Tuberculosis (TB) in humans and animals remains a public health emergency and a global economic problem. TB causes more human deaths than any other infectious disease, and TB in livestock leads to a global estimated loss of three billion US dollars per year. The main agents of TB are a group of closely related bacteria known as the Mycobacterium tuberculosis Complex (MTBC), which differ in their host tropism. The MTBC Lineages 1 to 7 are human-adapted, and several other MTBC lineages, or ecotypes, mainly affect different wild and domestic animal species. Among the latter, Mycobacterium bovis and Mycobacterium caprae are the most important agents of TB in livestock and also the most important causes of zoonotic TB in humans. Recently, there has been an increase in cases of Mycobacterium orygis reported among immigrants from South Asia, indicating that this ecotype has the potential to cause zoonotic disease in humans. During the last few years, analyses of large globally representative collections of whole genome sequences from the human-adapted MTBC lineages have enhanced our understanding of the global population structure, phylogeography and evolutionary history of these pathogens. By contrast, little corresponding data exist for the various animal-adapted ecotypes of the MTBC. This is particularly true for M. bovis, M. caprae and M. orygis, for which most available data comes from convenience samples generated in high-income countries such as UK, USA and New Zealand. As a result, the global population structure, phylogeography and evolutionary history of these ecotypes remain unknown. Moreover, although we and others have shown that M. bovis, M. caprae and M. orygis are phylogenetically separate entities, whether these phylogenetic distances reflect ecological separation with respect to host tropism is unclear. The general view is that M. bovis has a broader host tropism compared to other members of the MTBC, but in M. caprae and M. orygis, the main maintenance host, and host tropism in general, are poorly defined, particularly with respect to their zoonotic potential. The overall goal of this project is to define the global population structure, phylogeography, evolutionary history and genomic diversity of M. bovis, M. caprae and M. orygis, and to explore the genomic and phenotypic determinants of host tropism in these animal-adapted members of the MTBC. We hypothesize that i) M. bovis, M. caprae and M. orygis exhibit a phylogeographical population structure that reflects events in domestication, animal husbandry, human migration and trade, ii) M. bovis, M. caprae and M. orygis differ in their host tropism and zoonotic potential, iii) these differences in host tropism are reflected in evolutionary signatures in the genomes of these pathogens, and iv) manifest in the phenotypic features of the corresponding host-pathogen interactions measured ex vivo. We will test these hypotheses by addressing the following three Specific Objectives:1.Determine the global population structure, phylogeography and evolutionary history of M. bovis, M. caprae and M. orygis;2.Explore the genomic basis of host tropism in M. bovis, M. caprae and M. orygis;3.Identify phenotypic correlates of host tropism in matched- and mismatched host macrophages infected with M. bovis, M. caprae or M. orygis.We propose an integrated and multidisciplinary approach, in which we combine i) detailed analyses of large, globally representative collections of M. bovis, M. caprae or M. orygis genome sequences generated using both short- and long sequencing read technologies, ii) application of state-of-the-art population genomic, phylogenomic, phylogeographic and molecular dating methods, and iii) functional assays based on host-pathogen matched- and mismatched macrophage infections analysed for intracellular bacterial growth, host-pathogen cross-talk using dual RNAseq, and host innate immune responses. In addition, we will study the relative zoonotic potential of these three ecotypes by infecting human macrophages. This project will generate fundamental new insights into the global genomic population structure, phylogeography and evolutionary history of the main agents of TB in livestock and the most important causes of zoonotic TB in humans. Moreover, it will shed new light into the genomic and phenotypic basis of host tropism in the animal-adapted MTBC, and thereby, contribute to an improved understanding of the biology of host-pathogen interaction in one of the world’s most important infectious diseases.