Biology subjects

Mizrahi, V.

Publications and source records attributed to Mizrahi, V..

4 recordsLinked to original sources

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↗