Phase variation as a major mechanism of adaptation inMycobacterium tuberculosiscomplex

Abstract

Phase variation induced by insertions and deletions (INDELs) in genomic homopolymeric tracts (HT) can silence and regulate genes in pathogenic bacteria but this process is not characterized in MTBC adaptation. We leverage 31,428 diverse clinical isolates to identify genomic regions including phase-variants under positive selection. Of 87,651 INDEL events that emerge repeatedly across the phylogeny, 12.4% are phase-variants within HTs (0.02% of the genome by length). We estimated the in-vitro frameshift rate in a neutral HT at 100x the neutral substitution rate at 1.1 × 10−5 frameshifts/HT/year. Using neutral evolution simulations, we identified 4,098 substitutions and 45 phase-variants to be putatively adaptive to MTBC (P<0.002). We experimentally confirm that a putatively adaptive phase-variant alters the expression of espA, a critical mediator of ESX-1 dependent virulence. Our evidence supports a new hypothesis that phase variation in the ESX-1 system of MTBC can act as a toggle between antigenicity and survival in the host.

The discovery of genome-wide mutational dependence in naturally evolving populations

“…Evolutionary events in M. tuberculosis. We estimated the evolutionary history of 31,435 diverse MTBC strains using maximum likelihood phylogeny and ancestral sequence reconstruction, with 2,815, 8,090, 3,398, 16,931, 98, and 96 strains belonging to Lineages 1-6, respectively as recently described (16). Restricting our analysis here to single nucleotide polymorphisms, we observe 4,776 sites in the genome to have evolved away from the pan-susceptible ancestral state (17) at least five times independently (Supplementary Data 1).…”

“…We use a dataset that reconstructed the evolutionary history of the 31,428 strains by building maximum likelihood phylogenies and performing maximum likelihood ancestral reconstruction ( 60 ) to infer mutational events over time in the population of strains ( 16 ). We consider only single nucleotide substitutions (SNS) in the coding and non-coding portions of the genome.…”

“…We use a previously curated dataset of 31,428 strains, representing six major M. tuberculosis lineages (16). Whole genome sequence data is processed using a previously validated pipeline (52,53) Briefly, reads are aligned to the H37Rv reference genome using BWA-MEM after trimming and filtering with PRINSEQ and contaminant removal with Kraken (54)(55)(56).…”

Roles for mycobacterial DinB2 in frameshift and substitution mutagenesis

“…Transient Mtb drug tolerance can be conferred by an FS mutation in a run of C (between 7C and 10C depending on the Mtb strain) of the glpK gene, encoding glycerol kinase ( Bellerose et al, 2019 ; Safi et al, 2019 ). In addition, recent in silico analyses of genomes of Mtb clinical isolates identified frequent indels in homopolymeric tracts, some of which may have important virulence functions, revealing that reversible gene silencing mediated by FS is not restricted to the glpK -dependent antibiotic tolerance pathway ( Gupta and Alland, 2021 ; Vargas et al, 2022 ). Based on the level of FS frequency we detected in homo-oligonucleotide runs, we believe that reversible gene silencing through frameshifting in long runs is a prevalent phenomenon in mycobacteria and that DinB1 and DinB2 could contribute to these indels.…”

Single nucleotide variation catalogue from clinical isolates mapped on tertiary and quaternary structures of ESX-1 related proteins reveals critical regions as putative Mtb therapeutic targets

“…First, we provide evidence illustrating the variation tolerance of the ESX-1 GOI, confirming that it is the most SNV-dense group of genes within the MTB genome. Next, we to identified several genomic regions including variants that arose independently under positive selection as done by Vargas et al (2022). We then examined the amino-acid locations that bear abundant SNV-counts.…”