Analysis of the intracellular fate of Legionella pneumophila mutants.

L. pneumophila is a model organism for investigating the mechanisms by which intracellular pathogens acquire the metabolites needed for replication while evading the microbicidal mechanisms of the macrophage. We determined that intracellular L. pneumophila replicate in close association with the endoplasmic reticulum and suggest that L. pneumophila exploits the macrophage autophagy pathway to establish this specialized vacuole. To identify the bacterial factors required at this step as well as the factors important for other stages of the intracellular pathway, we isolated a collection of bacterial mutants that are defective for growth in macrophages. The ability of the mutant strains to evade fusion with the lysosomes and to establish replication vacuoles was examined by fluorescence microscopic localization of markers for the late endosomes (lgp 120), lysosomes (Texas Red-ovalbumin), endoplasmic reticulum (BiP), and L. pneumophila. By this approach, we identified mutants with distinct intracellular fates; one type does not evade the endocytic pathway, another forms replication vacuoles less efficiently than does wild-type, and a third type forms replication vacuoles but replicates poorly. These mutants are likely to facilitate identification and characterization of the bacterial factors required by L. pneumophila to establish a protected niche for intracellular replication.