Independent genomic polymorphisms in the PknH serine threonine kinase locus during evolution of the Mycobacterium tuberculosis Complex affect virulence and host preference

Species belonging to the Mycobacterium tuberculosis Complex (MTBC) show more than 99% genetic identity but exhibit distinct host preference and virulence. The molecular genetic changes that underly host specificity and infection phenotype within MTBC members have not been fully elucidated. Here, we analysed RD900 genomic region across MTBC members using whole genome sequences from 60 different MTBC strains so as to determine its role in the context of MTBC evolutionary history. The RD900 region comprises two homologous genes, pknH1 and pknH2, encoding a serine/threonine protein kinase PknH flanking the tbd2 gene. Our analysis revealed that RD900 has been independently lost in different MTBC lineages and different strains, resulting in the generation of a single pknH gene. Importantly, all the analysed M. bovis and M. caprae strains carry a conserved deletion within a proline rich-region of pknH, independent of the presence or absence of RD900. We hypothesized that deletion of pknH proline rich-region in M. bovis may affect PknH function, having a potential role in its virulence and evolutionary adaptation. To explore this hypothesis, we constructed two M. bovis ‘knock-in’ strains containing the M. tuberculosis pknH gene. Evaluation of their virulence phenotype in mice revealed a reduced virulence of both M. bovis knock-in strains compared to the wild type, suggesting that PknH plays an important role in the differential virulence phenotype of M. bovis vs M. tuberculosis.