Unravelling the physiological roles of mazEF toxin-antitoxin system on clinical MRSA strain by CRISPR RNA-guided cytidine deaminase.
Unravelling the physiological roles of mazEF toxin-antitoxin system on clinical MRSA strain by CRISPR RNA-guided cytidine deaminase.
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DOI：
10.1186/s12929-022-00810-5
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发表时间：
2022-05-07
影响因子：
11
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中科院分区：
文献类型：
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近年来，毒素-抗毒素模块的抗体急剧增加，因为它普遍存在于许多细菌基因组中，包括耐甲氧西林金黄色葡萄球菌（MRSA）等病原体。TA系统的几种细胞功能已经被提出，然而，它们在细胞生理学中的确切作用仍然没有得到解决。本研究旨在通过使用CRISPR-cas9碱基编辑质粒（pnCasSA-BEC）构建迷宫和mazF突变体，了解MazEF毒素-抗毒素模块对分离的临床ST 239 MRSA菌株中生物膜形成、发病机制和抗生素耐药性的影响。对迷宫抗毒素突变体进行转录组分析（RNA-seq）以鉴定差异调节的基因。还评估了突变菌株的生物膜形成。对产生的两种突变体进行抗生素谱分析，然后进行小鼠实验以确定构建的菌株的致病性。我们的工作第一次表明，MazF促进了用于生物膜形成的MAZIA介导的细胞死亡和裂解，而在宿主毒力中不起任何显著作用，如鼠实验所示。有趣的是，在迷宫突变株中，激活的MazF毒素显著降低了对苯唑西林、达托霉素和万古霉素的敏感性。我们的研究表明，激活的MazF毒素导致对苯唑西林、达托霉素和万古霉素等抗生素的耐药性。因此，在未来，任何潜在的抗菌药物都可以被设计成靶向MazF毒素对抗有问题的多药耐药性细菌。在线版本包含补充材料，可通过10.1186/s12929-022-00810-5获得。
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. 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. 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. 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. The online version contains supplementary material available at 10.1186/s12929-022-00810-5.
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