Mycobacterium tuberculosis (Mtb) remains a leading cause of infectious death worldwide. Although tuberculosis generally remains confined to the lung, pathogenic mycobacteria also can disseminate to other tissues, resulting in some of the most dangerous manifestations of the infection. The interplay of bacterial and host factors that contribute to dissemination is incompletely understood. Beginning with an outbreak strain presenting with high rates of extrapulmonary dissemination and an extremely high rate of tuberculous bone disease, we identified a full-length vavriant of the Type VII secretion system substrate EsxM as an important component of the clinical phenotype. EsxM was intact in the outbreak strain and so-called “ancestral lineages” but truncated in modern Mtb strains. The active version of EsxM modulates the motility of infected macrophages leading to dissemination, but has been compromised in “modern-lineage” Mtb strains. The work proposed here will 1) interrogate the mechanisms by which the bacterial secreted effector EsxM modulates the behavior of infected macrophages; 2) identify how EsxM and its closely related paralogs are regulated in the bacteria during infection, including investigating non-redundant roles for the closely-related paralogs; and 3) translate these findings into murine and zebrafish models of dissemination and bone disease. These studies will provide insights into the bacterial genetic regulators of dissemination of infection, a fundamentally important question in our understanding of tuberculosis and other infectious diseases.