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Gene conversion is a key driver of diversity hotspots in M. tuberculosis antigens and virulence-associated loci

Maximillian G. MariniD, Natalia Quinones‐OlveraiD, Hu JiniD, Michael A. HarrisiD, Brendan M. JeffreyiD, Alex RosenthaliD, Kenan C. MurphyiD, Christopher M. SassettiiD, Heng LiiD, Maha R FarhatiD

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Abstract

Despite the long-held view of Mycobacterium tuberculosis (Mtb) as a genetically conserved pathogen, many genomic regions remain poorly resolved due to high sequence homology and repetitive content. Using complete genome assemblies generated from long-read sequencing of 151 globally representative clinical isolates, we comprehensively analyzed genome-wide patterns of genetic diversity and evolution across the Mtb genome. Our analysis uncovers pronounced diversity hotspots within paralogous regions generated by recurrent gene conversion between homologous genes. In many cases, these hotspots exhibit more than an order of magnitude greater genetic diversity than the rest of the Mtb genome, which is otherwise characterized by remarkably low variation. Mutations within these regions display clustered substitution patterns, excess paralog-matching variants, and distinct mutational spectra consistent with ongoing gene conversion. Our analysis identifies over 300 individual gene conversion events distributed throughout the Mtb phylogeny. These gene conversion events occur predominantly within gene families associated with virulence and host-pathogen interactions, including the PE, PPE, and ESX families. Several of the most pronounced diversity hotspots occur in antigens encoded within paralogous regions. Among these, the vaccine candidate PPE18 harbors mutations in validated epitope sequences and predicted alterations in HLA-II binding. Together, these findings demonstrate that gene conversion actively shapes antigenic and virulence-associated diversity in Mtb.

In-depth summary

Claude Opus 5.5 · xhigh effort · 8 Nov 2026 · from the full text of version 4 (22 Sept 2026) · CC BY 4.0

Complete genomes from 151 clinical M. tuberculosis isolates show gene conversion between paralogs creating diversity hotspots in PE/PPE/ESX genes and T cell antigens, including PPE18.

Repetitive and paralogous regions make up nearly 10% of the Mtb genome and are usually excluded from short-read analyses, so Mtb has been treated as clonal and genetically conserved. Complete long-read assemblies let the authors measure diversity and recombination inside these regions across all major lineages.

Key results

  1. ShownA small set of 1-kb windows shows extreme diversity and sits almost entirely in paralogous regions, enriched for PE/PPE, ESX and REP13E12 genes. Assembly error does not explain them.

    37 hotspot windows with modified Z-score above 10 (12 to 83 times the genome median of 0.2 substitutions/kb); 36 of 37 lie in paralogous regions; none of 24,463 hotspot SNP calls overlapped a NucFlag-flagged region. · Hotspots of extreme genetic diversity are found…

  2. SuggestedParalogous regions carry mutation patterns consistent with gene conversion: dense substitution clusters, substitutions matching other paralogs, and an altered substitution spectrum.

    Median inter-substitution distance 8 bp in paralogous regions vs 1016 bp elsewhere; 60 of 157 tested regions biased toward paralog-matching variants, covering 33 of 37 hotspots. Separately, no loss-of-function mutations were found in the 13 SDSA/HR genes, supporting that these repair pathways are functional. · Paralogous regions of the Mtb genome harbor…

  3. ShownThe phylogenetic workflow detects hundreds of gene conversion events, concentrated in a few paralog networks, on both ancestral and terminal branches. The rate differs between lineages.

    324 events (median 101 bp, 6 SNPs); 91% in paralogous regions; Gini 0.85 across 71 networks; 5 high-burden networks hold 55% of events; 1.37 events per 100 non-event substitutions; Lineage 4 elevated, Lineages 2 and 6 reduced (q < 0.05). · Frequent gene conversion between a small subset…; Gene conversion is widespread throughout both…

  4. ShownCandidate events found in an independent short-read cohort were confirmed by long-read resequencing. Short-read data recovered only a small share of the events the long-read assemblies detected.

    937 isolates screened, 27 candidate events; 12 events resequenced (22 isolates, PacBio HiFi); 86/86 SNPs confirmed; short reads recovered 86 of 199 SNPs; recall 12/84 events (14.3%). · Validation of individual gene conversion events…

  5. SuggestedAntigens encoded in paralogous regions carry a higher non-synonymous mutation burden than unique-region antigens, and gene conversion events preferentially alter T cell epitopes. PPE18 is a recurrent example.

    Median 4.1 vs 1.0 mutations/100 aa (PR vs unique antigens, 53 antigens); 25 of 295 in-PR events (8.5%) changed an epitope, a 5.0-fold enrichment; PPE18: 44 of 61 non-synonymous mutations in epitopes, but epitope enrichment 1.73-fold, p = 0.055. · Gene conversion drives elevated diversity in a…; Recurrent gene conversion generates sequence…

Methods, data and reproducibility

The authors built hybrid long- and short-read assemblies, computed nucleotide diversity in 1-kb windows and called hotspots with a modified Z-score. They detected gene conversion events with Gubbins on a SNP phylogeny and mapped them to donor paralogs. Findings were validated by PacBio HiFi resequencing and linked to curated T cell epitope data and HLA-II binding predictions.

  • Complete hybrid genome assemblies of clinical Mtb isolates, lineages 1-6 and 8 (Mtb151); PacBio CLR, ONT R9.4.1 and HiFi long reads plus Illumina · Mycobacterium tuberculosis (human-adapted) · n = 151 isolates
  • Independent cohort of clinical isolates with short-read WGS, screened for gene conversion signatures · Mycobacterium tuberculosis · n = 937 isolates
  • PacBio HiFi resequencing of target and matched control isolates for 12 candidate events · Mycobacterium tuberculosis · n = 22 isolates
  • CD4+ T cell peptide screening data from two published studies, used to define validated antigens and epitopes · Mycobacterium tuberculosis peptides; donors with TB or latent TB · n = 18,371 unique peptides, 3,840 proteins; 53 antigens, 226 epitopes
Methods & tools
Flye, Pilon, Medaka, PolyPolish, Bakta, minimap2, vcftools, FastTree, Gubbins, NucFlag, netMHCpanII-EL, Snakemake
Software & environment
Snakemake v7.32.4; Gubbins v3.2.1; FastTree v2.1.10; bcftools v1.20; vcftools v0.1.16; Flye v2.6 or v2.9; Pilon v1.23; Medaka v1.5.0; PolyPolish v0.5.0; Bakta v4.8.
Limitations (5)
  • Events between nearly identical paralogs are likely underdetected, because detection relies on clustered substitutions. · stated by the authors
  • Older events may be eroded by later mutation, which weakens the clustering signal and complicates inference of the donor paralog. · stated by the authors
  • Reliance on a single reference genome may bias event localization and paralog assignment. Some geographic regions and sub-lineages are underrepresented. · stated by the authors
  • In silico HLA-II binding predictions cannot establish immunological consequences; T cell receptor recognition and vaccine efficacy were not tested. · stated by the authors
  • The 151 assemblies come from several long-read platforms and pipeline variants. Independent validation covered only 12 of 27 candidate events, chosen by DNA availability. · noted in this summary

Read this if you analyze Mtb or other clonal bacteria and need to know how recombination between paralogs affects diversity, dN/dS and antigen variation.

Citations
1

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