Mycobacterium avium causes disseminated disease in patients with AIDS and other immunosuppressive conditions and pulmonary infections in individuals with chronic lung diseases. Much still need to be learn about the mechanisms of M. avium pathogenesis. Using a mouse model of disseminated M. avium disease, we applied an in vivo expression technology system and identified M. avium genes up-regulated in different organs of mice during early stage of infection. The M. avium oppA gene, involved in an active transport of oligopeptides across the cell membrane, was found highly expressed in lung, liver and spleen of mice. Mutation in the transport domain of the oppA gene resulted in bacterial attenuation in both macrophages and in mice. Using protein-protein interaction assay, it was determined that two hypothetical small proteins, MAV_2941 (73aa) and MAV_4320 (45aa), interact with OppA. MAV_2941 was shown to be secreted by the bacterium into the macrophage cytoplasm. Mutations in MAV_2941 was associated with significant impairment of growth in macrophages. Understanding the mechanisms involved in the functions of MAV_2941 and MAV_4320 is warranted.