Affiliation:
1. Tufts University Schools of Medicine1 and
2. Veterinary Medicine,2 Boston, Massachusetts 02111
3. Howard Hughes Medical Institute3 and
Abstract
ABSTRACT
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.
Publisher
American Society for Microbiology
Subject
Infectious Diseases,Immunology,Microbiology,Parasitology