Background Mycobacterium tuberculosis, the causative agent of Tuberculosis (TB) is a notorious pathogen that is responsible for the highest mortalities from a single bacterial pathogen worldwide. Many studies have revealed that cholesterol contribute to M. tuberculosis pathogenesis with unique transcriptome changes implicated to cholesterol metabolism in genetically diverse clinical strains of M. tuberculosis complex (MTBC). Hence, the current study was aimed at investigating epigenetic changes associated with cholesterol metabolism since these changes may provide novel targets for development of TB treatments.

Methods The laboratory M. tuberculosis strain, H37Rv together with the recently identified Lineage 8 clinical strain were cultured in 7H9 broth and minimal media supplemented with cholesterol as the main carbon source. DNA was extracted using the Cetyltrimethylammonium bromide method followed by clean-up using Zymo DNA concentrator kit. Long read whole genome sequencing was performed in a PacBio SMART sequencer for complete methylome characterization using the RS Modification and Motif Analysis protocol and annotated further using DistAMo by selecting methylated genes with a significant z score (≥2 or ≤-2).

Results The highest significantly methylated motifs, CTCCAG, CTGGAG and VNCYGVNYR coding for Rv2060, rseA and Rv1175 genes, respectively, were detected in H37Rv grown in normal 7H9 broth while an additional CYGVNYR motif was detected during growth in cholesterol-rich media. This was in contrast to RNCYGVNYR motif detected in the Rv3632 gene for Lineage 8 strain during grown in 7H9 broth compared to CBBV, CTACCCGVC, GATNNNNRTAC, GNCTACSCA, GTAYNNNNATC, GVGGYMVCR and CACGCAGHNH motifs detected for pks8, Rv2459, PE_PGRS16, vapC22, fadD2, sseA, ackA genes, respectively.

Conclusion These findings suggest “unique” epigenetic regulation in clinical strains of MTBC compared to the laboratory H37Rv, which may explain their virulence traits. The precise characterization of MTBC methylation profiles in cholesterol-rich environments could open new avenues for the development of treatments since cholesterol is essential during M. tuberculosis pathogenesis.

Funding: EDCTP