Shared Pathogenomic Patterns Characterize a New Phylotype, Revealing Transition toward Host-Adaptation Long before Speciation of Mycobacterium tuberculosis

Abstract

Tuberculosis remains one of the deadliest infectious diseases of humanity. To better understand the evolutionary history of host-adaptation of tubercle bacilli (MTB), we sought for mycobacterial species that were more closely related to MTB than the previously used comparator species Mycobacterium marinum and Mycobacterium kansasii. Our phylogenomic approach revealed some recently sequenced opportunistic mycobacterial pathogens, Mycobacterium decipiens, Mycobacterium lacus, Mycobacterium riyadhense, and Mycobacterium shinjukuense, to constitute a common clade with MTB, hereafter called MTB-associated phylotype (MTBAP), from which MTB have emerged. Multivariate and clustering analyses of genomic functional content revealed that the MTBAP lineage forms a clearly distinct cluster of species that share common genomic characteristics, such as loss of core genes, shift in dN/dS ratios, and massive expansion of toxin-antitoxin systems. Consistently, analysis of predicted horizontal gene transfer regions suggests that putative functions acquired by MTBAP members were markedly associated with changes in microbial ecology, for example adaption to intracellular stress resistance. Our study thus considerably deepens our view on MTB evolutionary history, unveiling a decisive shift that promoted conversion to host-adaptation among ancestral founders of the MTBAP lineage long before Mycobacterium tuberculosis has adapted to the human host.

Keywords: evolutionary transition; host adaption; mycobacteria; pathogen evolution; pathogenomic; tuberculosis.

Fig. 1.

—Phylogenetic organization of MTB and closely related SGM species. (A) SGM phylogenetic tree. For tree construction, concatenated conserved protein sequences from 107 universally conserved bacterial genes, as defined by the bcgTree software, were extracted from 28 mycobacterial species. For data analysis, a similarity matrix was calculated using the JTT_DCmut+I+G model. The phylogentic tree was constructed using ML estimations. Bootstrap values were calculated from 500 replicates. Red branches: members of the MTBAP lineage. (B) Genetic distance of MTBAP lineage and MGS–MKM outgroup members relative to M. tuberculosis H37Rv. dS distribution of 923 core-genome genes compared with those of M. tuberculosis H37Rv. Synonymous mutation rates (dS) for each gene were determined using the PAML algorithm. y-Axis: logarithmic scale. Mcan, M. canettii STB-K; Mdec, M. decipiens; Mshi, M. shinjukuense; Mlac, M. lacus; Mriy, M. riyadhense; Mkan, M. kansasii; Mmar, M. marinum E11; Mszu, M. szulgai; Mgor, M. gordonae. Bold bar: median. Box edges: 25th and 75th percentiles. Whiskers: extreme values.

Fig. 2.

—Genomic evolution in MTBAP lineage. (A) Color coded table representing a heatmap for which the rows and columns were sorted by hierarchical clustering approaches. The row tree shows the clustering of MICFAM protein families based on species profile similarity calculated by the Ward agglomerative hierarchical clustering algorithm. The column tree represents the clustering of species based on MICFAM profile. Red: under-represented gene families. Green: over-represented gene families. Color-key for Row Z-scores is shown together with a histogram indicating the number of MICFAM protein families associated with each of the Z-scores. (B) Estimated gene gain and loss in the MTBAP lineage and the MGS–MKM outgroup. Variable genome parts of the MTBAP lineage and the MGS–MKM outgroup were computed using the MICFAM tool with a 50% amino-acid identity threshold and 80% alignment coverage. A table representing presence or absence of gene families for each species was then analyzed by Gain Loss Mapping Engine. Red: branches showing more gene loss than gene gain. (C) dN/dS distribution of core-genome orthologs as compared with the M. gordonae outgroup. Bold bars indicate the median dN/dS values for each species. Notch estimates correspond to 95% confidence intervals for median values. Box edges represent 25th and 75th percentiles. Whiskers represent estimated extreme values. Mcan, M. canettii STB-K; Mdec, M. decipiens; Mshi, M. shinjukuense; Mlac, M. lacus; Mriy, M. riyadhense; Mkan, M. kansasii; Mmar, M. marinum E11; Mszu, M. szulgai; Mgor, M. gordonae.

Fig. 3.

—Multivariate and clustering analyses of PFAM domain contributions to the MTBAP lineage. (A) PCA of species from the MTBAP lineage and the MGS–MKM outgroup, based on occurrence of PFAM domains in each genome (only PFAM domains present in more than two species were conserved). Ellipse: 95% confidence value ellipse of MTBAP lineage members. (B) PFAM domain contribution to MTBAP lineage. x-Axis: PFAM domain scores determined by BCA (MTBAP lineage vs. MGS–MKM outgroup). y-Axis: PFAM domain differences in average occurrence for each class (MTBAP lineage vs. MGS–MKM outgroup). Red: toxin-associated domains. Green: antitoxin-associated domains.

Fig. 4.

—Toxin–antitoxin content of mycobacterial species in the MTBAP lineage and other SGM. (A) Number of putative toxin genes bearing PIN and MazF domain in SGM. Toxins from toxin–antitoxin systems were identified from whole proteome data sets, using HMMER (PF02452.16—PemK_toxin; PF01850.20 PIN domain) with a 0.01 threshold e value. Orange: VapC. Blue MazF. **: Values above two standard deviation levels from SGM (outside MTBAP lineage and MGS–MKM outgroup) average. (B) Putative orthologs of M. tuberculosis VapC and MazF toxins. Yellow: BBH 50% translated sequence identity, BBH 80% query cover. Red: BBH and synteny. Mcan, M. canettii STB-K; Mdec, M. decipiens; Mshi, M. shinjukuense; Mlac, M. lacus; Mriy, M. riyadhense; Mkan, M. kansasii; Mmar, M. marinum E11; Mszu, M. szulgai; Mgor, M. gordonae.

Fig. 5.

—Representation of selected genomic islands. The genomic regions depict the fumarate reductase locus (Rv1552-1555), the Myma locus (Rv3082c-3087), or the sulfolipid synthesis locus (Rv3820c-3826), and the surrounding genomic regions in M. tuberculosis, M. decipiens, and M. kansasii. Pink links: homologous genomic regions. Filled arrows: genes in genomic islands.

Fig. 6.

—Hypothetical evolutionary scenario of the members of the MTBAP lineage. In this schematic representation, the likely evolutionary breakpoint is marked, beyond which the concerned members depict shared host-adapted traits.