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      Legionella Eukaryotic-Like Type IV Substrates Interfere with Organelle Trafficking

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          Legionella pneumophila, the causative agent of Legionnaires' disease, evades phago-lysosome fusion in mammalian and protozoan hosts to create a suitable niche for intracellular replication. To modulate vesicle trafficking pathways, L. pneumophila translocates effector proteins into eukaryotic cells through a Type IVB macro-molecular transport system called the Icm-Dot system. In this study, we employed a fluorescence-based translocation assay to show that 33 previously identified Legionella eukaryotic-like genes ( leg) encode substrates of the Icm-Dot secretion system. To assess which of these proteins may contribute to the disruption of vesicle trafficking, we expressed each gene in yeast and looked for phenotypes related to vacuolar protein sorting. We found that LegC3-GFP and LegC7/YlfA-GFP caused the mis-secretion of CPY-Invertase, a fusion protein normally restricted to the yeast vacuole. We also found that LegC7/YlfA-GFP and its paralog LegC2/YlfB-GFP formed large structures around the yeast vacuole while LegC3-GFP localized to the plasma membrane and a fragmented vacuole. In mammalian cells, LegC2/YlfB-GFP and LegC7/YlfA-GFP were found within large structures that co-localized with anti-KDEL antibodies but excluded the lysosomal marker LAMP-1, similar to what is observed in Legionella-containing vacuoles. LegC3-GFP, in contrast, was observed as smaller structures which had no obvious co-localization with KDEL or LAMP-1. Finally, LegC3-GFP caused the accumulation of many endosome-like structures containing undigested material when expressed in the protozoan host Dictyostelium discoideum. Our results demonstrate that multiple Leg proteins are Icm/Dot-dependent substrates and that LegC3, LegC7/YlfA, and LegC2/YlfB may contribute to the intracellular trafficking of L. pneumophila by interfering with highly conserved pathways that modulate vesicle maturation.

          Author Summary

          Legionella pneumophila is a Gram-negative bacterial species that causes a severe pneumonia known as Legionnaires' disease. Inhalation of L. pneumophila–contaminated aerosols results in the infection of lung macrophages. Following infection, the bacteria use a Type IVB secretion system to deliver multiple effector proteins into the macrophages to create a membrane-bound replicative compartment called the Legionella-containing vacuole, or LCV. The LCV is defined by its recruitment of early secretory vesicles and avoidance of the bactericidal lysosomes. We identified several effector proteins that contain eukaryotic domains and share significant homology with eukaryotic organelle trafficking proteins. We demonstrate that 33 Legionella eukaryotic-like genes ( leg) encode proteins that are translocated into host cells. When artificially expressed in yeast, three Leg proteins (LegC2, LegC3, and LegC7) were able to disrupt normal vesicle trafficking and vacuole morphology. In addition, the Leg proteins induced the formation of, and were localized within, distinct structures when expressed in mammalian cells. In the protozoan host Dictyostelium discoideum, expression of LegC3 resulted in the accumulation of membranous compartments containing partially digested material. Thus, LegC3, LegC2, and LegC7 represent novel effector proteins that may contribute to the intracellular lifestyle of L. pneumophila by disrupting normal vacuolar trafficking pathways in host cells.

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          Most cited references 77

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          A new vital stain for visualizing vacuolar membrane dynamics and endocytosis in yeast

           SD Emr (1995)
          We have used a lipophilic styryl dye, N-(3-triethylammoniumpropyl)-4- (p-diethylaminophenyl-hexatrienyl) pyridinium dibromide (FM 4-64), as a vital stain to follow bulk membrane-internalization and transport to the vacuole in yeast. After treatment for 60 min at 30 degrees C, FM 4- 64 stained the vacuole membrane (ring staining pattern). FM 4-64 did not appear to reach the vacuole by passive diffusion because at 0 degree C it exclusively stained the plasma membrane (PM). The PM staining decreased after warming cells to 25 degrees C and small punctate structures became apparent in the cytoplasm within 5-10 min. After an additional 20-40 min, the PM and cytoplasmic punctate staining disappeared concomitant with staining of the vacuolar membrane. Under steady state conditions, FM 4-64 staining was specific for vacuolar membranes; other membrane structures were not stained. The dye served as a sensitive reporter of vacuolar dynamics, detecting such events as segregation structure formation during mitosis, vacuole fission/fusion events, and vacuolar morphology in different classes of vacuolar protein sorting (vps) mutants. A particularly striking pattern was observed in class E mutants (e.g., vps27) where 500-700 nm organelles (presumptive prevacuolar compartments) were intensely stained with FM 4- 64 while the vacuole membrane was weakly fluorescent. Internalization of FM 4-64 at 15 degrees C delayed vacuolar labeling and trapped FM 4- 64 in cytoplasmic intermediates between the PM and the vacuole. The intermediate structures in the cytoplasm are likely to be endosomes as their staining was temperature, time, and energy dependent. Interestingly, unlike Lucifer yellow uptake, vacuolar labeling by FM 4- 64 was not blocked in sec18, sec14, end3, and end4 mutants, but was blocked in sec1 mutant cells. Finally, using permeabilized yeast spheroplasts to reconstitute FM 4-64 transport, we found that delivery of FM 4-64 from the endosome-like intermediate compartment (labeled at 15 degrees C) to the vacuole was ATP and cytosol dependent. Thus, we show that FM 4-64 is a new vital stain for the vacuolar membrane, a marker for endocytic intermediates, and a fluor for detecting endosome to vacuole membrane transport in vitro.
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            Legionnaires' disease: description of an epidemic of pneumonia.

            An explosive, common-source outbreak of pneumonia caused by a previously unrecognized bacterium affected primarily persons attending an American Legion convention in Philadelphia in July, 1976. Twenty-nine of 182 cases were fatal. Spread of the bacterium appeared to be air borne. The source of the bacterium was not found, but epidemiologic analysis suggested that exposure may have occurred in the lobby of the headquarters hotel or in the area immediately surrounding the hotel. Person-to-person spread seemed not to have occurred. Many hotel employees appeared to be immune, suggesting that the agent may have been present in the vicinity, perhaps intermittently, for two or more years.
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              Receptor downregulation and multivesicular-body sorting.

              The sorting of proteins into the inner vesicles of multivesicular bodies is required for many key cellular processes, which range from the downregulation of activated signalling receptors to the proper stimulation of the immune response. Recent advances in our understanding of the multivesicular-body sorting pathway have resulted from the identification of ubiquitin as a signal for the efficient sorting of proteins into this transport route, and from the discovery of components of the sorting and regulatory machinery that directs this complex process.

                Author and article information

                Role: Editor
                PLoS Pathog
                PLoS Pathogens
                Public Library of Science (San Francisco, USA )
                August 2008
                August 2008
                1 August 2008
                : 4
                : 8
                [1 ]Integrated Program in Cellular, Molecular, and Biophysical Studies, Columbia University Medical Center, New York, New York, United States of America
                [2 ]Department of Microbiology, Columbia University Medical Center, New York, New York, United States of America
                [3 ]Division of Biology and Paleo Environment, Lamont-Doherty Earth Observatory, Columbia University, Palisades, New York, United States of America
                Tufts University School of Medicine, United States of America
                Author notes

                Conceived and designed the experiments: KSdF RTG XC ORA CDP HAS. Performed the experiments: KSdF RTG XC ORA MR CDP. Analyzed the data: KSdF RTG XC ORA MR HAS. Contributed reagents/materials/analysis tools: KSdF XC ORA CDP. Wrote the paper: KSdF RTG HAS.

                de Felipe et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
                Pages: 16
                Research Article
                Cell Biology/Membranes and Sorting
                Infectious Diseases/Bacterial Infections
                Infectious Diseases/Protozoal Infections
                Infectious Diseases/Respiratory Infections
                Microbiology/Cellular Microbiology and Pathogenesis
                Microbiology/Microbial Evolution and Genomics

                Infectious disease & Microbiology


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