Rec A Disruption Unveils Cross Talk Between Dna Repair And Membrane Damage, Efflux Pump Activity, Biofilm Formation In Mycobacterium Smegmatis

Tuberculosis (TB) caused by Mycobacterium tuberculosis (MTB) has emerged in recent decades as one of the leading causes of mortality worldwide. The burden of TB is alarmingly high, with one third affected global population as reported by WHO. Short-course treatment with an antibiotic is a powerful weapon to treat infection of susceptible MTB strain, however; MTB has developed resistance to anti-TB drugs, which is an escalating global health crisis. Thus there is urgent need to identify new drug targets. RecA is a 38 kilodalton protein required for the repair and maintenance of DNA and regulation of the SOS response. The objective of this study is to understand the effect of disruption of RecA gene (deletion mutant Delta disA from previous study) in a surrogate model for MTB, Mycobacterium smegmatis. This study demonstrated that disruption of RecA causes enhanced susceptibility towards rifampicin and generation of ROS leading to lipid peroxidation and impaired membrane homeostasis as depicted by altered cell membrane permeability and efflux pump activity. Mass spectrometry based lipidomic analysis revealed decreased mycolic acid moieties, phosphatidylinositol mannosides (PIM), Phthiocerol dimycocerosate (DIM). Furthermore, biofilm formation was considerably reduced. Additionally, we have validated all the disrupted phenotypes by RT-PCR which showed a good correlation with the biochemical assays. Lastly, RecA mutant displayed reduced infectivity in Caenorhabditis elegans illustrating its vulnerability as antimycobacterial target. Together, present study establishes a link between DNA repair, drug efflux and biofilm formation and validates RecA as an effective drug target. Intricate studies are needed to further understand and exploit this therapeutic opportunity.