Biology subjects

Cook, G. M.

Publications and source records attributed to Cook, G. M..

At least 19 recordsLinked to original sources

bioRxiv2025

Flexibility and modulation of translation initiation in enterovirus genomes

Enteroviruses comprise a large group of mammalian pathogens that often utilize two open reading frames (ORFs) to encode their proteins: the upstream protein (UP) and the main polyprotein. In some enteroviruses, in addition to the canonical upstream AUG (uAUG), there is another AUG that may represent an alternative upstream initiation site. An analysis of enterovirus sequences containing additional upstream AUGs identified several clusters, including strains of pathogenic Enterovirus alphacoxsackie and E. coxsackiepol. Using ribosome profiling on coxsackievirus CVA-13 (E. coxsackiepol), we demonstrate that both upstream AUG codons can be used for translation initiation in infected cells. Moreover, we confirm translation from both upstream AUGs using a reporter system. Mutating the additional upstream AUG in the context of CVA-13 did not result in phenotypic changes in immortalized cell lines. However, the wild-type virus outcompeted this mutant in human intestinal organoids and differentiated neuronal systems, representing an advantage in physiologically relevant infection sites. Mutation of the stop codon of the shorter upstream ORF led to dysregulated translation of the other ORFs in the reporter system, suggesting a potential role for the additional uORF in modulating the expression level of the other ORFs. These findings demonstrate the remarkable plasticity of enterovirus IRES-mediated initiation and the competitive advantage of double-upstream-AUG-containing viruses in terminally differentiated intestinal organoids and neuronal systems.

microbiology↗

bioRxiv2025

Hydrogenase-driven ATP synthesis from air

All cells require a continuous supply of the universal energy currency, adenosine triphosphate (ATP), to drive countless cellular reactions. The universally conserved F1Fo-ATP synthase regenerates ATP from ADP and Pi by harnessing a transmembrane electrochemical proton gradient (pmf). Bacteria have evolved a wide diversity of pmf-forming proteins to make ATP using light, organic, and inorganic energy sources. Recently, we proposed that many bacteria survive using atmospheric trace gases to produce ATP when limited for other energy sources. However, there is no direct proof that atmospheric energy sources are sufficient to generate pmf or drive ATP synthesis. Here, we show that the membrane-associated hydrogen:quinone oxidoreductase Huc from Mycobacterium smegmatis enables ATP synthesis from air. Purified Huc couples H2 oxidation to the reduction of various ubiquinone and menaquinone analogues. We designed and optimised a minimal respiratory chain in which Huc is reconstituted into liposomes with a pmf-generating terminal oxidase and the ATP-generating F1Fo-ATP synthase. Our experiments show that passive hydrogen exchange from air to solution is sufficient for the electron transfer and pmf generation required to accumulate ATP. Finally, by combining continuous culture bioenergetics measurements with theoretical calculations, we show this process is sufficient for mycobacteria to sustain pmf and ATP synthesis (two ATP molecules per H2 oxidised) for maintenance energy requirements during nutrient starvation. These findings prove that atmospheric energy sources are dependable lifeline substrates that enable continuous energy conservation during nutrient starvation. In addition, this work provides a new tool for ATP production in synthetic applications, which unlike other approaches is traceless without by-product accumulation.

microbiology↗

bioRxiv2025

Verapamil and its metabolite norverapamil inhibit the Mycobacterium tuberculosis MmpS5L5 efflux pump to potentiate the activity bedaquiline and other antitubercular drugs

Bedaquiline is the cornerstone of a new regimen for the treatment of drug-resistant tuberculosis. However, its clinical use is threatened by the emergence of bedaquiline-resistant strains of Mycobacterium tuberculosis. Bedaquiline targets mycobacterial ATP synthase but the predominant route to clinical bedaquiline resistance is via upregulation of the MmpS5L5 efflux pump due to mutations that inactivate the transcriptional repressor Rv0678. Here, we show that the MmpS5L5 efflux pump reduces susceptibility to bedaquiline as well as its new, more potent derivative TBAJ-876 and other antimicrobial substrates, including clofazimine and the DprE1 inhibitors PBTZ-169 and OPC-167832. Furthermore, the increased resistance of Rv0678 mutants stems entirely from increased MmpS5L5 activity. These results highlight the potential of a pharmacological MmpS5L5 inhibitor to increase drug efficacy. Verapamil, primarily used as a calcium channel inhibitor, is known to inhibit diverse efflux pumps and to potentiate bedaquiline and clofazimine activity in M. tuberculosis. Here, we show that verapamil potentiates the activity of multiple diverse MmpS5L5 substrates. Using biochemical approaches, we demonstrate that verapamil does not exert this effect by acting as a disruptor of the protonmotive force used to power MmpS5L5, as previously proposed, suggesting that verapamil inhibits the function of the MmpS5L5 pump. Finally, norverapamil, the major verapamil metabolite, which has greatly reduced calcium channel activity, has equal potency in reducing resistance to MmpS5L5 substrates. Our findings highlight verapamils potential for enhancing bedaquiline TB treatment, for preventing acquired resistance to bedaquiline and other MmpS5L5 substrates, while also providing the impetus to identify additional MmpS5L5 inhibitors. Significance StatementBedaquiline, an antitubercular drug targeting ATP synthase, forms the backbone of highly efficacious treatment regimens for drug-resistant tuberculosis. Bedaquiline resistance is emerging as a significant problem and is most commonly caused by mutations in Rv0678 that result in upregulation of the MmpS5L5 efflux pump. Here we define the contribution of the MmpS5L5 efflux pump to drug resistance in wild-type and Rv0678 mutant strains and show that the commonly used drug verapamil can inhibit MmpS5L5 activity. This suggests that this safe and inexpensive drug may be useful in enhancing bedaquiline treatment of TB and to help prevent bedaquiline acquired resistance.

microbiology↗

bioRxiv2025

Structure of mycobacterial NDH-2 bound to a 2-mercapto-quinazolinone inhibitor

The mycobacterial type II NADH dehydrogenase (NDH-2) is a promising drug target because of its central role in energy metabolism in Mycobacterium tuberculosis and other pathogens, and because it lacks a known mammalian homologue. However, the absence of structural information on how the enzyme binds inhibitors has made optimization of lead compounds challenging. We used electron cryomicroscopy (cryo-EM) to determine the structure of NDH-2 from Mycobacterium smegmatis, a fast-growing non-pathogenic model for respiration in M. tuberculosis, both alone and in complex with a 2-mercapto-quinazolinone inhibitor. The structure shows that active mycobacterial NDH-2 is dimeric, with the dimerization interface stabilized by an extended C-terminal helix not found in NDH-2 from other bacterial genera. The arrangement of monomers in the dimer differs from the arrangement described for other prokaryotic NDH-2 dimers, instead resembling dimers formed by NDH-2 in eukaryotes. Density for the 2-mercapto-quinazolinone in the menaquinone-binding site shows that the inhibitor blocks menaquinone reduction through direct interaction with the flavin adenine dinucleotide cofactor. These results reveal structural elements of NDH-2 that could be used to design specific inhibitors of the mycobacterial enzyme.

biochemistry↗

bioRxiv2024

Remission spectroscopy resolves the mode of action of bedaquiline within living mycobacteria

Bedaquiline, an ATP synthase inhibitor, is the spearhead of transformative therapies against drug-resistant Mycobacterium tuberculosis. Here, remission spectroscopy is used to measures the energy-transducing cytochromes within unperturbed, respiring suspensions of mycobacterial and human cells, allowing spectroscopic measurements of electron transport chains as they power living cells and respond to bedaquiline. No evidence is found for protonophoric or ionophoric uncoupling. Rather, by directly inhibiting ATP synthase, bedaquiline slows the respiratory supercomplex (Qcr:Cta; bcc:aa3) by increasing the proton-motive force, causing sub-second redirection of electron flux through the cytochrome bd oxidase (Cyd) to O2. Electron flux redirection explains the idiosyncratic bedaquiline-induced increase in O2 consumption rates previously observed. Redirection occurs as Cyd is present even in cells grown in plentiful O2. Applying the same approach to human cells did not detect bedaquiline-induced inhibition of mitochondrial function despite such inhibition being seen in isolated systems. Overall, we clarify how bedaquiline works, why different models for its action developed, and the mechanisms underlying the synergy of bedaquiline in combination regimes.

biophysics↗

bioRxiv2024

Cryo-EM of native membranes reveals an intimate connection between the Krebs cycle and respiration in mycobacteria

Imaging of endogenous protein complexes in their native membranes can reveal protein-protein interactions that are lost upon detergent solubilization. To investigate interactions in the mycobacterial oxidative phosphorylation machinery, we prepared inverted membrane vesicles from Mycobacterium smegmatis and enriched for vesicles containing complexes of interest by affinity chromatography. Electron cryomicroscopy (cryo-EM) of these vesicles revealed that malate-quinone oxidoreductase (Mqo), an enzyme from the Krebs cycle, physically associates with the electron transport chain Complex III2IV2 (CIII2CIV2) supercomplex. Analysis of the Mqo:CIII2CIV2 interaction shows that CIII2CIV2 is necessary for malate-driven, but not NADH- driven, electron transport chain activity and oxygen consumption. Further, the association of Mqo with CIII2CIV2 enables electron transfer from malate to CIII2CIV2 with millisecond kinetics. Together, these findings indicate a connection between the Krebs cycle and respiration that directs electrons along a single branch of the mycobacterial electron transport chain.

biochemistry↗

bioRxiv2024

Extended blood stage sensitivity profiles of Plasmodium vivax to doxycycline and tafenoquine using Plasmodium cynomolgi as a model

Testing Plasmodium vivax antimicrobial sensitivity is limited to ex vivo schizont maturation assays, which precludes determining the IC50s of delayed action antimalarials such as doxycycline. Using Plasmodium cynomolgi as a model for P. vivax, we determined the physiologically significant delayed death effect induced by doxycycline (IC50(96h), 1401 {+/-} 607 nM). As expected, IC50(96 h) to chloroquine (20.4 nM), piperaquine (12.6 {micro}M) and tafenoquine (1424 nM) were not affected by extended exposure.

microbiology↗

bioRxiv2024

Establishment of an Inhalation Administration Noninvasive Murine Model for Rapidly Testing Drug Activity against Mycobacterium tuberculosis

The efficacy of many compounds against Mycobacterium tuberculosis is often limited when administered via conventional oral or injection routes due to suboptimal pharmacokinetic characteristics. Inhalation delivery methods have been investigated to achieve high local therapeutic doses in the lungs. However, previous models, typically employing wild-type M. tuberculosis strains, were intricate, time-consuming, labor-intensive, and with poor repeatability. In this study, we developed an autoluminescence-based inhalation administration model to evaluate drug activity by quantifying relative light units (RLUs) emitted from live mice infected with autoluminescent M. tuberculosis. This novel approach has several improvements: it eliminates the need for anesthesia in mice during administration and simplifies the instrument manipulation; it is cost-effective by utilizing mice instead of larger animals; it shortens time from several months to 16 or 17 days for obtaining result; it is non-invasive by measuring the live RLUs of mice; up to six mice can be administrated daily and simultaneously, even 2-3 times/day; results are relatively objective and repeatable minimizing human factors. Proof-of-concept experiments demonstrated that inhalable rifampicin, isoniazid, and ethambutol showed anti-M. tuberculosis activity at concentrations as low as 0.5, 0.5, and 0.625 mg/mL, respectively, as evidenced by comparing the live RLUs of mice. Furthermore, consistency between RLUs and colony-forming units of the lungs reaffirms the reliability of RLUs as an indicator of drug efficacy, highlighting the potential of this approach for accurately assessing anti-M. tuberculosis activity in vivo. This autoluminescence-based and non-invasive inhalation model offers a substantial reduction in the time, effort, and cost required for evaluating the efficacy of screening new drugs and repurposing old drugs in vivo via inhalation administration.

microbiology↗

bioRxiv2024

A marine sponge-associated mycobacterium closely related to Mycobacterium tuberculosis

Reconstructing the evolutionary origins of Mycobacterium tuberculosis, the causative agent of human tuberculosis, has helped identify bacterial factors that have led to the tubercle bacillus becoming such a formidable human pathogen. Here we report the discovery and detailed characterization of an exceedingly slow growing mycobacterium that is closely related to M. tuberculosis for which we have proposed the species name Mycobacterium spongiae sp. nov., (strain ID: FSD4b-SM). The bacterium was isolated from a marine sponge, taken from the waters of the Great Barrier Reef in Queensland, Australia. Comparative genomics revealed that, after the opportunistic human pathogen Mycobacterium decipiens, M. spongiae is the most closely related species to the M. tuberculosis complex reported to date, with 80% shared average nucleotide identity and extensive conservation of key M. tuberculosis virulence factors, including intact ESX secretion systems and associated effectors. Proteomic and lipidomic analyses showed that these conserved systems are functional in FSD4b-SM, but that it also produces cell wall lipids not previously reported in mycobacteria. We investigated the virulence potential of FSD4b-SM in mice and found that, while the bacteria persist in lungs for 56 days after intranasal infection, no overt pathology was detected. The similarities with M. tuberculosis, together with its lack of virulence, motivated us to investigate the potential of FSD4b-SM as a vaccine strain and as a genetic donor of the ESX-1 genetic locus to improve BCG immunogenicity. However, neither of these approaches resulted in superior protection against M. tuberculosis challenge compared to BCG vaccination alone. The discovery of M. spongiae adds to our understanding of the emergence of the M. tuberculosis complex and it will be another useful resource to refine our understanding of the factors that shaped the evolution and pathogenesis of M. tuberculosis.

microbiology↗

bioRxiv2023

KatG inactivation generates vulnerabilities in isoniazid resistant strains of Mycobacterium tuberculosis

Drug-resistant strains of Mycobacterium tuberculosis are a major global health problem. Resistance to the front-line antibiotic isoniazid is often associated with mutations in the katG encoded bifunctional catalase-peroxidase. We hypothesised that perturbed KatG activity would generate collateral vulnerabilities in INH-resistant katG mutants, providing new pathways to combat isoniazid resistance. Here, we used whole genome CRISPRi screens, transcriptomics, and metabolomics to generate a genome-wide map of cellular vulnerabilities in a M. tuberculosis katG mutant. We discovered that metabolic and transcriptional remodelling compensates for the loss of KatG but in doing so generates vulnerabilities in ribosome biogenesis, and nucleotide and amino acid metabolism. These vulnerabilities were more sensitive to inhibition in an isoniazid-resistant katG mutant under in vitro and host-relevant conditions and translated to clinical populations. These findings provide an experimental framework for developing novel strategies to combat antimicrobial resistance in M. tuberculosis and other bacterial pathogens.

microbiology↗

bioRxiv2022

The two-component system CroRS regulates isoprenoid flux to mediate antimicrobial tolerance in the bacterial pathogen Enterococcus faecalis

Antimicrobial tolerance is the ability of a microbial population to survive, but not proliferate, during antimicrobial exposure. Significantly, it has been shown to precede the development of bona fide antimicrobial resistance. We have previously identified the two-component system CroRS as a critical regulator of tolerance to antimicrobials like teixobactin in the bacterial pathogen Enterococcus faecalis. To understand the molecular mechanism of this tolerance, we carried out RNA-seq analyses in the E. faecalis wild-type and isogenic {Delta}croRS mutant to determine the teixobactin-induced CroRS regulon. We identified a 132 gene CroRS regulon and show CroRS upregulates expression of all major components of the enterococcal cell envelope in response to teixobactin challenge. To gain further insight into the function of this regulon we isolated and characterized {Delta}croRS mutants recovered for wild-type growth and tolerance. We show introduction of a single stop codon in a heptaprenyl diphosphate synthase (hppS), a key enzyme in the synthesis of the quinone electron carrier demethylmenaquinone (DMK), is sufficient to rescue loss of cell envelope integrity in the croRS deletion strain. Based on these findings, we propose a model where CroRS acts as a gate-keeper of isoprenoid biosynthesis, mediating flux of isoprenoids needed for cell wall synthesis (undecaprenyl pyrophosphate; UPP) and respiration (DMK) to maintain cell wall homeostasis upon antimicrobial challenge. Dysregulation of this flux in the absence of croRS leads to a loss of tolerance, which is rescued by loss of function mutations in HppS, allowing an increase in isoprenoid flow to UPP and subsequently cell wall synthesis. ImportanceAntimicrobial tolerance is the ability of a microorganism to survive, but not grow upon antimicrobial challenge, and is an important precursor to the development of antimicrobial resistance (the ability to profilerate). Understanding the molecular mechanisms that underpin tolerance will therefore aid in hampering the development of resistance to novel antimicrobials such as teixobactin. CroRS is an essential two-component regulator of antimicrobial tolerance in the bacterial pathogen Enterococcus faecalis. We have determined the antimicrobial-induced CroRS regulon and identified key mutations in a heptaprenyl diphosphate synthase to uncover a novel mechanism of antimicrobial tolerance.

microbiology↗

bioRxiv2022

Energy extraction from air: structural basis of atmospheric hydrogen oxidation

Diverse aerobic bacteria use atmospheric H2 as an energy source for growth and survival. This recently discovered yet globally significant process regulates the composition of the atmosphere, enhances soil biodiversity, and drives primary production in certain extreme environments. Atmospheric H2 oxidation has been attributed to still uncharacterised members of the [NiFe]-hydrogenase superfamily. However, it is unresolved how these enzymes overcome the extraordinary catalytic challenge of selectively oxidizing picomolar levels of H2 amid ambient levels of the catalytic poison O2, and how the derived electrons are transferred to the respiratory chain. Here we determined the 1.52 [A] resolution CryoEM structure of the mycobacterial hydrogenase Huc and investigated its mechanism by integrating kinetics, electrochemistry, spectroscopy, mass spectrometry, and molecular dynamics simulations. Purified Huc is an oxygen-insensitive enzyme that couples the oxidation of atmospheric H2 at its large subunit to the hydrogenation of the respiratory electron carrier menaquinone at its small subunit. The enzyme uses a narrow hydrophobic gas channel to selectively bind atmospheric H2 at the expense of O2, while three [3Fe-4S] clusters and their unusual ligation by a D-histidine modulate the electrochemical properties of the enzyme such that atmospheric H2 oxidation is energetically feasible. Huc forms an 833 kDa complex composed of an octamer of catalytic subunits around a membrane-associated central stalk, which extracts and transports menaquinone a remarkable 94 [A] from the membrane, enabling its reduction. These findings provide a mechanistic basis for the biogeochemically and ecologically critical process of atmospheric H2 oxidation. Through the first characterisation of a group 2 [NiFe]-hydrogenase, we also uncover a novel mode of energy coupling dependent on long-range quinone transport and pave way for the development of biocatalysts that oxidize H2 in ambient air.

biochemistry↗

bioRxiv2021

Ribosome profiling of porcine reproductive and respiratory syndrome virus reveals novel features of viral gene expression

Porcine reproductive and respiratory syndrome virus (PRRSV) is an arterivirus which causes significant economic losses to the swine industry worldwide. Here, we use ribosome profiling (RiboSeq) and parallel RNA sequencing (RNASeq) to characterise the transcriptome and translatome of both species of PRRSV and analyse the host response to infection. We quantified viral gene expression over a timecourse of infection, and calculated the efficiency of programmed ribosomal frameshifting (PRF) at both sites on the viral genome. At the nsp2 frameshift site (a rare example of protein-stimulated frameshifting), -2 PRF efficiency increases over time, likely facilitated by accumulation of the PRF- stimulatory viral protein (nsp1{beta}) during infection. This marks arteriviruses as the second example of temporally regulated PRF. Surprisingly, we also found PRF efficiency at the canonical ORF1ab frameshift site increases over time, in apparent contradiction of the common assumption that RNA structure-directed frameshift sites operate at a fixed efficiency. This has potential implications for the numerous other viruses with canonical PRF sites. Furthermore, we discovered several highly translated additional viral ORFs, the translation of which may be facilitated by multiple novel viral transcripts. For example, we found a 125-codon ORF overlapping nsp12, which is expressed as highly as nsp12 itself at late stages of replication, and is likely translated from novel subgenomic (sg) RNA transcripts that overlap the 3' end of ORF1b. Similar transcripts were discovered for both PRRSV-1 and PRRSV- 2, suggesting a potential conserved mechanism for temporal regulation of expression of the 3'-proximal region of ORF1b. In addition, we identified a highly translated, short upstream ORF (uORF) in the 5' UTR, the presence of which is highly conserved amongst PRRSV-2 isolates. This is the first application of RiboSeq to arterivirus-infected cells, and reveals new features which add to the complexity of gene expression programmes in this important family of nidoviruses.

pathology↗

bioRxiv2021

RNase HI depletion strongly potentiates cell killing by rifampicin in mycobacteria

Multidrug resistant (MDR) tuberculosis (TB) is defined by the resistance of Mycobacterium tuberculosis, the causative organism, to the first-line antibiotics rifampicin and isoniazid. Mitigating or reversing resistance to these drugs offers a means of preserving and extending their use in TB treatment. R-loops are RNA/DNA hybrids that are formed in the genome during transcription, and can be lethal to the cell if not resolved. RNase HI is an enzyme that removes R-loops, and this activity is essential in M. tuberculosis: knockouts of rnhC, the gene encoding RNase HI, are non-viable. This essentiality supports it as a candidate target for the development of new antibiotics. In the model organism Mycolicibacterium smegmatis, RNase HI activity is provided by two RNase HI enzymes, RnhA and RnhC. We show that the partial depletion of RNase HI activity in M. smegmatis, by knocking out either of the genes encoding RnhA or RnhC, led to the accumulation of R-loops. The sensitivity of the knockout strains to the antibiotics moxifloxacin, streptomycin and rifampicin was increased, with sensitivity to the transcriptional inhibitor rifampicin strikingly increased by nearly 100-fold. We also show that R-loop accumulation accompanies partial transcriptional inhibition, suggesting a mechanistic basis for the synergy between RNase HI depletion and transcriptional inhibition. A model of how transcriptional inhibition can potentiate R-loop accumulation is presented. Finally, we identified four small molecules that inhibit recombinant RnhC activity and that also potentiated rifampicin activity in whole-cell assays against M. tuberculosis, supporting an on-target mode of action, and providing the first step in developing a new class of anti-mycobacterial drug. ImportanceThis study validates mycobacterial RNase HI as a druggable, vulnerable candidate for a new therapeutic treatment of M. tuberculosis with a novel mode of action. RNase HI depletion shows synergistic bacterial killing with some current first- and second-line antibiotics, suggesting that RNase HI inhibitors would combine well with these regimens, and could potentially accelerate the clearance of drug-sensitive strains. RNase HI inhibitors also have the potential to reduce the effective dose of rifampicin, with the comcommitant reduction in side effects. The potentiation of rifampicin efficacy conferred by RNase HI deficiency suggests that RNase HI inhibitors may be able to mitigate against development of rifampicin resistance. The synergy may also be able to reverse rifampicin resistance, rescuing this antibiotic for therapy. The surprising finding that low levels of transcriptional inhibition potentiate R-loop formation provides a key new insight into R-loop metabolism.

microbiology↗

bioRxiv2021

Impaired succinate oxidation prevents growth and influences drug susceptibility in Mycobacterium tuberculosis

Succinate is a major focal point in mycobacterial metabolism and respiration, serving as both an intermediate of the 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 bi-directional 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 + 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 + 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 several antitubercular drugs against M. tuberculosis, including potentiating the activity of bioenergetic inhibitors and attenuating the activity of cell wall inhibitors. Together, these data provide fundamental insights into mycobacterial physiology, energy metabolism, and antimicrobial susceptibility. ImportanceNew 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 anti-tuberculosis drugs. Combined, these findings provide fundamental insights into mycobacterial physiology and drug susceptibility that will be useful in the continued development of bioenergetic inhibitors.

microbiology↗

bioRxiv2021

Sterilizing Effects of Novel Regimens Containing TB47, Clofazimine and Linezolid in a Murine Model of Tuberculosis.

Toxicity and inconvenience associated with the use of injectable drug-containing regimens for tuberculosis (TB) have made all-oral regimens a preferred alternative. Widespread resistance to fluoroquinolones and pyrazinamide makes it essential to identify new drug candidates and study their effects on current regimens for TB. TB47 is a pyrazolo[1,5-a]pyridine-3-carboxamide with powerful synergistic in vitro and in vivo activities against mycobacteria, especially with clofazimine. Here, we investigated the bactericidal and sterilizing activities of novel oral regimens containing TB47 + clofazimine + linezolid, and the potential roles of levofloxacin and/or pyrazinamide in such drug combinations. Using a well-established mouse model, we assessed the effect of these regimens on bacterial burden in the lung during treatment and relapse (4 months after stopping treatment + immunosuppression). Our findings indicate that the TB47 + clofazimine + linezolid + pyrazinamide, with/without levofloxacin, regimens had fast-acting (4 months) sterilizing activity and no relapse was observed. When pyrazinamide was excluded from the regimen, treatment times were longer (5-6 months) to achieve sterilizing conditions. We propose that TB47 + clofazimine + linezolid can form a highly sterilizing block for use as an alternative pan-TB regimen.

microbiology↗

bioRxiv2020

Cellular and structural basis of synthesis of the unique intermediate dehydro-F420-0 in mycobacteria

F420 is a low-potential redox cofactor used by diverse bacteria and archaea. In mycobacteria, this cofactor has multiple roles, including adaptation to redox stress, cell wall biosynthesis, and activation of the clinical antitubercular prodrugs pretomanid and delamanid. A recent biochemical study proposed a revised biosynthesis pathway for F420 in mycobacteria; it was suggested that phosphoenolpyruvate served as a metabolic precursor for this pathway, rather than 2-phospholactate as long proposed, but these findings were subsequently challenged. In this work, we combined metabolomic, genetic, and structural analyses to resolve these discrepancies and determine the basis of F420 biosynthesis in mycobacterial cells. We show that, in whole cells of Mycobacterium smegmatis, phosphoenolpyruvate rather than 2-phospholactate stimulates F420 biosynthesis. Analysis of F420 biosynthesis intermediates present in M. smegmatis cells harboring genetic deletions at each step of the biosynthetic pathway confirmed that phosphoenolpyruvate is then used to produce the novel precursor compound dehydro-F420-0. To determine the structural basis of dehydro-F420-0 production, we solved high-resolution crystal structures of the enzyme responsible (FbiA) in apo, substrate, and product bound forms. These data show the essential role of a single divalent cation in coordinating the catalytic pre-complex of this enzyme and demonstrate that dehydro-F420-0 synthesis occurs through a direct substrate transfer mechanism. Together, these findings resolve the biosynthetic pathway of F420 in mycobacteria and have significant implications for understanding the emergence of antitubercular prodrug resistance.

biochemistry↗

bioRxiv2019

Two uptake hydrogenases differentially interact with the aerobic respiratory chain during mycobacterial growth and persistence

Aerobic soil bacteria metabolize atmospheric hydrogen (H2) to persist when nutrient sources are limited. This process is the primary sink in the global H2 cycle and supports the productivity of microbes in oligotrophic environments. To mediate this function, bacteria possess [NiFe]-hydrogenases capable of oxidising H2 to subatmospheric concentrations. The soil saprophyte Mycobacterium smegmatis has two such [NiFe]-hydrogenases, designated Huc and Hhy, which belong to different phylogenetic subgroups. Huc and Hhy exhibit similar characteristics: both are oxygen-tolerant, oxidise H2 to subatmospheric concentrations, and enhance survival during hypoxia and carbon limitation. These shared characteristics pose the question: Why does M. smegmatis require two hydrogenases mediating a seemingly similar function? In this work we resolve this question by showing that Huc and Hhy are differentially expressed, localised, and integrated into the respiratory chain. Huc is active in late exponential and early stationary phase, supporting energy conservation during mixotrophic growth and the transition into dormancy. In contrast, Hhy is most active during long-term persistence, providing energy for maintenance processes when carbon sources are depleted. We show that Huc and Hhy are obligately linked to the aerobic respiratory chain via the menaquinone pool and are differentially affected by respiratory uncouplers. Consistent with their distinct expression profiles, Huc and Hhy interact differentially with the terminal oxidases of the respiratory chain. Huc exclusively donates electrons to, and possibly physically associates with, the proton pumping cytochrome bcc-aa3 supercomplex. In contrast, the more promiscuous Hhy can also provide electrons to the cytochrome bd oxidase complex. These data demonstrate that, despite their similar characteristics, Huc and Hhy perform distinct functions during mycobacterial growth and survival.

microbiology↗