Identification of linked Legionella pneumophila genes essential for intracellular growth and evasion of the endocytic pathway

Legionella pneumophila replicates within a specialized phagosome in cultured cells, a function necessary for its pathogenicity. The replicative phagosome lacks membrane marker proteins, such as the glycoprotein LAMP-1, that are indicators of the normal endocytic pathway. We describe the isolation of several Legionella genes essential for intracellular growth and evasion of the endocytic pathway, using a genetic and cell biological approach. We screened 4,960 ethyl methanesulfonate-mutagenized colonies for defects in intracellular growth and trafficking to the replicative phagosome. Six mutant strains of L. pneumophila that had severe intracellular growth defects in mouse bone marrow-derived macrophages were identified. All six mutants were found in phagosomes that colocalized with LAMP-1, indicating defects in intracellular trafficking. The growth defects of two of these strains were complemented by molecular clones from a bank constructed from a wild-type L. pneumophila strain. The inserts from these clones are located in a region of the chromosome contiguous with several other genes essential for intracellular growth. Three mutants could be complemented by single open reading frames placed in trans , one mutant by a gene termed dotH and two additional mutants by a gene termed dotO . A deletion mutation was created in a third gene, dotI , which is located directly upstream of dotH . The Δ dotI strain was also defective for intracellular growth in macrophages, and this defect was complemented by a single open reading frame in trans . Based on sequence analysis and structural predictions, possible roles of dotH , dotI , and dotO in intracellular growth are discussed.