Unravelling the physiological roles of mazEF toxin–antitoxin system on clinical MRSA strain by CRISPR RNA-guided cytidine deaminase

Abstract

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.

Role of VapBC4 toxin-antitoxin system ofSulfolobus acidocaldariusin heat stress adaptation

“…The presence of a large number of vapBC toxin-antitoxin (TA) systems within the group of thermoacidocphilic archaea, including S. acidocaldarius , implies a possible role in adapting to their specific environments. Because bacterial TA systems are involved in programmed cell death or persister cell formation during stress conditions [2], we speculated that depending on the stress conditions, S. acidocaldarius might activate or deactivate specific vapBC systems to determine the fate of the cells. In our earlier research, we have demonstrated how the vapBC system plays a part in adapting to various stresses.…”

“…In recent years, the investigation of toxin-antitoxin (TA) systems has gained attention due to their essential roles in regulating bacterial growth, aiding survival under stressful conditions, and their potential implications in bacterial virulence and antibiotic resistance [1, 2]. TA systems consist of a pair of closely linked genes encoding a stable toxin and a labile antitoxin, wherein the antitoxin neutralizes the toxin’s effects, maintaining cellular equilibrium [3].…”

Genome Editing of Veterinary Relevant Mycoplasmas Using a CRISPR-Cas Base Editor System

“…In the era of synthetic biology, with the fast expansion of the toolbox for modifying the genomes of living organisms, base-editing tools have attracted interest because of specific features: independence of endogenous cellular DNA repair pathways, lower cytotoxic effects than with other Cas9-based tools, high efficacy, which means that there is no need for selection markers, possibility of multitargeting, and the lack of a scar at the edited locus. These properties make BE promising tools for clinical usage in human therapeutics ( 60 ) and for multiple applications in plants ( 51 , 61 ) and microorganisms of medical or biotechnological relevance ( 62 – 64 ). Because of their high efficacy, BE are particularly attractive for bacteria that are difficult to transform, as, ultimately, one single transformant can be enough to obtain a mutant.…”

The Benefits of Toxicity: M. smegmatis VapBC TA Module Is Induced by Tetracycline Exposure and Promotes Survival

“…The demonstrated deceleration in protein synthesis under VapC expression and bacterial growth slowdown resultant from tRNA slicing reveals the toxin role as the accurate tuner of the translation process and contributes to stress response development, in particular, under treatment with antibiotics inhibiting ribosome activity [15]. In turn, the mazEF module is associated with the formation of persisting and dormant bacteria [16,17] and affects antibiotic resistance [18] as well as susceptibility to different environmental stresses [9,19].…”