Evidence that Dot-dependent and -independent factors isolate the Legionella pneumophila phagosome from the endocytic network in mouse macrophages

Legionella pneumophila survives within macrophages by evading phagosome-lys osome fusion, To determine whether L, pneumophila resides in gn intermediat e endosomal compartment or is isolated from the endosomal pathway and to in vestigate what bacterial factors contribute to establishment of its vacuole , we applied a series of fluorescence microscopy assays. The majority of va cuoles, aged 2.5 min to 4 h containing post-exponential phase (PE) L. pneum ophila, appeared to be separate from the endosomal pathway, as judged by th e absence of transferrin receptor, LAMP-1,cathepsin D and each of four fluo rescent probes used to label the endocytic pathway either before or after i nfection, In contrast, more than 70% of phagosomes that contained Escherich ia coli, polystyrene beads, or exponential phase (E) L, pneumophila matured to phagolysosomes, as judged by co-localization with LAMP-1, cathepsin D a nd fluorescent endosomal probes, Surprisingly, neither bacterial viability nor the putative Dot/Icm transport complex was absolutely required for vacu ole isolation; although phagosomes containing either formalin-killed PE wil d-type or live PE dotA or dotB mutant L, pneumophila rapidly accumulated LA MP-1, less than 20% acquired lysosomal cathepsin D or fluorescent endosomal probes. Therefore, a Dot-dependent factor(s) isolates the L. pneumophila p hagosome from a LAMP-1-containing compartment, and a formalin-resistant Dot -independent activity inhibits vacuolar accumulation of endocytosed materia l and delivery to the degradative lysosomes.