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Infect. Immun. doi:10.1128/IAI.00292-07
Copyright (c) 2007, American Society for Microbiology and/or the Listed Authors/Institutions. 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, KY 40292

^* To whom correspondence should be addressed. Email: abukwaik{at}louisville.edu.

	Abstract

The Legionella pneumophila-containing phagosome evades endocytic fusion and intercepts ER-to-golgi vesicle traffic, which is believed to be mediated by the Dot/Icm type IV secretion system. Although phagosomes harboring the 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 their phagosomes within 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 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 the 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 integrity of the phagosome harboring the parental strain of L. pneumophila. We conclude that the Dot/Icm transporter is involved in maintaining 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.

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