Biology subjects

Mizrahi, V.

Publications and source records attributed to Mizrahi, V..

13 recordsLinked to original sources

bioRxiv2025

Chlorotonils exhibit potent activity against Mycobacterium tuberculosis, while resistance is mediated by MmpR5-MmpL5

Treatment of Mycobacterium tuberculosis (Mtb) is challenging and requires administration of at least four different antibiotics. Unfortunately, multi-drug resistant Mtb strains continue to emerge, undermining the effectiveness of current treatment regimens and highlighting the urgent need for new therapeutics. In this study, we evaluated the potential of natural product-derived chlorotonils as anti-Mtb agents. We demonstrate that chlorotonils exhibit nanomolar potency against a range of attenuated and virulent Mtb strains. Mechanistic studies and resistance profiling in Mtb revealed that chlorotonils affect both lipid and energy metabolism. Through systems biology approaches, including the construction of an Mtb CRISPRi library specifically designed for chemical-genomic profiling, we identified MmpR5/MmpL5 as major driver of chlorotonil-resistance in Mtb leading also to cross-resistance with bedaquiline. Our findings highlight chlorotonils as valuable chemical tools to further dissect the role and function of the MmpS5-MmpL5 efflux pump in drug-resistant Mtb.

microbiology↗

bioRxiv2024

Disruption of riboflavin biosynthesis in mycobacteria establishes 5-amino-6-D-ribitylaminouracil (5-A-RU) as key precursor of MAIT cell agonists

Mucosal-associated invariant T (MAIT) cells exhibit an intrinsic ability to recognize and respond to microbial infections. The semi-invariant antigen recognition receptor of MAIT cells specifically detects the non-polymorphic antigen-presenting molecule, major histocompatibility complex class I-related protein 1 (MR1), which primarily binds riboflavin-derived metabolites of microbial origin. To further interrogate the dependence of these antigens on riboflavin biosynthesis in mycobacteria, we deleted individual genes in the riboflavin biosynthesis pathways in Mycobacterium smegmatis (Msm) and Mycobacterium tuberculosis (Mtb) and evaluated the impact thereof on MAIT cell activation. Blocking the early steps of the pathway by deletion of RibA2 or RibG profoundly reduced, but did not completely ablate, MAIT cell activation by Msm or Mtb, whereas deletion of RibC, which catalyzes the last step in the pathway, had no significant effect. Interestingly, deletion of RibH specifically enhanced MAIT cell recognition of Mtb whereas loss of lumazine synthase (RibH) activity had no impact on MAIT cell activation by Msm. MAIT cell activation by Msm was likewise unaffected by blocking the production of the MAIT cell antagonist, Fo (by inhibiting its conversion from the riboflavin pathway intermediate, 5-amino-6-D-ribitylaminouracil (5-A-RU), through the deletion of fbiC). Together, these results confirm a central role for 5-AR-U in generating mycobacterial MR1 ligands and reveal similarities and differences between Msm and Mtb in terms of the impact of riboflavin pathway disruption on MAIT cell activation. Author summaryMucosal-associated invariant T (MAIT) cells are an abundant population of innate-like T-cells which respond to microbial infections. These specialized cells recognize the MR1 molecule, which presents microbial metabolites derived from riboflavin (vitamin B2) biosynthesis. These cells are enriched in the airways and in some cases reduced in the peripheral blood of tuberculosis (TB) infected individuals suggestive of a role in the early response to infection by Mycobacterium tuberculosis. In this study, we investigated the effect of deleting individual genes in the riboflavin biosynthesis pathway on MAIT cell activation by Mycobacterium tuberculosis or Mycobacterium smegmatis. Our findings revealed that disrupting early stages in the pathway profoundly reduced but did not eliminate MAIT cell activation by both mycobacterial species. However, blocking the penultimate step in the pathway, catalyzed by the enzyme, lumazine synthase, led specifically to increased MAIT cell recognition of M. tuberculosis. Our results confirm the pivotal role of the riboflavin pathway intermediate, 5-A-RU, in generating mycobacterial ligands that serve as MAIT cell agonists. By enhancing our understanding of how MAIT cells recognize mycobacterial infections, the results of this study could inform strategies for the development of vaccines and/or immunotherapies for TB.

immunology↗

bioRxiv2023

Modulation of riboflavin biosynthesis and utilization in mycobacteria

Riboflavin (vitamin B2) is the precursor of the flavin coenzymes, FAD and FMN, which play a central role in cellular redox metabolism. While humans must obtain riboflavin from dietary sources, certain microbes, including Mycobacterium tuberculosis (Mtb), can biosynthesize riboflavin de novo. Riboflavin precursors have also been implicated in the activation of mucosal-associated invariant T (MAIT) cells which recognize metabolites derived from the riboflavin biosynthesis pathway complexed to the MHC-I-like molecule, MR1. To investigate the biosynthesis and function of riboflavin and its pathway intermediates in mycobacterial metabolism, physiology and MAIT cell recognition, we constructed conditional knockdowns (hypomorphs) in riboflavin biosynthesis and utilization genes in Mycobacterium smegmatis (Msm) and Mtb by inducible CRISPR interference. Using this comprehensive panel of hypomorphs, we analyzed the impact of gene silencing on viability, on the transcription of (other) riboflavin pathway genes, on the levels of the pathway proteins and on riboflavin itself. Our results revealed that (i) despite lacking a canonical transporter, both Msm and Mtb assimilate exogenous riboflavin when supplied at high concentration; (ii) there is functional redundancy in lumazine synthase activity in Msm; (iii) silencing of ribA2 or ribF is profoundly bactericidal in Mtb; and (iv) in Msm, ribA2 silencing results in concomitant knockdown of other pathway genes coupled with RibA2 and riboflavin depletion and is also bactericidal. In addition to their use in genetic validation of potential drug targets for tuberculosis, this collection of hypomorphs provides a useful resource for investigating the role of pathway intermediates in MAIT cell recognition of mycobacteria. IMPORTANCEThe pathway for biosynthesis and utilization of riboflavin, precursor of the essential coenzymes, FMN and FAD, is of particular interest in the flavin-rich pathogen, Mycobacterium tuberculosis (Mtb), for two important reasons: (i) the pathway includes potential tuberculosis (TB) drug targets; and (ii) intermediates from the riboflavin biosynthesis pathway provide ligands for mucosal associated invariant T (MAIT) cells, which have been implicated in TB pathogenesis. However, the riboflavin pathway is poorly understood in mycobacteria, which lack canonical mechanisms to transport this vitamin and to regulate flavin coenzyme homeostasis. By conditionally disrupting each step of the pathway and assessing the impact on mycobacterial viability and on the levels of the pathway proteins as well as riboflavin, our work provides genetic validation of the riboflavin pathway as a target for TB drug discovery and offers a resource for further exploring the association between riboflavin biosynthesis, MAIT cell activation and TB infection and disease.

molecular biology↗

bioRxiv2021

The mycobacterial mutasome: composition and recruitment in live cells

A DNA damage-inducible mutagenic gene cassette has been implicated in the emergence of drug resistance in Mycobacterium tuberculosis during anti-tuberculosis (TB) chemotherapy. However, the molecular composition and operation of the encoded "mycobacterial mutasome" - minimally comprising DnaE2 polymerase and ImuA' and ImuB accessory proteins - remain elusive. Following exposure of mycobacteria to DNA damaging agents, we observe that DnaE2 and ImuB co-localize with the DNA polymerase III {beta} subunit ({beta} clamp) in distinct intracellular foci. Notably, genetic inactivation of the mutasome in an imuBAAAAGG mutant containing a disrupted {beta} clamp-binding motif abolishes ImuB-{beta} clamp focus formation, a phenotype recapitulated pharmacologically by treating bacilli with griselimycin and in biochemical assays in which this {beta} clamp-binding antibiotic collapses pre-formed ImuB-{beta} clamp complexes. These observations establish the essentiality of the ImuB-{beta} clamp interaction for mutagenic DNA repair in mycobacteria, identifying the mutasome as target for adjunctive therapeutics designed to protect anti-TB drugs against emerging resistance.

microbiology↗

bioRxiv2021

DNA-dependent binding of nargenicin to DnaE1 inhibits replication in Mycobacterium tuberculosis

Natural products provide a rich source of potential antimicrobials for use in treating infectious diseases for which drug resistance has emerged. Foremost among these is tuberculosis. Assessment of the antimycobacterial activity of nargenicin, a natural product that targets the replicative DNA polymerase of Staphylococcus aureus, revealed that it is a bactericidal genotoxin that induces a DNA damage response in Mycobacterium tuberculosis (Mtb) and inhibits growth by blocking the replicative DNA polymerase, DnaE1. Cryo-electron microscopy revealed that binding of nargenicin to Mtb DnaE1 requires the DNA substrate such that nargenicin is wedged between the terminal base pair and the polymerase and occupies the position of both the incoming nucleotide and templating base. Comparative analysis across three bacterial species suggests that the activity of nargenicin is partly attributable to the DNA binding affinity of the replicative polymerase. This work has laid the foundation for target-led drug discovery efforts focused on Mtb DnaE1.

microbiology↗

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

Flow cytometry method for absolute counting and single-cell phenotyping of mycobacteria

Detection and accurate quantitation of viable Mycobacterium tuberculosis is fundamental to understanding mycobacterial pathogenicity, tuberculosis (TB) disease progression and outcomes; TB transmission; drug action, efficacy and drug resistance. Despite this importance, methods for determining numbers of viable bacilli are limited in accuracy and precision owing to inherent characteristics of mycobacterial cell biology - including the tendency to clump, and "differential" culturability - and technical challenges consequent on handling an infectious pathogen under biosafe conditions. We developed an absolute counting method for mycobacteria in liquid cultures using a bench-top flow cytometer, and the low-cost fluorescent dyes Calcein-AM (CA) and SYBR-gold (SG). During exponential growth CA+ cell counts are highly correlated with CFU counts and can be used as a real-time alternative to simplify the accurate standardisation of inocula for experiments. In contrast to CFU counting, this method can detect and enumerate cell aggregates in samples, which we show are a potential source of variance and bias when using established methods. We show that CFUs comprise a sub-population of intact, metabolically active mycobacterial cells in liquid cultures, with CFU-proportion varying by growth conditions. A pharmacodynamic application of the flow cytometry method, exploring kinetics of fluorescent probe defined subpopulations compared to CFU is demonstrated. Flow cytometry derived Mycobacterium bovis BCG time-kill curves differ for rifampicin and kanamycin versus isoniazid and ethambutol, as do the relative dynamics of discrete morphologically-distinct subpopulations of bacilli revealed by this high-throughput single-cell technique.

microbiology↗

bioRxiv2020

De novo cobalamin biosynthesis, transport and assimilation and cobalamin-mediated regulation of methionine biosynthesis in Mycobacterium smegmatis

Cobalamin is an essential co-factor in all domains of life, yet its biosynthesis is restricted to some bacteria and archaea. Mycobacterium smegmatis, an environmental saprophyte frequently used as surrogate for the obligate human pathogen, M. tuberculosis, carries approximately 30 genes predicted to be involved in de novo cobalamin biosynthesis. M. smegmatis also encodes multiple cobalamin-dependent enzymes, including MetH, a methionine synthase which catalyses the final reaction in methionine biosynthesis. In addition to metH, M. smegmatis possesses a cobalamin-independent methionine synthase, metE, suggesting that enzyme selection - MetH or MetE - is regulated by cobalamin availability. Consistent with this notion, we previously described a cobalamin-sensing riboswitch controlling metE expression in M. tuberculosis. Here, we apply a targeted mass spectrometry-based approach to confirm de novo cobalamin biosynthesis in M. smegmatis during aerobic growth in vitro. We also demonstrate that M. smegmatis transports and assimilates exogenous cyanocobalamin (CNCbl; a.k.a. vitamin B12) and its precursor, dicyanocobinamide ((CN)2Cbi). Interestingly, the uptake of CNCbl and (CN)2Cbi appears restricted in M. smegmatis and dependent on the conditional essentiality of the cobalamin-dependent methionine synthase. Using gene and protein expression analyses combined with single-cell growth kinetics and live-cell time-lapse microscopy, we show that transcription and translation of metE are strongly attenuated by endogenous cobalamin. These results support the inference that metH essentiality in M. smegmatis results from riboswitch-mediated repression of MetE expression. Moreover, differences observed in cobalamin-dependent metabolism between M. smegmatis and M. tuberculosis provide some insight into the selective pressures which might have shaped mycobacterial metabolism for pathogenicity. IMPORTANCEAccumulating evidence suggests that alterations in cobalamin-dependent metabolism marked the evolution of Mycobacterium tuberculosis from an environmental ancestor to an obligate human pathogen. However, the roles of cobalamin in mycobacterial physiology and pathogenicity remain poorly understood. We used the non-pathogenic saprophyte, M. smegmatis, to investigate the production of cobalamin, transport and assimilation of cobalamin precursors, and the potential role of cobalamin in regulating methionine biosynthesis. We provide biochemical and genetic evidence confirming constitutive de novo cobalamin biosynthesis in M. smegmatis under standard laboratory conditions, in contrast with M. tuberculosis, which appears to lack de novo cobalamin biosynthetic capacity. We also demonstrate that the uptake of cyanocobalamin (vitamin B12) and its precursors is restricted in M. smegmatis, apparently depending on the need to service the co-factor requirements of the cobalamin-dependent methionine synthase. These observations support the utility of M. smegmatis as a model to elucidate key metabolic adaptations enabling mycobacterial pathogenicity.

microbiology↗

bioRxiv2020

Chemical validation of Mycobacterium tuberculosis phosphopantetheine adenylyltransferase using fragment linking and CRISPR interference

The coenzyme A (CoA) biosynthesis pathway has attracted attention as a potential target for much-needed novel antimicrobial drugs, including for the treatment of tuberculosis (TB), the lethal disease caused by Mycobacterium tuberculosis (Mtb). Seeking to identify the first inhibitors of Mtb phosphopantetheine adenylyltransferase (MtbPPAT), the enzyme that catalyses the penultimate step in CoA biosynthesis, we performed a fragment screen. In doing so, we discovered three series of fragments that occupy distinct regions of the MtbPPAT active site, presenting a unique opportunity for fragment linking. Here we show how, guided by X-ray crystal structures, we could link weakly-binding fragments to produce an active site binder with a KD < 20 M and on-target anti-Mtb activity, as demonstrated using CRISPR interference. This study represents a big step toward validating MtbPPAT as a potential drug target and designing a MtbPPAT-targeting anti-TB drug. O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

biochemistry↗

bioRxiv2020

Arrayed CRISPRi and Quantitative Imaging Describe the Morphotypic Landscape of Essential Mycobacterial Genes

Mycobacterium tuberculosis possesses a large number of genes of unknown or merely predicted function, undermining fundamental understanding of pathogenicity and drug susceptibility. To address this challenge, we developed a high-throughput functional genomics approach combining inducible CRISPR-interference and image-based analyses of morphological features and sub-cellular molecular localizations in the related non-pathogen, M. smegmatis. Applying automated imaging and analysis to an arrayed library of 272 essential gene knockdown mutants, we derive robust, quantitative descriptions of bacillary morphologies consequent on gene silencing. Leveraging statistical-learning, we demonstrate that functionally related genes cluster by morphotypic similarity and that this information can be used to infer gene function. Exploiting this observation, we reveal a previously unknown restriction-modification system, and identify filamentation as a defining mycobacterial response to histidine starvation. Our results support the application of large-scale image-based analyses for mycobacterial functional genomics, simultaneously establishing the utility of this approach for drug mechanism-of-action studies.

microbiology↗

bioRxiv2019

Capture and visualization of live Mycobacterium tuberculosis bacilli from tuberculosis bioaerosols

The tuberculosis (TB) pandemic demands urgent interventions such as those designed to interrupt Mycobacterium tuberculosis (Mtb) transmission, a challenge exacerbated by our poor understanding of the events enabling successful transfer of infectious bacilli between hosts. To address this problem, we developed the Respiratory Aerosol Sampling Chamber (RASC), a personal clean-room equipped with high-efficiency filtration and sampling technologies that allow biosafe capture and isolation of particulate matter - including Mtb bacilli - released by patients during natural breathing and (non-induced) cough. Here, we demonstrate the use of DMN-trehalose labelling to detect and quantify live Mtb bacilli among complex bioaerosol samples arrayed in a bespoke nanowell device following capture in the RASC. A pilot study identified Mtb in more than 85 % of known TB patients, improving significantly on previous work which has relied on animal infection and cough sampling to estimate transmission events. Moreover, intra-patient comparisons of bioaerosol and sputum samples indicated that Mtb aerosols likely derive from a compartment other than sputum. These results support the utility of the RASC platform for research aimed at interrupting Mtb transmission, including the non-invasive detection of Mtb-infected individuals who are predicted to contribute to bacillary spread despite the absence of clinical symptoms.

microbiology↗

bioRxiv2019

Inhibiting Mycobacterium tuberculosis CoaBC by targeting a new allosteric site.

Coenzyme A (CoA) is a fundamental co-factor for all life, involved in numerous metabolic pathways and cellular processes, and its biosynthetic pathway has raised substantial interest as a drug target against multiple pathogens including Mycobacterium tuberculosis. The biosynthesis of CoA is performed in five steps, with the second and third steps being catalysed in the vast majority of prokaryotes, including M. tuberculosis, by a single bifunctional protein, CoaBC. Depletion of CoaBC was found to be bactericidal in M. tuberculosis. Here we report the first structure of a full-length CoaBC, from the model organism Mycobacterium smegmatis, describe how it is organised as a dodecamer and regulated by CoA thioesters. A high-throughput biochemical screen focusing on CoaB identified two inhibitors with different chemical scaffolds. Hit expansion led to the discovery of potent inhibitors of M. tuberculosis CoaB, which we show to bind to a novel cryptic allosteric site within CoaB.

biochemistry↗

bioRxiv2019

Developing synergistic drug combinations to restore antibiotic sensitivity in drug-resistant Mycobacterium tuberculosis

Tuberculosis (TB) is a leading global cause of mortality owing to an infectious agent, accounting for almost one-third of antimicrobial resistance (AMR) deaths annually. We aimed to identify synergistic anti-TB drug combinations with the capacity to restore therapeutic efficacy against drug-resistant mutants of the causative agent, Mycobacterium tuberculosis. We investigated combinations containing the known translational inhibitors, spectinomycin (SPT) and fusidic acid (FA), or the phenothiazine, chlorpromazine (CPZ), which disrupts mycobacterial energy metabolism. Potentiation of whole-cell drug efficacy was observed in SPT-CPZ combinations. This effect was lost against an M. tuberculosis mutant lacking the major facilitator superfamily (MFS) efflux pump, Rv1258c. Notably, the SPT-CPZ combination restored SPT efficacy against an SPT-resistant mutant carrying a g1379t point mutation in rrs, encoding the mycobacterial 16S ribosomal RNA. Combinations of SPT with FA, which targets the mycobacterial elongation factor G, exhibited potentiating activity against wild-type M. tuberculosis. Moreover, this combination produced a marginal potentiating effect against both FA-monoresistant and SPT-monoresistant mutants. Finally, combining SPT with the frontline anti-TB agents, rifampicin (RIF) and isoniazid, resulted in enhanced activity in vitro and ex vivo against both drug-susceptible M. tuberculosis and a RIF-monoresistant rpoB S531L mutant.These results support the utility of novel potentiating drug combinations in restoring antibiotic susceptibility of M. tuberculosis strains carrying genetic resistance to any one of the partner compounds.

microbiology↗