Regulation of the sRNA ncBCG427 on mycobacterial stress adaptation

Mycobacterium tuberculosis complex (MTBC) comprises the primary pathogens responsible for tuberculosis in humans and animals, including the virulent Mycobacterium tuberculosis (M. tb) and Mycobacterium bovis (M. bovis). As a group of intracellular bacteria, MTBC has developed intricate mechanisms for infection and survival within host cells. Among these mechanisms, small non-coding RNAs (sRNAs) play essential roles in regulating post-transcriptional pathways that may aid in stress adaptation in mycobacteria. In our previous research, we identified a novel sRNA, ncBCG427, in BCG, the attenuated strain of M. bovis. This sRNA is differentially expressed under various stress conditions, and homologs exist in both M. tb and M. bovis.

To explore the regulatory capabilities of ncBCG427 on bacterial stress adaptation in the original BCG strain, we constructed overexpression (BCG_ncBCG427) and control (BCG_Vector) strains. Our findings demonstrated that ncBCG427 significantly enhanced BCG survival under carbon starvation and acid stress conditions, while it resulted in decreased survival under iron starvation conditions. Transcriptomic analyses revealed that overexpression of ncBCG427 substantially altered gene expression profiles in these three stress environments, with differentially expressed genes (DEGs) in the same pathways enriched for different stressors. Notably, we identified 22 DEGs that responded to two or three stress conditions, implicating their involvement in pathways related to the sulfur relay system, ribosome function, folate biosynthesis, and the biosynthesis of cofactors. This suggested that these genes may served as key regulators in ncBCG427-mediated stress adaptation. Furthermore, quantitative PCR (qPCR) was employed to verify the expression levels of 16 DEGs in BCG, with 14 of them matching homologous genes in M. tb. We then created a ncBCG427 overexpression strain in M. tb (MTB_ncBCG427) alongside a control strain (MTB_Vector). Interestingly, 4 of the 14 genes displayed consistent expression trends in both M. tb and BCG under different stress conditions.

In conclusion, our study underscored the significant role of the sRNA ncBCG427 in enhancing mycobacterial survival under stress conditions and proposed potential target genes for further exploration into the mechanisms of stress adaptation in mycobacteria.