate dehydrogenase protein E) and YgfX (a DUF1434 protein). SdhE was required for growth on succinate as a sole carbon source and for the function, but not stability, of succinate dehydrogenase, an important component of the electron transport chain and the tricarboxylic acid cycle. SdhE interacted with the flavoprotein SdhA, directly bound the flavin adenine dinucleotide co-factor, and was required for the flavinylation of SdhA. This is the first demonstration of a protein required for FAD incorporation in bacteria. Furthermore, the loss of SdhE was highly pleiotropic, suggesting that SdhE might flavinylate other flavoproteins. Our findings are of wide importance to central metabolism because SdhE homologues are present in α-, β-, and γ-proteobacteria and multiple eukaryotes, including humans and yeast.

Serratia sp. strain ATCC 39006 produces the red-pigmented antibiotic prodigiosin. Regulation of prodigiosin biosynthesis involves a complex hierarchy, with PigP a master transcriptional regulator of multiple genes involved in prodigiosin production. The focus of this study was a member of the PigP regulon, pigS, which encodes an ArsR/SmtB family transcriptional repressor. Mutations in pigS reduced production of prodigiosin by decreasing the transcription of the biosynthetic operon. The pigS gene is the first in a four-gene operon, which also encodes three membrane proteins (pmpABC) of the COG2391 (DUF395; YedE/YeeE) and COG0730 (DUF81; TauE/SafE) families that we propose constitute transport components for sulfur-containing compounds. We provide the first experimental evidence confirming the membrane localization of a COG2391 protein, that of PmpB. Divergently transcribed from pigS-pmpABC is a bicistronic operon (blhA-orfY), which encodes a metallo-β-lactamase and a coenzyme A-disulfide reductase containing a rhodanese homology domain, both of which may participate in reactions with sulfur-containing compounds. The overproduction of the BlhA and OrfY enzymes and the PmpABC membrane proteins differentially affected pigmentation. We have dissected the contributions of these various proteins and determined their importance in the control of prodigiosin production. PigS-mediated control of prodigiosin occurred via binding directly to a short inverted repeat sequence in the intergenic region overlapping the predicted -10 regions of both pigS and blhA promoters and repressing transcription. PigP was required for the activation of these promoters, but only in the absence of PigS-mediated repression.