An integrated whole genome analysis of Mycobacterium tuberculosis reveals insights into relationship between its genome, transcriptome and methylome

Abstract

Human tuberculosis disease (TB), caused by Mycobacterium tuberculosis (Mtb), is a complex disease, with a spectrum of outcomes. Genomic, transcriptomic and methylation studies have revealed differences between Mtb lineages, likely to impact on transmission, virulence and drug resistance. However, so far no studies have integrated sequence-based genomic, transcriptomic and methylation characterisation across a common set of samples, which is critical to understand how DNA sequence and methylation affect RNA expression and, ultimately, Mtb pathogenesis. Here we perform such an integrated analysis across 22 M. tuberculosis clinical isolates, representing ancient (lineage 1) and modern (lineages 2 and 4) strains. The results confirm the presence of lineage-specific differential gene expression, linked to specific SNP-based expression quantitative trait loci: with 10 eQTLs involving SNPs in promoter regions or transcriptional start sites; and 12 involving potential functional impairment of transcriptional regulators. Methylation status was also found to have a role in transcription, with evidence of differential expression in 50 genes across lineage 4 samples. Lack of methylation was associated with three novel variants in mamA, likely to cause loss of function of this enzyme. Overall, our work shows the relationship of DNA sequence and methylation to RNA expression, and differences between ancient and modern lineages. Further studies are needed to verify the functional consequences of the identified mechanisms of gene expression regulation.

Drivers and sites of diversity in the DNA adenine methylomes of 93 Mycobacterium tuberculosis complex clinical isolates

“…If mntR bound to that site, it could prevent HsdM from methylating it. Meanwhile, genotyping mntR revealed that all 18 IO isolates shared the variant mntR Q131* ( Figure 5B ), a nonsense mutation found previously in IO isolates ( Gomez-Gonzalez et al, 2019 ) that truncates MntR. This truncation could explain why cobK 304 was methylated in all 18 IO isolates.…”

“…This contrasts with the approach of prior M. tuberculosis methylome studies ( Chiner-Oms et al, 2019 ; Gomez-Gonzalez et al, 2019 ; Phelan et al, 2018 ; Shell et al, 2013 ; Zhu et al, 2016 ), which examined only the MTase motifs identified by PacBio MotifMaker ( https://github.com/PacificBiosciences/MotifMaker ). This systematic approach revealed MTase genotypes with partial function, previously mischaracterized as completely inactive ( Gomez-Gonzalez et al, 2019 ). Third, we used phylogenetic analysis to identify convergent MTase activity and heterogeneity analysis to characterize epigenomic diversity in vitro.…”

“…However, subsequent SMRT-sequencing studies reported contradictory findings. Three studies reported a lack of methylation at all MamA motif sites in mamA E270A isolates ( Gomez-Gonzalez et al, 2019 ; Phelan et al, 2018 ; Zhu et al, 2016 ) while another reported partial methylation in one mamA E270A isolate, and a complete lack of methylation in two mamA E270A isolates ( Chiner-Oms et al, 2019 ). In the 34 isolates with mamA E270A , MamA motif sites had intermediate IPD ratios ( Figure 2A , top), indicating partial activity.…”

“…Throughout this manuscript we have referred to the DNA adenine methyltransferase encoded by Rv2756c as HsdM ( hsdM for the gene) and its specificity subunit encoded by Rv2761, as HsdS ( hsdS for the gene) to be consistent with previous work ( Shell et al, 2013 ). It appears that Rv2756c was originally referred to as HsdM based on homology to hsdM in R-M systems—before the existence of its restriction component had been investigated—and has propagated through subsequent studies ( Chiner-Oms et al, 2019 ; Gomez-Gonzalez et al, 2019 ; Phelan et al, 2018 ; Zhu et al, 2016 ). However, it has since been determined that Rv2756c lacks a functional HsdR component ( Zhu et al, 2016 ).…”

“…Previous interrogation of cis-regulation by DNA methylation in M. tuberculosis through SMRT-sequencing identified seven differentially methylated sites upstream of differentially expressed genes ( Gomez-Gonzalez et al, 2019 ). However, only Euro-American and Indo-Oceanic (IO) isolates were analyzed, and only the 200 base pairs (bp) upstream of differentially expressed genes were examined for methylation, rather than a window more rigorously informed by mapped promoters and potential mechanisms of cis-regulation.…”