Mycobacterium avium subsp. hominissuis (MAH) is a pervasive environmental bacterium that can cause opportunistic infections in humans. Among the most robust and hardy members of the Mycobacterium genus, M. avium can persist and thrive in a range of challenging environments, including many which place it in direct contact with humans. Surface-exposed proteins are central to the bacterial processes involved in both environmental persistence and pathogenesis. These proteins also play a critical role in how the immune system of the host recognizes and responds to pathogens. Mycobacteria have evolved a specialized mechanism for protein export, a Type VII Secretion System (T7SS), in order to transport their proteins through their thick and impermeable cell envelope. This system is responsible for the export of several classes of proteins, many of which play an integral role in virulence. A central focus of this dissertation is the characterization of a conserved element of the T7SSs in pathogenic mycobacteria, a PPE family protein, whose deletion attenuates virulence in M. avium. Specifically, we examined the localization of this PPE protein (MAV_2928) within the bacterium, screened potential protein-protein interactions with other conserved elements in the adjacent T7SS loci and analyzed the transcriptional regulation of the gene in response to environmental changes. Seeking to more thoroughly characterize the surface-exposed proteome of M. avium, particularly in the context of early infection, we then developed a method, based on selective biotinylation and affinity purification, to profile the of surface-exposed proteome of the bacterium. We employed this method to analyze the surface-exposed proteomes of M. avium 109 that had been exposed to macrophages to those of M. avium 109 that had been cultured in media. This comparison detected several proteins whose presence at the bacterial surface appeared to be dependent on particular growth conditions. Lastly, in order to establish a more efficient method to isolate biotinylated surface proteins from complex mixtures, we developed a testing paradigm to identify modifications to the original method that might improve our coverage of identified proteins. Through this process, we developed a more robust methodology that yielded improved coverage and depth. We then utilized this technology to profile the surface-exposed proteome of another clinical isolate of M. avium subsp. hominissuis, M. avium 104. Beyond improving our understanding of the basic biology of M. avium, this new data provides independent evidence that PPE family proteins are indeed exported to the surface of M. avium, where they remain associated with the bacterial cell envelope. In total, this analysis represents the most comprehensive profile of the surface-exposed proteins of M. avium generated to date. / Graduation date: 2012