Investigating the role of small RNAS in transcriptome regulation of genetically diverse clinical strains of mycobacterium tuberculosis.

Abstract

Tuberculosis (TB), caused by the human adapted members of the Mycobacterium tuberculosis complex (MTBC), is a threat to global health. Understanding the regulatory network of the MTBC members may reveal novel vaccine candidates and drug targets. The small RNAs (sRNAs) have only recently been investigated for their role in Mycobacterium tuberculosis (M. tb) transcriptome regulation with none being explored in clinical strains or within the MTBC lineages. The present study aimed to investigate the regulatory role of sRNAs on the M. tb transcriptome in a lineage-specific manner, with emphasis on the clinical strains most prevalent in South Africa. In silico whole genome sequence alignment of strains belonging to the eight MTBC lineages was performed to identify sRNAs containing lineage-specific mutations and their respective potential targets. To elucidate transcriptome regulation in clinical strains of M. tb belonging to the Beijing and F15/LAM4/KZN lineages, mRNA and sRNA sequencing were performed followed by Hisat-Ballgown Bioinformatics analysis to identify novel sRNAs and their respective targets. The sRNAs discovered from sRNA sequencing were confirmed through real time qPCR. The in silico data revealed several sRNAs that may play a role in transcriptome regulation at a lineage-specific level, such as those involved in macrophage entry, lipid biosynthesis pathway, adaptation mechanisms during antibiotic exposure, and environmental stress. They may also be able to disrupt genes that are detrimental and restore functions to those that are beneficial. The mutated and consensus sRNAs were identified to target the same function, but one pathway may be more efficient than the other. Novel sRNAs were discovered from sRNA sequencing of the Beijing and F15/LAM4/KZN clinical strains, with their predicted targets absent from the mRNA sequencing results, indicating these sRNAs may elicit an inhibitory function. Real time-PCR analysis revealed significant fold change differences between the clinical strains belonging to the Beijing, F15/LAM4/KZN, F11 and Unique families suggesting an underlying regulation of these transcripts at a family level. This data could explain the underlying phenotypic differences observed within the MTBC and understanding of the regulatory function of these sRNAs, may identify novel alternative strategies in the fight against M. tb.