Members of the Mycobacterium tuberculosis complex (MTBC) inflict tremendous morbidity and mortality on humans and animals worldwide. These bacteria have co-evolved alongside humans and spread out of Africa with us. How environmental mycobacteria grew to become dedicated human pathogens, and how they adapted to infect domesticated livestock and beyond are unclear. Clues to their origins can be found in their genomes; gain and loss of genes, mutations, and genetic transfers between species can reflect specialization or diversification for new niches. Using whole genome sequencing projects from around the world, these hidden marks of adaptation can help explore pathways involved in host specificity, antigenic indicators of host immune subversion, and the footprints of evolution through analysis of conservation and exchange of CRISPR/Cas systems.A genome-wide association study of MTBC genomes identified 120 loci associated with classification of M. tuberculosis variants bovis vs. others, which overlapped with an identical set associated with isolation from bovine hosts vs. others. These markers may be useful for SNP-based classification of MTBC variants, but more importantly, some are in genes involved in cholesterol and fatty acid catabolism, including genes known essential to grow on cholesterol. Adapting to new host lipid profiles may have allowed for a human host-specialist like M. tuberculosis to switch host reservoirs and expand to broadly infecting mammals as M. bovis. By using validated epitopes from the MTBC from the Immune Epitope Database, the genome sequence of a uniquely attenuated strain of M. bovis – strain Ravenel – was analyzed for mutations in epitope-producing regions to identify changes. Such changes were rare among a reference virulent strain and an outbreak strain we also sequenced, but more commonplace in a reference attenuated strain and strain Ravenel. No changes were predicted in silico to destabilize the mutant proteins, indicating the substitutions likely allow similar protein functionality, but may confer differential immune recognition by the host, a process known to be critical to mycobacterial infection and persistence and which could contribute to observed attenuation.Finally, MTBC members are believed to be some of the few species of mycobacteria that carry “bacterial immune system” of CRISPR/Cas systems, and the MTBC and strains of closely related M. canettii are reported to be the only Mycobacterium to possess Type III-A systems. By searching for homologous cas genes and genetic contexts, previously unreported Cas systems of 4 classified types were found across a range of pathogenic and saprophytic mycobacteria, the closest related species outside the MTBC like M. lacus, M. shinjukuense showed only non-Type III-A systems, but the Type III-A system was identified in an unusual environmental Mycobacterium. The frequency of Cas systems and the rare nature of the MTBC Cas Type III-A support searching for it as a marker for the evolutionary origins of tuberculosis. The work herein demonstrates the utility of existing datasets and software for discovery and provides three distinct paths forward for researchers to study the evolution and adaptation of Mycobacterium species to new hosts and environments: over 100 genetic loci associated with differentiation of specialist M. tuberculosis vs. generalist M. bovis, an understudied path for pathogen attenuation by epitope variation in the unique background of the MTBC, and the expansion of the mycobacterial CRISPR/Cas repertoire but continued rarity of the Type III-A system supports its use as a marker to trace the evolutionary history of the MTBC.
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