SummaryPathogenic mycobacteria have the ability to persist in phagocytic cells and to suppress the immune system. The glycolipid lipoarabinomannan (LAM), in particular its mannose cap, has been shown to inhibit phagolysosome fusion and to induce immunosuppressive IL-10 production via interaction with the mannose receptor or DC-SIGN. Hence, the current paradigm is that the mannose cap of LAM is a crucial factor in mycobacterial virulence. However, the above studies were performed with purified LAM, never with live bacteria. Here we evaluate the biological properties of capless mutants of Mycobacterium marinum and M. bovis BCG, made by inactivating homologues of Rv1635c. We show that its gene product is an undecaprenyl phosphomannose-dependent mannosyltransferase. Compared with parent strain, capless M. marinum induced slightly less uptake by and slightly more phagolysosome fusion in infected macrophages but this did not lead to decreased survival of the bacteria in vitro, nor in vivo in zebra fish. Loss of caps in M. bovis BCG resulted in a sometimes decreased binding to human dendritic cells or DC-SIGN-transfected Raji cells, but no differences in IL-10 induction were observed. In mice, capless M. bovis BCG did not survive less well in lung, spleen or liver and induced a similar cytokine profile. Our data contradict the current paradigm and demonstrate that mannose-capped LAM does not dominate the Mycobacterium-host interaction.

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...

Mycobacterium marinum, a natural pathogen of fish and frogs and an occasional pathogen of humans, is capable of inducing actin tail formation within the cytoplasm of macrophages, leading to actinbased motility and intercellular spread. Actin tail formation by M. marinum is markedly reduced in macrophages deficient in the Wiskott-Aldrich syndrome protein (WASP), which still contain the closely related and ubiquitously expressed protein N-WASP (neuronal WASP). In fibroblasts lacking both WASP and N-WASP, M. marinum is incapable of efficient actin polymerization and of intercellular spread. By reconstituting these cells, we find that M. marinum is able to use either WASP or N-WASP to induce actin polymerization. Inhibition or genetic deletion of tyrosine phosphorylation, Nck, WASP-interacting protein, and Cdc42 does not affect M. marinum actin tail formation, excluding the participation of these molecules as upstream activators of N-WASP in the initiation of actin-based motility. In contrast, deletion of the phosphatidylinositol 4,5-bisphosphate-binding basic motif in N-WASP eliminates M. marinum actin tail formation. Together, these data demonstrate that M. marinum subversion of host actin polymerization is most similar to distantly related Gram-negative organisms but that its mechanism for activating WASP family proteins is unique. Binding of specific molecules including phosphatidylinositol 4,5-bisphosphate (PIP 2 ), Cdc42, Nck, and Grb2 to N-WASP disrupts this inhibition and unmasks the WASP homology 2 and acidic (WA) domain, allowing the Arp2͞3 complex to initiate de novo actin polymerization (2-6). WASP, a protein found only in hematopoietic cells, is closely related to N-WASP, sharing 50% sequence similarity. The two proteins are similarly organized and are controlled by the same host cell inputs. Less related to N-WASP are the WAVE proteins that contain the WA domain output region but have different activating inputs (7).Diverse pathogens have evolved mechanisms to hijack this Arp2͞ 3-dependent pathway of actin polymerization for their own benefit (reviewed in ref. 8). For vaccinia virus, actin polymerization is required for viral egress from infected cells. Enterohemorrhagic and enteropathogenic Escherichia coli species' actin polymerization leads to pedestal formation, causing attachment and effacement lesions on gut epithelia. For Listeria, Shigella, Burkholderia, Rickettsia, and Mycobacterium marinum, actin polymerization on the surface of cytoplasmic bacteria results in actin tails, intracellular motility, and direct intercellular spread. Interestingly, these pathogens use independently evolved proteins to target different steps leading to Arp2͞3 activation. Listeria, Rickettsia, and perhaps Burkholderia mimic host cell N-WASP to activate the Arp2͞3 complex directly, and actin tail formation by these species is independent of host cell N-WASP (9-14). The other pathogens depend on the WASP family for actin polymerization: Shigella and enterohemorrhagic E. coli (EHEC) have molecules that directly recruit...