IDENTIFICATION OF LINKED LEGIONELLA-PNEUMOPHILA GENES ESSENTIAL FOR INTRACELLULAR GROWTH AND EVASION OF THE ENDOCYTIC PATHWAY

Legionella pneumophila replicates within a specialized phagosome in cu ltured cells, a function necessary for its pathogenicity. The replicat ive phagosome lacks membrane marker proteins, such as the glycoprotein LAMP-1, that are indicators of the normal endocytic pathway. We descr ibe the isolation of several Legionella genes essential for intracellu lar growth and evasion of the endocytic pathway, using a genetic and c ell biological approach. We screened 4,960 ethyl methanesulfonate-muta genized colonies for defects in intracellular growth and trafficking t o 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 traf ficking. The growth defects of two of these strains were complemented by molecular clones from a bank constructed from a wild-type L. pneumo phila strain. The inserts from these clones are located in a region of the chromosome contiguous with several other genes essential for intr acellular growth. Three mutants could be complemented by single open r eading frames placed in trans, one mutant by a gene termed dotH and tw o additional mutants by a gene termed dotO. A deletion mutation was cr eated in a third gene, dotI, which is located directly upstream of dot H. The Delta dotI strain was also defective for intracellular growth i n macrophages, and this defect was complemented bg a single open readi ng frame in trans. Based an sequence analysis and structural predictio ns, possible roles of dotH, dotI, and dotO in intracellular growth are discussed.