sRNA ncBCG427 activates the expression of target gene MSMEG_4757 to enhance the survival of Mycobacterium smegmatis through lipid metabolism in adverse environments

The capability of mycobacteria to survive in adverse environments is crucial for successful infection, yet the underlying mechanisms remain unclear. A novel sRNA, ncBCG427, was previously identified in intracellular versus extracellular mycobacteria, with predicted targets clustering in lipid metabolism pathways in Mycobacterium smegmatis (M. sm). This study aimed to investigate how ncBCG427 regulates the survival of M. sm through lipid metabolism. Using lipidomics, metabolites from the ncBCG427-expressing strain (MS_ncBCG427) and the control strain (MS_Vector) were screened, revealing enrichment in lipid-associated pathways. The gene MSMEG_4757 (Fas) was identified as critical to this pathway and confirmed as a target of ncBCG427. Western blot analysis demonstrated that ncBCG427 increased Fas expression in THP-1 cells post-infection. Additionally, Oil Red O staining indicated that both ncBCG427 and MSMEG_4757 enhanced lipid droplet formation in A549 cells. Both MS_ncBCG427 and MS_4757 exhibited increased biofilm formation and enhanced survival under various adverse conditions, including carbon starvation, acid stress, membrane stress, and exposure to drugs such as rifampicin and streptomycin. In contrast, low-expression strains (MS_sh4757 and MS_ncBCG427_sh4757) showed reduced survival. In conclusion, ncBCG427 targets MSMEG_4757 to regulate lipid metabolism, enhancing biofilm formation and survival in adverse environments, revealing a novel mechanism of mycobacterial survival and potential antimicrobial targets.