Virulence hierarchies within the <i>Mycobacterium tuberculosis</i> complex

The <i>Mycobacterium tuberculosis</i> complex (MTBC) includes <i>M. tuberculosis</i> (<i>M. tb</i>), the primary cause of human tuberculosis, <i>M. bovis,</i> the classical zoonotic pathogen and cause of bovine tuberculosis, and <i>M. orygis,</i> a recently recognized multihost pathogen. Given that <i>M. tb, M. bovis,</i> and <i>M. orygis</i> pose significant threats to the health of humans and animals, we sought to understand fundamental differences in pathogenicity among these closely related organisms. Building upon historical observations, we conducted a comparative virulence assessment of these pathogens using both bovine and murine infection models. Holstein calves were infected via aerosol with <i>M. tb, M. bovis,</i> or <i>M. orygis</i>, and pathology was analyzed through macroscopic and microscopic assessments of lungs and lymph nodes, along with quantitative tissue bacterial burden measurements. In C57BL/6 mice, we compared virulence using three readouts, namely survival, lethal dose determination, and detailed pathological assessments. Despite genomic similarity, animal-adapted MTBCs consistently showed dramatically enhanced virulence compared to <i>M. tb</i> with distinct immunopathology and, in the murine model, mortality within 24 days. Using gene disruption studies guided by proteomic comparisons, we determined that these infection outcomes were dependent on shared (ESAT-6) and lineage-associated (MPT70) virulence factors, the route of infection, and prior infection or immunization. Our findings reveal unexpected virulence hierarchies within the MTBC, with fundamental and translational implications for tuberculosis research.