Early Events in Phagosome Establishment Are Required for Intracellular Survival ofLegionella pneumophila

Abstract

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 ordotA. By using quantitative confocal fluorescence microscopy, PLF could be scored on a per-bacterium basis by determining the extent to which fluorescein-labeled L. pneumophilacolocalized 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 icmand 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.

Icm/Dot‐dependent upregulation of phagocytosis by Legionella pneumophila

“…In contrast, . 90% of the phagosomes containing either dead bacteria or viable icm/dot mutant bacteria fuse and form phagolysosomes (Horwitz, 1987;Roy et al, 1998;Wiater et al, 1998). Thus, the ability of the bacteria to influence the fusion properties of the phagosome is key to the ultimate fate of the bacteria.…”

“…Legionella pneumophila wild-type but not icm/dot mutant bacteria block maturation of the phagosome within minutes after internalization (Roy et al, 1998;Wiater et al, 1998). This finding prompted us to investigate whether phagocytosis was also affected by the pathogen.…”

Intracellular innate resistance to bacterial pathogens

“…Epithelial cell -chlamydial inclusion Avoids lysosome fusion by diverting chlamydial inclusion from normal vesicular trafficking pathway (Hackstadt et al, 1997) Coxiella burnetii Macrophage, epithelial cell, fibroblast -acidic vacuole with markers of autophagy Exploits autophagic pathway, acid tolerant (Raoult et al, 1990;Gutierrez et al, 2005) Legionella pneumophila Macrophage -initially found in autophagosome, then in lysosome Modulates autophagic pathway, delays fusion with lysosomes (Andrews et al, 1998;Wiater et al, 1998;Amer and Swanson, 2005;) Listeria monocytogenes Macrophage, epithelial cell, fibroblast -cytosol Delays fusion with lysosomes, escapes from phagosome (Gaillard et al, 1987;Tilney and Portnoy, 1989;Henry et al, 2006) Mycobacterium tuberculosis Macrophage -modified early endosome Inhibits phagolysosome fusion, resistant to RNI (Flynn and Chan, 2003) Salmonella enterica ser. Typhimurium Macrophage, epithelial cell -Salmonella-containing vacuole (acquires LAMP1 but inhibits fusion with lysosomes) Modulates fusion with lysosomes, inhibits recruitment of phagocyte oxidase, resistance to antimicrobial peptides (Vazquez-Torres et al, 2000;Ernst et al, 2001;Holden, 2002) innate immune functions (Miyauchi et al, 1985;Radons et al, 1994).…”

A yeast genetic system for the identification and characterization of substrate proteins transferred into host cells by the Legionella pneumophila Dot/Icm system

“…A type IVb secretion system encoded by 24 different dot and icm genes plays a central role in the modulation of host cell functions by Legionella (Segal and Shuman, 1997;Vogel et al ., 1998). Mutant Legionella that have a non-functional Dot/Icm system are unable to modulate vacuole transport (Marra et al ., 1992;Berger and Isberg, 1993;Swanson and Isberg, 1996;Andrews et al ., 1998;Roy et al ., 1998;Wiater et al ., 1998). Most Legionella dot and icm mutants reside in vacuoles that undergo endocytic maturation and fail to recruit ER-derived vesicles, resulting in a severe intracellular growth defect.…”