Mycobacterium marinum actin-based motility

Mycobacterium tuberculosis infects more than one billion people and causes millions of deaths per year, but the mechanisms by which this organism causes disease are poorly understood. M. marinum causes a tuberculosis-like disease in fish and has been used increasingly as a model to understand the molecular pathogenesis of tuberculosis. Pathogenic mycobacteria are classical intracellular pathogens of macrophages and are widely believed not to enter the cytoplasm, but rather to exist exclusively within phagosomes. Contrary to this accepted paradigm, we found by using various microscopy techniques that intracellular M. marinum not only enters the cytoplasm of infected macrophages, but also is propelled by induction of actin polymerization leading to direct intercellular spread. This process of actin-based motility, although never observed previously for mycobacteria, is utilized by many bacterial intracellular pathogens such as Listeria monocytogenes and Shigella flexneri and is required for full virulence of these species in vivo. To polymerize actin, these pathogens and M. marinum exploit a host cell signaling pathway by recruiting cytoskeletal factors leading to Arp2/3 complex actin nucleation. Using deficient cell lines and pharmacological inhibition, we demonstrate that the WASP family of proteins, including WASP and N-WASP, is critical for efficient M. marinum actin polymerization, whereas factors upstream of the WASP family such as tyrosine phosphorylation, Nck, WIP, and Cdc42 are not important. Through detailed analysis of the N-WASP domains able to restore M. marinum actin polymerization in N-WASP-/- cells, we found that the basic motif of N-WASP to which host cell lipids bind is required. These data indicate that M. marinum utilizes a mechanism distinct from other pathogens to initiate actin polymerization perhaps by using the distinct cell surface lipids present in the mycobacterial cell wall.