f Understanding dissemination of Mycobacterium tuberculosis from the lungs during primary infection

Understanding how inhaled Mycobacterium tuberculosis achieves dramatic replication and crosses the alveolar barrier to establish systemic latent infection, before adaptive immunity is elicited in humans, is limited by the small infecting inoculum carried in aerosol droplets (1–5 μm diameter) and the inability to identify the time of infection. M. tuberculosis is believed to disseminate via infected macrophages. However, like other invasive bacterial pathogens, M. tuberculosis could also cross the barrier directly using adhesins and toxins. An in vitro alveolar barrier mimicking the gas-exchange regions of the alveolus was devised comprising monolayers of human alveolar epithelial and endothelial cells cultured on opposing sides of a basement membrane. Migration of dissemination-competent strains of M. tuberculosis, and dissemination-attenuated M. tuberculosis and Mycobacterium bovis mutant strains lacking adhesin/toxin ESAT-6 and adhesin HBHA were tested for macrophage-free migration across the barrier. Strains that disseminate similarly in vivo migrated similarly across the in vitro alveolar barrier. Strains lacking ESAT-6 expression/secretion were attenuated, and absence of both ESAT-6 and HBHA increased attenuation of bacterial migration across the barrier. Thus, as reported for other bacteria, M. tuberculosis utilizes adhesins and toxins for macrophage-independent crossing of the alveolar barrier. This in vitro model will allow identification and characterization of molecules/mechanisms employed by M. tuberculosis to establish systemic latent tuberculosis infection during primary infection.