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Infect Immun, March 1998, p. 950-958, Vol. 66, No. 3
0019-9567/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Identification of Linked Legionella pneumophila Genes Essential for Intracellular Growth and Evasion of the Endocytic Pathway

Helene L. Andrews,1,2 Joseph P. Vogel,1 and Ralph R. Isberg1,3,*

Howard Hughes Medical Institute3 and Tufts University Schools of Medicine1 and Veterinary Medicine,2 Boston, Massachusetts 02111

Received 2 October 1997/Returned for modification 13 November 1997/Accepted 10 December 1997

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.

* Corresponding author. Mailing address: Department of Molecular Biology and Microbiology, Howard Hughes Medical Institute, Tufts University School of Medicine, 136 Harrison Ave., Boston, MA 02111. Phone: (617) 636-7393. Fax: (617) 636-0337. E-mail: risberg{at}opal.tufts.edu.




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