Browsing by Subject "CRISPR–Cas9"

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    Unravelling the physiological roles of mazEF toxin–antitoxin system on clinical MRSA strain by CRISPR RNA-guided cytidine deaminase
    (Springer Nature, 2022-05-07) Jain, Sonia; Bhowmick, Arghya; Jeong, Bohyun; Bae, Taeok; Ghosh, Abhrajyoti; Microbiology and Immunology, School of Medicine
    Background: Curiosity on toxin-antitoxin modules has increased intensely over recent years as it is ubiquitously present in many bacterial genomes, including pathogens like Methicillin-resistant Staphylococcus aureus (MRSA). Several cellular functions of TA systems have been proposed however, their exact role in cellular physiology remains unresolved. Methods: This study aims to find out the impact of the mazEF toxin-antitoxin module on biofilm formation, pathogenesis, and antibiotic resistance in an isolated clinical ST239 MRSA strain, by constructing mazE and mazF mutants using CRISPR-cas9 base-editing plasmid (pnCasSA-BEC). Transcriptome analysis (RNA-seq) was performed for the mazE antitoxin mutant in order to identify the differentially regulated genes. The biofilm formation was also assessed for the mutant strains. Antibiogram profiling was carried out for both the generated mutants followed by murine experiment to determine the pathogenicity of the constructed strains. Results: For the first time our work showed, that MazF promotes cidA mediated cell death and lysis for biofilm formation without playing any significant role in host virulence as suggested by the murine experiment. Interestingly, the susceptibility to oxacillin, daptomycin and vancomycin was reduced significantly by the activated MazF toxin in the mazE mutant strain. Conclusions: Our study reveals that activated MazF toxin leads to resistance to antibiotics like oxacillin, daptomycin and vancomycin. Therefore, in the future, any potential antibacterial drug can be designed to target MazF toxin against the problematic multi-drug resistant bug.