Genome-wide estimation of recombination, mutation and positive selection enlightens diversification drivers of Mycobacterium bovis

Abstract

Genome sequencing has reinvigorated the infectious disease research field, shedding light on disease epidemiology, pathogenesis, host–pathogen interactions and also evolutionary processes exerted upon pathogens. Mycobacterium tuberculosis complex (MTBC), enclosing M. bovis as one of its animal-adapted members causing tuberculosis (TB) in terrestrial mammals, is a paradigmatic model of bacterial evolution. As other MTBC members, M. bovis is postulated as a strictly clonal, slowly evolving pathogen, with apparently no signs of recombination or horizontal gene transfer. In this work, we applied comparative genomics to a whole genome sequence (WGS) dataset composed by 70 M. bovis from different lineages (European and African) to gain insights into the evolutionary forces that shape genetic diversification in M. bovis. Three distinct approaches were used to estimate signs of recombination. Globally, a small number of recombinant events was identified and confirmed by two independent methods with solid support. Still, recombination reveals a weaker effect on M. bovis diversity compared with mutation (overall r/m = 0.037). The differential r/m average values obtained across the clonal complexes of M. bovis in our dataset are consistent with the general notion that the extent of recombination may vary widely among lineages assigned to the same taxonomical species. Based on this work, recombination in M. bovis cannot be excluded and should thus be a topic of further effort in future comparative genomics studies for which WGS of large datasets from different epidemiological scenarios across the world is crucial. A smaller M. bovis dataset (n = 42) from a multi-host TB endemic scenario was then subjected to additional analyses, with the identification of more than 1,800 sites wherein at least one strain showed a single nucleotide polymorphism (SNP). The majority (87.1%) was located in coding regions, with the global ratio of non-synonymous upon synonymous alterations (dN/dS) exceeding 1.5, suggesting that positive selection is an important evolutionary force exerted upon M. bovis. A higher percentage of SNPs was detected in genes enriched into “lipid metabolism”, “cell wall and cell processes” and “intermediary metabolism and respiration” functional categories, revealing their underlying importance in M. bovis biology and evolution. A closer look on genes prone to horizontal gene transfer in the MTBC ancestor and included in the 3R (DNA repair, replication and recombination) system revealed a global average negative value for Taijima’s D neutrality test, suggesting that past selective sweeps and population expansion after a recent bottleneck remain as major evolutionary drivers of the obligatory pathogen M. bovis in its struggle with the host.

A critical evaluation of Mycobacterium bovis pangenomics, with reference to its utility in outbreak investigation

“…Recombination events inferred on terminal branches can be unreliable because they only involve one genome. In addition to recombination analysis done on the core genome alignment, fastGEAR was used to investigate recombination on individual gene alignments with recombination events implicated in Reis and Cunha [7], and to confirm the recombination events detected by Gubbins. The fastGEAR analysis confirmed recombination events identified in Reis and Cunha [7] in narX and pks12 .…”

“…In addition to recombination analysis done on the core genome alignment, fastGEAR was used to investigate recombination on individual gene alignments with recombination events implicated in Reis and Cunha [7], and to confirm the recombination events detected by Gubbins. The fastGEAR analysis confirmed recombination events identified in Reis and Cunha [7] in narX and pks12 . There is a high density of SNPs in a 100 bp locus in narX in18 monophyletic genomes.…”

“…Raw M. bovis sequence files from Almaw et al [10], Loiseau et al [11], Orloski et al [12], Reis and Cunha [7], Rodrigues et al [13], Zwyer et al [14] and from BioProject PRJNA769553 were downloaded from the NCBI Sequence Read Archive. A full list of downloaded samples can be found in Supplemental File 1.…”

“…Since this resulting alignment is conservative, some potential recombination events may be excised from the alignment before it is fed to Gubbins. Therefore, in addition to the recombination analysis on the core genome alignment, individual gene alignments that had previously been implicated in recombination events in M. bovis [7] were also assessed for recombination using fastGEAR [32]. The individual gene alignments were created using the built-in MAFFT aligner included in Panaroo [22,33].…”

“…Experimental evidence has shown that while the closely related free-living Mycobacterium canettii could accept donor DNA via homologous recombination, MTBC could not [2]. However, despite this lack of experimental evidence, computational methods sometimes identify recombination in MTBC genomes [4,7,8]. Some of the recombination in MTBC species identified through computational methods stems from poor quality data or unreliable alignments.…”