Biology subjects

Rifat, D.

Publications and source records attributed to Rifat, D..

4 recordsLinked to original sources

bioRxiv2026

Rv2061c, a Putative F420-Dependent Oxidoreductase, is Required for Tolerance to Malachite Green and Other Antimicrobials and Oxidative Stress Protection in Mycobacterium tuberculosis

The pathogenicity of Mycobacterium tuberculosis relies on its ability to withstand hostile host environments. Coenzyme F420 is critical for redox metabolism in Mycobacterium tuberculosis an organism distinguished by its abundant use of this low-redox-potential cofactor and a repertoire of F420-dependent enzymes. While F420-dependent mechanisms are known to confer protection against antimicrobials and oxidative stress, the specific enzymes responsible remain largely unidentified. In this study, using the F420-dependent detoxification of malachite green as a phenotypic marker, we performed a genome-wide screen in Mycobacterium tuberculosis using transposon mutagenesis and whole genome sequencing. The results of transposon mutagenesis and whole genome sequencing revealed that, among the 28 genes encoding F420-dependent enzymes, only mutations in fgd (essential for F420H2 re-generation) and Rv2061c, a gene encoding a putative F420-dependent oxidoreductase, were highly susceptible to malachite green. Subsequent experiments confirmed that Rv2061c and its homolog MT2120 are required for detoxifying malachite green and crystal violet. Furthermore, the Rv2061c mutant exhibited increased susceptibility to isoniazid and pretomanid; as well as menadione-induced oxidative stress. We also identified mutations in the transcriptional repressor Rv0678 as a key determinant of resistance to malachite green and crystal violet. This study establishes Rv2061c as a pivotal F420-dependent oxidoreductase that protects Mycobacterium tuberculosis from malachite green and other antimicrobial compounds as well as oxidative stress and further supports Rv0678 as a master regulator of multidrug resistance. These findings expand our knowledge of Mycobacterium tuberculosis defense mechanisms and highlight F420-dependent pathways as a potential target for new drug development.

microbiology↗

bioRxiv2024

In vitro and in vivo validation of cwlM and pbpB essentiality for viability and resistance to imipenem in Mycobacterium abscessus

Mycobacterium abscessus lung infection is notoriously difficult to treat due, in part, to the intrinsic resistance of this pathogen to most marketed antibiotics. {beta}-Lactams, namely imipenem and cefoxitin, are first-line drugs in combination regimens used to treat this infection; and there is growing interest in dual-{beta}-lactam-based regimens. Better understanding of the molecular basis of {beta}-lactam activity through study of the genetic determinants of {beta}-lactam susceptibility and tolerance would enable more rational drug combinations and guide discovery of novel drug targets to complement {beta}-lactams. We recently used an inducible CRISPR interference (CRISPRi) system to silence cwlM and pbpB and confirm their essentiality for in vitro growth and resistance to sub-MIC concentrations of imipenem. Here, we extend those findings to show that silencing either gene alone is bactericidal and augments the bactericidal activity of imipenem in vitro. Furthermore, using CRISPRi in a mouse model of M. abscessus lung infection for the first time, we confirm the essentiality of each gene for in vivo survival. These results validate cwlM and pbpB as essential genes and promising drug targets in this pathogen, including for potentiation of carbapenem activity. The results further establish CRISPRi as a powerful method for validating drug targets and studying gene-gene and gene-drug interactions in vitro and in vivo.

microbiology↗

bioRxiv2020

Mutations in fbiD(Rv2983) as a novel determinant of resistance to pretomanid and delamanid in Mycobacterium tuberculosis

The nitroimidazole pro-drugs delamanid and pretomanid comprise one of only two new antimicrobial classes approved to treat tuberculosis (TB) in 50 years. Prior in vitro studies suggest a relatively low barrier to nitroimidazole resistance in Mycobacterium tuberculosis, but clinical evidence is limited to date. We selected pretomanid-resistant M. tuberculosis mutants in two mouse models of TB using a range of pretomanid doses. The frequency of spontaneous resistance was approximately 10-5 CFU. Whole genome sequencing of 161 resistant isolates from 47 mice revealed 99 unique mutations, 91% of which occurred in 1 of 5 genes previously associated with nitroimidazole activation and resistance: fbiC (56%), fbiA (15%), ddn (12%), fgd (4%) and fbiB (4%). Nearly all mutations were unique to a single mouse and not previously identified. The remaining 9% of resistant mutants harbored mutations in Rv2983, a gene not previously associated with nitroimidazole resistance but recently shown to be a guanylyltransferase necessary for cofactor F420 synthesis. Most mutants exhibited high-level resistance to pretomanid and delamanid, although Rv2983 and fbiB mutants exhibited high-level pretomanid resistance, but relatively small changes in delamanid susceptibility. Complementing an Rv2983 mutant with wild-type Rv2983 restored susceptibility to pretomanid and delamanid. By quantifying intracellular F420 and its precursor Fo in overexpressing and loss-of-function mutants, we provide further evidence that Rv2983 is necessary for F420 biosynthesis. Finally, Rv2983 mutants and other F420H2-deficient mutants displayed hypersusceptibility to some antibiotics and to concentrations of malachite green found in solid media used to isolate and propagate mycobacteria from clinical samples.

microbiology↗

bioRxiv2018

Mutations in Rv2983 as a novel determinant of resistance to nitroimidazole drugs in Mycobacterium tuberculosis

Delamanid represents one of two novel antimicrobial classes approved to treat tuberculosis in over 40 years. Pretomanid is another promising nitroimidazole pro-drug in clinical development. Characterization of the full spectrum of mutations conferring resistance to nitroimidazoles and their related phenotypes in Mycobacterium tuberculosis will inform development of suitable genotypic and phenotypic drug susceptibility tests. Here, we used a range of pretomanid doses to select pretomanid-resistant mutants in two pathologically distinct murine TB models. The frequency of spontaneous pretomanid resistance mutations was approximately 10-5 CFU. Pretomanid demonstrated dose-dependent bactericidal activity and selective amplification of resistant mutants. Whole genome sequencing of 161 resistant isolates from 47 mice revealed 99 unique mutations, 90% of which were found in 1 of 5 genes previously associated with nitroimidazole activation and resistance. The remaining 10% harbored isolated mutations in Rv2983. Complementing an Rv2983 mutant with a wild-type copy of Rv2983 restored wild-type susceptibility to pretomanid and delamanid, confirming that loss of Rv2983 function causes nitroimidazole resistance. By quantifying F420 and its precursor Fo in Mycobacterium smegmatis overexpressing Rv2983 and an M. tuberculosis Rv2983 mutant, we provide evidence that Rv2983 is necessary for F420 biosynthesis and nitroimidazole activation, perhaps as the guanylyltransferase CofC. F420H2-deficient mutants displayed hypersusceptibility to malachite green (MG), a selective decontaminant present in solid media used to isolate and propagate mycobacteria from clinical samples. The wide diversity of mutations causing high-level pretomanid resistance and MG hypersusceptibility of most mutants poses significant challenges to clinical detection of nitroimidazole resistance using either genotypic or phenotypic methods.\n\nSignificanceNitroimidazole pro-drugs represent a promising new class of anti-tuberculosis drugs. Reliable methods to assure nitroimidazole susceptibility are critical to assure their optimal use. Yet, the spectrum of nitroimidazole resistance mutations remains incompletely characterized. Using 161 pretomanid-resistant Mycobacterium tuberculosis isolates selected in pretomanid-treated mice, we discovered a novel resistance determinant, Rv2983, required for cofactor F420 biosynthesis and characterized the remarkable diversity of mutations in this and 5 other genes involved in nitroimidazole activation. We show that F420H2-deficient nitroimidazole-resistant mutants are hypersusceptible to the selective decontaminant malachite green used in solid media to isolate mycobacteria and may evade detection on such media. These results have important implications for development and clinical use of genotypic and phenotypic methods for nitroimidazole susceptibility testing.

microbiology↗