Mycobacterium tuberculosis is the causative agent of tuberculosis, a primarily pulmonary disease that is estimated to infect approximately one third of the world population yet the mechanisms by which this bacterium causes disease are poorly understood. A key feature of M. tuberculosis pathogenesis is the parasitism of host macrophages following phagocytic entry, which is facilitated by the bacteria's manipulation of the host cell environment. The secretion of virulence proteins is a common mechanism used by pathogenic bacteria for altering the host environment. One such pathway, present in Mycobacterium and other Gram-positive genera, is ESX-1 (early secretory antigenic target 6 system 1). Although ESX-1 is required for multiple phenotypes related to the pathogenesis of infection, only three substrates of the secretion machine have been identified to date, and the mechanism by which these substrates are exported is not understood. In this work I identify a novel ESX-1 substrate and find that its delivery to the ESX-1 machine requires different protein interactions than previously identified substrates. I probe the role of this novel substrate in virulence and present data that the various ESX-1 substrates contribute additively to virulence. These results expand our understanding of a major virulence mechanism of Mycobacteria as well as mechanisms for the secretion of proteins through thick impermeable bacterial cell walls.