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 delta 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.
Andrews
H L
HL
Tufts University School of Medicine, Boston, Massachusetts 02111, USA.
Vogel
J P
JP
Isberg
R R
RR
D000818:Animals; D001426:Bacterial Proteins / Q000032:analysis; D001483:Base Sequence; D002478:Cells, Cultured; D004705:Endocytosis; D005260:Female; D005798:Genes, Bacterial; D005816:Genetic Complementation Test; D008040:Genetic Linkage; D016952:Legionella pneumophila / Q000235:genetics* / Q000254:growth & development; D008264:Macrophages / Q000382:microbiology; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D016366:Open Reading Frames
D001426:Bacterial Proteins