The ability to invade and grow in macrophages is necessary for Mycobacterium tuberculosis to cause disease. We have found a Mycobacterium marinum locus of two genes that is required for both invasion and intracellular survival in macrophages. The genes were designated iipA (mycobacterial invasion and intracellular persistence) and iipB. The iip mutant, which was created by insertion of a kanamycin resistance gene cassette at the 5 region of iipA, was completely avirulent to zebra fish. Expression of the M. tuberculosis orthologue of iipA, Rv1477, fully complemented the iip mutant for infectivity in vivo, as well as for invasion and intracellular persistence in macrophages. In contrast, the iipB orthologue, Rv1478, only partially complemented the iip mutant in vivo and restored invasion but not intracellular growth in macrophages. While IipA and IipB differ at their N termini, they are highly similar throughout their C-terminal NLPC_p60 domains. The p60 domain of Rv1478 is fully functional to replace that of Rv1477, suggesting that the N-terminal sequence of Rv1477 is required for full virulence in vivo and in macrophages. Further mutations demonstrated that both Arg-Gly-Asp (RGD) and Asp-Cys-Ser-Gly (DCSG) sequences in the p60 domain are required for function. The iip mutant exhibited increased susceptibility to antibiotics and lysozyme and failed to fully separate daughter cells in liquid culture, suggesting a role for iip genes in cell wall structure and function. Altogether, these studies demonstrate an essential role for a p60-containing protein, IipA, in the pathogenesis of M. marinum infection.Mycobacterium tuberculosis is an extraordinarily successful human pathogen, with 2 to 3 billion people infected worldwide (12). Its success likely reflects its complex parasitic lifestyle with sophisticated mechanisms for combating host defense. For example, M. tuberculosis inhibits acidification of the bacterium-containing phagosome and its fusion to lysosomes (6,29,33). Within the "maturation-arrested" phagosome, M. tuberculosis proliferates and ultimately kills the host cells by apoptosis and/or necrosis (11,13,22). The ability to invade and grow inside host cells is an important virulence property for pathogenic mycobacteria, and infection of macrophages by M. tuberculosis plays a key role in initiating a primary infection in the lung (29). In recent years, some progress has been made toward understanding the M. tuberculosis molecules involved both in invasion and in intracellular growth. For example, the heparin-binding hemagglutinin adhesin has been shown to mediate M. tuberculosis adherence to epithelial cells and to potentiate extrapulmonary dissemination of M. tuberculosis (23). An exported repetitive protein (Erp) of M. tuberculosis and a Mycobacterium marinum homologue of M. tuberculosis Rv3881c both are required for intracellular growth in cultured macrophages and virulence in vivo (3, 16). However, as M. tuberculosis is known to utilize multiple mechanisms for invasion (14), there must be additional myco...