Molecular studies of mycobacterium tuberculosis complex (MTBC) virulence

The Mycobacterium tuberculosis complex subspecies (MTBCs) are closely related pathogens causing tuberculosis in mammalian hosts. Research has predominantly focused on Mycobacterium tuberculosis (M. tb, the causative agent of human tuberculosis) and Mycobacterium bovis (M. bovis, a causative agent of zoonotic tuberculosis). Notably, M. tb is a human-adapted pathogen whereas M. bovis is an animal-adapted member of the MTBC. Foundational work by Robert Koch and Theobald Smith established that M. tb and M. bovis have significantly different infection outcomes, with M. bovis infection resulting in more severe pathology in experimental infections. Globally, more than 10 million new cases of TB are reported each year; ~1% are estimated to be due to zoonotic tuberculosis (zTB), typically attributed to M. bovis. However, recent studies have highlighted that another organism, Mycobacterium orygis, may also contribute to the global TB burden. As this organism was first characterized in 2012, it has not been subject to thorough experimental investigation and as such, knowledge on its epidemiology and pathogenesis are limited. Current molecular tools have enabled researchers to translate natural observations from clinics into systematic evaluations in the laboratory, using in vitro, ex vivo, and in vivo techniques. To this end, in this thesis, I used mycobacterial engineering to develop isogenic strains of M. bovis Bacille Calmette-Guérin (BCG), M. bovis, and M. orygis. BCG strains were subsequently employed in clinical diagnostics whereas M. bovis and M. orygis isogenic strains were used to investigate infection outcomes using both ex vivo and in vivo models.In chapter II, I developed a panel of 6 mono-resistant strains of the attenuated BCG vaccine using oligo-mediated recombineering for laboratory quality control studies. In chapter III, I used the ORBIT (oligo-mediated recombineering followed by Bxb-1 integrase targeting) system to develop 8 deletion strains targeting either the canonical virulence factor ESAT-6, or the non-canonical virulence factors MPT70/MPT83 (in M. bovis and M. orygis) and characterized their effects on protein secretion and macrophage infection. In chapter IV I used a murine infection model to characterize the experimental outcome following aerosol exposure to M. tb, M. bovis, and for the first time, M. orygis, revealing that M. bovis and M. orygis are hypervirulent compared to M. tb following infection of the same host (based on comparative pathology, median survival, and lethal dose). Additionally, I established that MPT70/MPT83 are critical virulence factors in M. bovis but not M. orygis in this model. It was also observed that rapid mortality seen following M. orygis infection could be delayed, but not overcome, by vaccination with either BCG (a live, attenuated vaccine) or novel subunit vaccines. Finally, in chapter V I, in conjunction with researchers at the University of Saskatchewan, determined that M. bovis and M. orygis show significantly worse experimental outcomes compared to M. tb in a calf model of infection. Ultimately, this dissertation provides novel insights into the MTBCs and utilizes in vitro, ex vivo, and in vivo techniques to experimentally characterize M. orygis for the first time. This thesis established two recombineering systems in animal-associated lineages of the MTBCs and the products of the tools have been used in both clinical and research laboratories. Perhaps most importantly, this thesis has provided evidence that a third species should be considered in future pathogenesis research and may be used as a new ‘foundation’ for the study of MTBC virulence factors