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Infection and Immunity, July 2007, p. 3290-3304, Vol. 75, No. 7
0019-9567/07/$08.00+0     doi:10.1128/IAI.00292-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Rapid Escape of the dot/icm Mutants of Legionella pneumophila into the Cytosol of Mammalian and Protozoan Cells ^,

Department of Microbiology and Immunology, Room MS-410, University of Louisville College of Medicine, Louisville, Kentucky 40292

Received 21 February 2007/ Returned for modification 27 March 2007/ Accepted 3 April 2007

The Legionella pneumophila-containing phagosome evades endocytic fusion and intercepts endoplasmic reticulum (ER)-to-Golgi vesicle traffic, which is believed to be mediated by the Dot/Icm type IV secretion system. Although phagosomes harboring dot/icm mutants are thought to mature through the endosomal-lysosomal pathway, colocalization studies with lysosomal markers have reported contradictory results. In addition, phagosomes harboring the dot/icm mutants do not interact with endocytosed materials, which is inconsistent with maturation of the phagosomes in the endosomal-lysosomal pathway. Using multiple strategies, we show that the dot/icm mutants defective in the Dot/Icm structural apparatus are unable to maintain the integrity of their phagosomes and escape into the cytoplasm within minutes of entry into various mammalian and protozoan cells in a process independent of the type II secretion system. In contrast, mutants defective in cytoplasmic chaperones of Dot/Icm effectors and rpoS, letA/S, and letE regulatory mutants are all localized within intact phagosomes. Importantly, non-dot/icm L. pneumophila mutants whose phagosomes acquire late endosomal-lysosomal markers are all located within intact phagosomes. Using high-resolution electron microscopy, we show that phagosomes harboring the dot/icm transporter mutants do not fuse to lysosomes but are free in the cytoplasm. Inhibition of ER-to-Golgi vesicle traffic by brefeldin A does not affect the integrity of the phagosomes harboring the parental strain of L. pneumophila. We conclude that the Dot/Icm transporter is involved in maintaining the integrity of the L. pneumophila phagosome, independent of interception of ER-to-Golgi vesicle traffic, which is a novel function of type IV secretion systems.

Published ahead of print on 16 April 2007.

Supplemental material for this article may be found at http://iai.asm.org/.

Editor: W. A. Petri, Jr.

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