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Infection and Immunity, September 1998, p. 4450-4460, Vol. 66, No. 9
0019-9567/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Early Events in Phagosome Establishment Are Required for Intracellular Survival of Legionella pneumophila

Lawrence A. Wiater,1 Kenneth Dunn,2,dagger Frederick R. Maxfield,2,Dagger and Howard A. Shuman1,*

Departments of Microbiology1 and Pathology,2 College of Physicians and Surgeons, Columbia University, New York, New York 10032

Received 6 April 1998/Returned for modification 13 May 1998/Accepted 29 May 1998

During infection, the Legionnaires' disease bacterium, Legionella pneumophila, survives and multiplies within a specialized phagosome that is near neutral pH and does not fuse with host lysosomes. In order to understand the molecular basis of this organism's ability to control its intracellular fate, we have isolated and characterized a group of transposon-generated mutants which were unable to kill macrophages and were subsequently found to be defective in intracellular multiplication. These mutations define a set of 20 genes (19 icm [for intracellular multiplication] genes and dotA [for defect in organelle trafficking]). In this report, we describe a quantitative assay for phagosome-lysosome fusion (PLF) and its use to measure the levels of PLF in cells that have been infected with either wild-type L. pneumophila or one of several mutants defective in different icm genes or dotA. By using quantitative confocal fluorescence microscopy, PLF could be scored on a per-bacterium basis by determining the extent to which fluorescein-labeled L. pneumophila colocalized with host lysosomes prelabeled with rhodamine-dextran. Remarkably, mutations in the six genes that were studied resulted in maximal levels of PLF as quickly as 30 min following infection. These results indicate that several, and possibly all, of the icm and dotA gene products act at an early step during phagosome establishment to determine whether L. pneumophila-containing phagosomes will fuse with lysosomes. Although not ruled out, subsequent activity of these gene products may not be necessary for successful intracellular replication.

* Corresponding author. Mailing address: Department of Microbiology, College of Physicians and Surgeons, Columbia University, 701 West 168th St., New York, NY 10032. Phone: (212) 305-6913. Fax: (212) 305-1468. E-mail: shuman{at}cuccfa.ccc.columbia.edu.

dagger Present address: Department of Medicine, Nephrology Section, Indiana University Medical Center, Indianapolis, IN 46202.

Dagger Present address: Department of Biochemistry, Cornell University Medical College, New York, NY 10021.

Infection and Immunity, September 1998, p. 4450-4460, Vol. 66, No. 9
0019-9567/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


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