Genomic analyses of the ancestral Manila family of <i>Mycobacterium tuberculosis</i>

<div><p>With its airborne transmission and prolonged latency period, <i>Mycobacterium tuberculosis</i> spreads worldwide as one of the most successful bacterial pathogens and continues to kill millions of people every year. <i>M</i>. <i>tuberculosis</i> lineage 1 is inferred to originate ancestrally based on the presence of the 52-bp TbD1 sequence and analysis of single nucleotide polymorphisms. Previously, we briefly reported the complete genome sequencing of <i>M</i>. <i>tuberculosis</i> strains 96121 and 96075, which belong to the ancient Manila family and modern Beijing family respectively. Here we present the comprehensive genomic analyses of the Manila family in lineage 1 compared to complete genomes in lineages 2–4. Principal component analysis of the presence and absence of CRISPR spacers suggests that Manila isolate 96121 is distinctly distant from lineages 2–4. We further identify a truncated <i>whiB5</i> gene and a putative operon consisting of genes encoding a putative serine/threonine kinase PknH and a putative ABC transporter, which are only found in the genomes of Manila family isolates. Six single nucleotide polymorphisms are uniquely conserved in 38 Manila strains. Moreover, when compared to <i>M</i>. <i>tuberculosis</i> H37Rv, 59 proteins are under positive selection in Manila family isolate 96121 but not in Beijing family isolate 96075. The unique features further serve as biomarkers for Manila strains and may shed light on the limited transmission of this ancestral lineage outside of its Filipino host population.</p></div

CRISPR spacer rearrangements in Manila family 96121, Beijing family 96075, and H37Rv.

<p>Green highlights spacers conserved with other MTBC species but lost in modern Beijing family isolate 96075 and H37Rv. Red highlights spacers specific in Manila family isolate 96121. Blue highlights spacers present in H37Rv but absent in Manila family isolate 96121 and Beijing family isolate 96075.</p

Strain information list.

<p>Strain information list.</p

List of proteins under positive selection in Manila family 96121 but not Beijing family 96075.

<p>List of proteins under positive selection in Manila family 96121 but not Beijing family 96075.</p

List of Manila 96121 nonsynonymous SNPs in protein coding genes.

<p>List of Manila 96121 nonsynonymous SNPs in protein coding genes.</p

Evolution of CRISPR spacers in complete genomes of 21 <i>M</i>. <i>tuberculosis</i> strains.

<p>(A) Accumulation curve of spacers in complete genomes of 21 <i>M</i>. <i>tuberculosis</i> strains. (B) PCA of presence and absence of CRISPR spacers in complete genomes of 21 <i>M</i>. <i>tuberculosis</i> strains. The blue arrow indicates the position of Manila family 96121, illustrating a dramatic difference in the composition of spacers in Manila family 96121.</p

Clustering and spoligotype details of selected Manila and Manila-like isolates.

<p>Clustering and spoligotype details of selected Manila and Manila-like isolates.</p

Maximum parsimony phylogeny of Manila-like isolates.

<p>Evolutionary history inferred by MEGA7 using the maximum parsimony method and rooted by <i>Mycobacterium bovis</i> BCG P3. Branch support was determined by 1000 bootstrap repetitions (bootstrap values shown above each node). A model averaged phylogeny further supported this result (data not shown).</p

Family specific and characteristic virulence factor SNP mutations of the Manila family of <i>Mtb</i>.Family specific virulence factor mutations of the Manila family, non-silent.

<p>Family specific and characteristic virulence factor SNP mutations of the Manila family of <i>Mtb</i>.Family specific virulence factor mutations of the Manila family, non-silent.</p

Whole genome sequencing and SNP data.

<p>Whole genome sequencing and SNP data.</p