Functional Analysis of Host Factors that Mediate the Intracellular Lifestyle of Cryptococcus neoformans
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{"title"=>"Functional analysis of host factors that mediate the intracellular lifestyle of cryptococcus neoformans", "type"=>"journal", "authors"=>[{"first_name"=>"Qing Ming", "last_name"=>"Qin", "scopus_author_id"=>"24482222700"}, {"first_name"=>"Jijing", "last_name"=>"Luo", "scopus_author_id"=>"43861663200"}, {"first_name"=>"Xiaorong", "last_name"=>"Lin", "scopus_author_id"=>"8971753200"}, {"first_name"=>"Jianwu", "last_name"=>"Pei", "scopus_author_id"=>"7103299011"}, {"first_name"=>"Lei", "last_name"=>"Li", "scopus_author_id"=>"55584803700"}, {"first_name"=>"Thomas A.", "last_name"=>"Ficht", "scopus_author_id"=>"7003492038"}, {"first_name"=>"Paul", "last_name"=>"de Figueiredo", "scopus_author_id"=>"6602879807"}], "year"=>2011, "source"=>"PLoS Pathogens", "identifiers"=>{"pui"=>"362058354", "isbn"=>"1553-7374 (Electronic)\\r1553-7366 (Linking)", "issn"=>"15537366", "doi"=>"10.1371/journal.ppat.1002078", "scopus"=>"2-s2.0-79959848082", "pmid"=>"21698225", "sgr"=>"79959848082"}, "id"=>"9259e824-776e-3cab-bc33-552c794465e9", "abstract"=>"Cryptococcus neoformans (Cn), the major causative agent of human fungal meningoencephalitis, replicates within phagolysosomes of infected host cells. Despite more than a half-century of investigation into host-Cn interactions, host factors that mediate infection by this fungal pathogen remain obscure. Here, we describe the development of a system that employs Drosophila S2 cells and RNA interference (RNAi) to define and characterize Cn host factors. The system recapitulated salient aspects of fungal interactions with mammalian cells, including phagocytosis, intracellular trafficking, replication, cell-to-cell spread and escape of the pathogen from host cells. Fifty-seven evolutionarily conserved host factors were identified using this system, including 29 factors that had not been previously implicated in mediating fungal pathogenesis. Subsequent analysis indicated that Cn exploits host actin cytoskeletal elements, cell surface signaling molecules, and vesicle-mediated transport proteins to establish a replicative niche. Several host molecules known to be associated with autophagy (Atg), including Atg2, Atg5, Atg9 and Pi3K59F (a class III PI3-kinase) were also uncovered in our screen. Small interfering RNA (siRNA) mediated depletion of these autophagy proteins in murine RAW264.7 macrophages demonstrated their requirement during Cn infection, thereby validating findings obtained using the Drosophila S2 cell system. Immunofluorescence confocal microscopy analyses demonstrated that Atg5, LC3, Atg9a were recruited to the vicinity of Cn containing vacuoles (CnCvs) in the early stages of Cn infection. Pharmacological inhibition of autophagy and/or PI3-kinase activity further demonstrated a requirement for autophagy associated host proteins in supporting infection of mammalian cells by Cn. Finally, systematic trafficking studies indicated that CnCVs associated with Atg proteins, including Atg5, Atg9a and LC3, during trafficking to a terminal intracellular compartment that was decorated with the lysosomal markers LAMP-1 and cathepsin D. Our findings validate the utility of the Drosophila S2 cell system as a functional genomic platform for identifying and characterizing host factors that mediate fungal intracellular replication. Our results also support a model in which host Atg proteins mediate Cn intracellular trafficking and replication.", "link"=>"http://www.mendeley.com/research/functional-analysis-host-factors-mediate-intracellular-lifestyle-cryptococcus-neoformans-7", "reader_count"=>6, "reader_count_by_academic_status"=>{"Professor > Associate Professor"=>1, "Student > Doctoral Student"=>1, "Researcher"=>1, "Student > Ph. D. Student"=>1, "Student > Bachelor"=>2}, "reader_count_by_user_role"=>{"Professor > Associate Professor"=>1, "Student > Doctoral Student"=>1, "Researcher"=>1, "Student > Ph. D. Student"=>1, "Student > Bachelor"=>2}, "reader_count_by_subject_area"=>{"Unspecified"=>1, "Biochemistry, Genetics and Molecular Biology"=>1, "Agricultural and Biological Sciences"=>3, "Medicine and Dentistry"=>1}, "reader_count_by_subdiscipline"=>{"Medicine and Dentistry"=>{"Medicine and Dentistry"=>1}, "Agricultural and Biological Sciences"=>{"Agricultural and Biological Sciences"=>3}, "Biochemistry, Genetics and Molecular Biology"=>{"Biochemistry, Genetics and Molecular Biology"=>1}, "Unspecified"=>{"Unspecified"=>1}}, "reader_count_by_country"=>{"Brazil"=>1}, "group_count"=>1}

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Figshare

  • {"files"=>["https://ndownloader.figshare.com/files/765277"], "description"=>"<p>(<b>A</b>) and murine J774.A1 macrophages (<b>B</b>). Host cells were infected with the following Cn strains: AI100-dsRed, XL1601, HMCA1, MCD16, XX44, AI81 and Cap59. The infected cells were then extensively washed at 3 h.p.i. and then fresh medium supplemented with 20 µg/ml fluconazole was added to each well. The infected cells were continuously incubated at 28°C (S2 cells) or at 37°C, 5% CO<sub>2</sub> (J774.A1 cells). At 3 and 15 h.p.i., the number of Cn cells present in the supernatant or within host cells was determined as described in the <a href=\"http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002078#s4\" target=\"_blank\"><i>Materials and Methods</i></a> section. Cn mutants display similar internalization patterns (<b>A1</b> and <b>B1</b>), levels of Cn escape (<b>A2</b> and <b>B2</b>) and relative intracellular replication efficiencies (<b>A3</b> and <b>B3</b>) in S2 (<b>A</b>, left panel) and J774.A1 (<b>B</b>, right panel) cells. Data from the control (AI100-dsRed) were normalized to 100%. Data represent the means ± SD from at least three independent experiments. **, *** indicates significance at P<0.01 and P<0.001, respectively.</p>", "links"=>[], "tags"=>["mutants", "s2"], "article_id"=>435648, "categories"=>["Infectious Diseases"], "users"=>["Qing-Ming Qin", "Jijing Luo", "Xiaorong Lin", "Jianwu Pei", "Lei Li", "Thomas A. Ficht", "Paul de Figueiredo"], "doi"=>"https://dx.doi.org/10.1371/journal.ppat.1002078.g004", "stats"=>{"downloads"=>1, "page_views"=>5, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Cryptococcus_neoformans_mutants_behave_similarly_in_Drosophila_S2_cells_/435648", "title"=>"<i>Cryptococcus neoformans</i> mutants behave similarly in <i>Drosophila</i> S2 cells.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2011-06-16 01:34:08"}
  • {"files"=>["https://ndownloader.figshare.com/files/384683", "https://ndownloader.figshare.com/files/384743", "https://ndownloader.figshare.com/files/384801", "https://ndownloader.figshare.com/files/384906", "https://ndownloader.figshare.com/files/384933", "https://ndownloader.figshare.com/files/384972", "https://ndownloader.figshare.com/files/385012", "https://ndownloader.figshare.com/files/385053", "https://ndownloader.figshare.com/files/385087", "https://ndownloader.figshare.com/files/385120", "https://ndownloader.figshare.com/files/385156", "https://ndownloader.figshare.com/files/385200", "https://ndownloader.figshare.com/files/385225"], "description"=>"<div><p><em>Cryptococcus neoformans</em> (Cn), the major causative agent of human fungal meningoencephalitis, replicates within phagolysosomes of infected host cells. Despite more than a half-century of investigation into host-Cn interactions, host factors that mediate infection by this fungal pathogen remain obscure. Here, we describe the development of a system that employs <em>Drosophila</em> S2 cells and RNA interference (RNAi) to define and characterize Cn host factors. The system recapitulated salient aspects of fungal interactions with mammalian cells, including phagocytosis, intracellular trafficking, replication, cell-to-cell spread and escape of the pathogen from host cells. Fifty-seven evolutionarily conserved host factors were identified using this system, including 29 factors that had not been previously implicated in mediating fungal pathogenesis. Subsequent analysis indicated that Cn exploits host actin cytoskeletal elements, cell surface signaling molecules, and vesicle-mediated transport proteins to establish a replicative niche. Several host molecules known to be associated with autophagy (Atg), including Atg2, Atg5, Atg9 and Pi3K59F (a class III PI3-kinase) were also uncovered in our screen. Small interfering RNA (siRNA) mediated depletion of these autophagy proteins in murine RAW264.7 macrophages demonstrated their requirement during Cn infection, thereby validating findings obtained using the <em>Drosophila</em> S2 cell system. Immunofluorescence confocal microscopy analyses demonstrated that Atg5, LC3, Atg9a were recruited to the vicinity of Cn containing vacuoles (CnCvs) in the early stages of Cn infection. Pharmacological inhibition of autophagy and/or PI3-kinase activity further demonstrated a requirement for autophagy associated host proteins in supporting infection of mammalian cells by Cn. Finally, systematic trafficking studies indicated that CnCVs associated with Atg proteins, including Atg5, Atg9a and LC3, during trafficking to a terminal intracellular compartment that was decorated with the lysosomal markers LAMP-1 and cathepsin D. Our findings validate the utility of the <em>Drosophila</em> S2 cell system as a functional genomic platform for identifying and characterizing host factors that mediate fungal intracellular replication. Our results also support a model in which host Atg proteins mediate Cn intracellular trafficking and replication.</p> </div>", "links"=>[], "tags"=>["factors", "mediate", "intracellular"], "article_id"=>135949, "categories"=>["Cancer"], "users"=>["Qing-Ming Qin", "Jijing Luo", "Xiaorong Lin", "Jianwu Pei", "Lei Li", "Thomas A. Ficht", "Paul de Figueiredo"], "doi"=>["https://dx.doi.org/10.1371/journal.ppat.1002078.s001", "https://dx.doi.org/10.1371/journal.ppat.1002078.s002", "https://dx.doi.org/10.1371/journal.ppat.1002078.s003", "https://dx.doi.org/10.1371/journal.ppat.1002078.s004", "https://dx.doi.org/10.1371/journal.ppat.1002078.s005", "https://dx.doi.org/10.1371/journal.ppat.1002078.s006", "https://dx.doi.org/10.1371/journal.ppat.1002078.s007", "https://dx.doi.org/10.1371/journal.ppat.1002078.s008", "https://dx.doi.org/10.1371/journal.ppat.1002078.s009", "https://dx.doi.org/10.1371/journal.ppat.1002078.s010", "https://dx.doi.org/10.1371/journal.ppat.1002078.s011", "https://dx.doi.org/10.1371/journal.ppat.1002078.s012", "https://dx.doi.org/10.1371/journal.ppat.1002078.s013"], "stats"=>{"downloads"=>33, "page_views"=>14, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/Functional_Analysis_of_Host_Factors_that_Mediate_the_Intracellular_Lifestyle_of_Cryptococcus_neoformans_/135949", "title"=>"Functional Analysis of Host Factors that Mediate the Intracellular Lifestyle of <em>Cryptococcus neoformans</em>", "pos_in_sequence"=>0, "defined_type"=>4, "published_date"=>"2011-06-16 01:39:09"}
  • {"files"=>["https://ndownloader.figshare.com/files/764811"], "description"=>"<p>Live cell images were selected from a representative time-lapse movie (n = 3). Time-lapse images were taken every 2 min. Frames are labeled according to the start of the imaging process, which was initiated approximately 3.5 hrs post-infection. Before the imaging was initiated, the infected cells were washed three times with 1×PBS. Fresh serum-free medium supplemented with 20 µg/ml fluconazole was then added to the cells to inhibit the replication of the extracellular (but not intracellular) population of Cn cells. <b>A</b>. <i>Drosophila</i> S2 cells support Cn infection. Yellow arrows: Phagocytosis of Cn cells by S2 cells. Light green arrows: Cn replication precedes host cell lysis. Red arrows: Examples of Cn extrusion from host cells. White arrow: Cn cell-to-cell dissemination by host cell division. <b>B</b>. Cn direct cell-to-cell spread (red arrows) and intracellular replication (yellow arrows) in S2 cells.</p>", "links"=>[], "tags"=>["images", "s2", "cells", "intracellular", "cell-to-cell", "dissemination"], "article_id"=>435175, "categories"=>["Infectious Diseases"], "users"=>["Qing-Ming Qin", "Jijing Luo", "Xiaorong Lin", "Jianwu Pei", "Lei Li", "Thomas A. Ficht", "Paul de Figueiredo"], "doi"=>"https://dx.doi.org/10.1371/journal.ppat.1002078.g001", "stats"=>{"downloads"=>0, "page_views"=>3, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Live_cell_images_indicate_that_Drosophila_S2_cells_support_the_phagocytosis_intracellular_replication_cell_to_cell_dissemination_and_escape_of_Cryptococcus_neoformans_Cn_/435175", "title"=>"Live cell images indicate that <i>Drosophila</i> S2 cells support the phagocytosis, intracellular replication, cell-to-cell dissemination and escape of <i>Cryptococcus neoformans</i> (Cn).", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2011-06-16 01:26:15"}
  • {"files"=>["https://ndownloader.figshare.com/files/764999"], "description"=>"<p><b>A1</b> and <b>B1</b>. Host cell phagocytosis of Cn cells. Infected S2 and J774.A1 cells (on coverslips in 24-well plates) were extensively washed after coincubation with Cn (AI100-dsRed) cells for 1 or 3 hrs. The infected cells were fixed, stained with rhodamine-phalloidin and then analyzed by fluorescence microscopy. The numbers of Cn within host cells were quantified, and the percentages of host cells that had been phagocytosed at the indicated time points post-infection were determined. For each well in each experiment, at least 500 cells were analyzed. <b>A2</b> and <b>B2</b>. Host cells support Cn intracellular replication and escape from infected cells. At 3 h.p.i., Cn (AI100-dsRed) infected host cells were extensively washed. Fresh medium supplemented with 20 µg/ml fluconazole was added to each well containing infected host cells. The infected cells were then continuously incubated at 28°C (S2 cells) or 37°C, 5% CO<sub>2</sub> (J774.A1 macrophages). At the indicated time points, media were collected from each well to determine the number of Cn cells that had escaped. The number of CFUs recovered from each sample was then quantified, and the fold-increase of Cn cells (compared to the initial amount recovered at 3 h.p.i.) was calculated and plotted as a function of time. In parallel, at various times post-infection, the corresponding infected S2 and J774.A1 cells were lysed and the number of CFUs contained therein determined after plating and growth on solid medium. The fold-increase of Cn cells was then calculated and plotted (black line) as above. Data represent means ± standard deviation (SD) from at least three independent experiments in which triplicate wells were analyzed.</p>", "links"=>[], "tags"=>["s2", "cells", "murine", "macrophages", "patterns", "cn", "phagocytosis", "intracellular"], "article_id"=>435366, "categories"=>["Infectious Diseases"], "users"=>["Qing-Ming Qin", "Jijing Luo", "Xiaorong Lin", "Jianwu Pei", "Lei Li", "Thomas A. Ficht", "Paul de Figueiredo"], "doi"=>"https://dx.doi.org/10.1371/journal.ppat.1002078.g002", "stats"=>{"downloads"=>0, "page_views"=>2, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Drosophila_S2_cells_and_murine_J774_A1_macrophages_support_similar_patterns_of_Cn_phagocytosis_and_intracellular_replication_/435366", "title"=>"<i>Drosophila</i> S2 cells and murine J774.A1 macrophages support similar patterns of Cn phagocytosis and intracellular replication.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2011-06-16 01:29:26"}
  • {"files"=>["https://ndownloader.figshare.com/files/765440"], "description"=>"<p><i>Drosophila</i> S2 cells (<b>A</b>) and murine J774.A1 macrophages (<b>B</b>) were coincubated with the indicated compounds for 1 hr at the concentrations shown in <a href=\"http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002078#ppat.1002078.s010\" target=\"_blank\">Table S2</a>. Next, Cn cells were added to the compound-treated host cells and incubated (in the presence of compound) for 3 hrs. The infected host cells were then extensively washed to remove the extracellular population of Cn cells, and new media containing fresh compounds and fluconazole (20 µg/ml) were added. Phagocytosis of Cn cells by host cells (<b>A1</b> and <b>B1</b>) was determined at 3 h.p.i. by lysing the infected cells, plating the cell lysate on YPD solid medium, and determining the CFUs present after 24 hr incubation at 37°C. Finally, at 12 h.p.i., the escape (<b>A2</b> and <b>B2</b>) or intracellular replication (<b>A3</b> and <b>B3</b>) efficiencies of Cn cells were measured separately as described in the <a href=\"http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002078#s4\" target=\"_blank\"><i>Materials and Methods</i></a> section. *, **, *** indicates significance at P<0.05, 0.01 and 0.001, respectively, compared with the DMSO treated control. CytD, 3-MA and BAF indicate that host cells were treated with cytochalasin D, 3-Methyladenine and bafilomycin A1, respectively. Data represent the means ± standard deviations from at least three independent experiments.</p>", "links"=>[], "tags"=>["compounds"], "article_id"=>435811, "categories"=>["Infectious Diseases"], "users"=>["Qing-Ming Qin", "Jijing Luo", "Xiaorong Lin", "Jianwu Pei", "Lei Li", "Thomas A. Ficht", "Paul de Figueiredo"], "doi"=>"https://dx.doi.org/10.1371/journal.ppat.1002078.g005", "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Effect_of_selected_compounds_on_Cryptococcus_neoformans_infection_/435811", "title"=>"Effect of selected compounds on <i>Cryptococcus neoformans</i> infection.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2011-06-16 01:36:51"}
  • {"files"=>["https://ndownloader.figshare.com/files/765528"], "description"=>"<p><b>A</b>. Fifty-seven host factors that, when depleted, significantly altered Cn infection phenotypes were identified in an RNAi screen using <i>Drosophila</i> S2 host cells. The 57 host factors were analyzed using established gene ontology categories (<a href=\"http://flybase.org/\" target=\"_blank\">http://flybase.org/</a>). The results were plotted in a pie chart with the percentage of genes in each category indicated. <b>B</b>. Comparison of the 57 identified host factors with those that were reported to mediate bacterial or fungal pathogen infection of host cells. Among the 57 hits, twenty-nine (50.9%) had not been previously reported to function as host factors for intracellular fungal pathogens. Shared and unique host factors were determined by analyzing published data from screens performed in S2 cells infected with the following pathogens: <i>Brucella melitensis </i><a href=\"http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002078#ppat.1002078-Qin1\" target=\"_blank\">[41]</a>, <i>Chlamydia caviae </i><a href=\"http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002078#ppat.1002078-Derre1\" target=\"_blank\">[43]</a>, <i>Chlamydia trachomatis </i><a href=\"http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002078#ppat.1002078-Elwell1\" target=\"_blank\">[44]</a>, <i>Listeria monocytogenes </i><a href=\"http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002078#ppat.1002078-Cheng1\" target=\"_blank\">[38]</a>, <a href=\"http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002078#ppat.1002078-Agaisse1\" target=\"_blank\">[40]</a>, <i>Mycobacterium fortuitum </i><a href=\"http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002078#ppat.1002078-Agaisse1\" target=\"_blank\">[40]</a>, <a href=\"http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002078#ppat.1002078-Philips1\" target=\"_blank\">[42]</a> and <i>Candida albicans </i><a href=\"http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002078#ppat.1002078-StroscheinStevenson1\" target=\"_blank\">[39]</a>. The host factors that are shared with other human pathogens (49.1%) are listed in red (<i>C. albicans</i>), magenta (<i>C. albicans</i> and bacterial pathogens) and blue (bacterial pathogens). Each host factor mediates Cn phagocytosis and/or intracellular replication.</p>", "links"=>[], "tags"=>["factors", "regulating"], "article_id"=>435897, "categories"=>["Infectious Diseases"], "users"=>["Qing-Ming Qin", "Jijing Luo", "Xiaorong Lin", "Jianwu Pei", "Lei Li", "Thomas A. Ficht", "Paul de Figueiredo"], "doi"=>"https://dx.doi.org/10.1371/journal.ppat.1002078.g006", "stats"=>{"downloads"=>2, "page_views"=>5, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Identification_of_host_factors_regulating_Cryptococcus_neoformans_infection_/435897", "title"=>"Identification of host factors regulating <i>Cryptococcus neoformans</i> infection.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2011-06-16 01:38:17"}
  • {"files"=>["https://ndownloader.figshare.com/files/765733"], "description"=>"<p>Murine J774.A1 or RAW264.7 cells were seeded on 12-mm coverslips placed on the bottom of 24-well plates and then infected with Cn (AI100-dsRed) for 1 or 3 hr. At 3 h.p.i., the infected cells were wash three times and then continuously incubated in fresh medium supplemented with 20 µg/ml fluconazole. At the indicated time points, the infected cells were fixed and prepared for immunofluorescence microscopy using antibodies directed against mouse proteins LC3 (<b>A</b>, J774.A1 cells) or Atg9a (<b>C</b>, RAW264.7 cells. Inset in <b>C3</b>: a magnified section shows Atg9a in close apposition to CnCvs during an early stage of infection) and Atg5 (<b>D</b>, J774.A1 cells). Regions of colocalization appear yellow in the images (<b>C3</b> and <b>C6</b>). CnCVs surrounded by host proteins are indicated by white arrows. Scale bars indicate 5 µM. Images were taken from a representative experiment of at least three independent experiments. <b>B</b>. Quantification of Cn cells that resided in host LC3-positive compartments during a time course of infection. At each time point, the percent of total cells analyzed in which LC3 was tightly associated with CnCvs was calculated and plotted as a function of time. Data represent the means ± SD from at least three independent experiments. For each experiment, at least 300 internalized or replicative Cn cells were analyzed. <b>E</b>. Induction of LC3-I to LC3-II processing by Cn infection. Murine J774.A1 (<b>E1</b>), RAW264.7 (<b>E2</b>) macrophages or MEFs (<b>E3</b>) were infected with Cn strain AI100-dsRed or cultured in serum free DMEM (starvation) for 12 hr. At the indicated time points, Cn infected and serum starved cell samples were collected, and subjected to immunoblotting analysis using antibodies directed against mouse LC3. NS = non-starved. Representative images were taken from a single experiment. Three independent experiments gave similar results.</p>", "links"=>[], "tags"=>["interactions", "atg", "proteins"], "article_id"=>436101, "categories"=>["Infectious Diseases"], "users"=>["Qing-Ming Qin", "Jijing Luo", "Xiaorong Lin", "Jianwu Pei", "Lei Li", "Thomas A. Ficht", "Paul de Figueiredo"], "doi"=>"https://dx.doi.org/10.1371/journal.ppat.1002078.g008", "stats"=>{"downloads"=>1, "page_views"=>11, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Cryptococcus_neoformans_interactions_with_Atg_proteins_during_infection_/436101", "title"=>"<i>Cryptococcus neoformans</i> interactions with Atg proteins during infection.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2011-06-16 01:41:41"}
  • {"files"=>["https://ndownloader.figshare.com/files/765117"], "description"=>"<p>(<b>A</b>) and J774.A1 (<b>B</b>) cells. Host cells were seeded on 12-mm coverslips, placed on the bottom of 24-well plates and then infected with AI100-dsRed. The infected cells were washed three times with 1×PBS at 3 h.p.i., and fresh medium supplemented with 20 µg/ml fluconazole was added to each well containing infected host cells. The infected cells were then continuously incubated at 28°C (S2 cells) or 37°C, 5% CO<sub>2</sub> (J774.A1 cells). At the indicated time points, the infected cells were fixed and stained for analysis by immunofluorescence microscopy using antibodies directed against the indicated proteins. In Panels <b>A</b> and <b>B</b>, the localizations of the early endosome marker EEA1 (1), late endosome marker M6PR (4), lysosome marker cathepsin D (7), endoplasmic reticulum (ER) marker calreticulin (10) and Golgi marker Grasp65 (13) in host cells are shown in green. Internalized or replicative Cn cells are shown in red (2, 5, 8, 11, 14). Cn cells in vacuoles that are surrounded by EEA1 (3), decorated with antibodies directed against the late endosome marker M6PR (6) or the lysosome marker cathepsin D (9), or surrounded by calreticulin (12) are shown in the merged images (white arrows). Tight association between Cn and Grasp65 was not observed at the indicated time point (15). Representative images from a single representative experiment are shown. At least three independent experiments were performed and each experiment gave similar results. Scale bars indicate 5 µM.</p>", "links"=>[], "tags"=>["intracellular", "trafficking"], "article_id"=>435488, "categories"=>["Infectious Diseases"], "users"=>["Qing-Ming Qin", "Jijing Luo", "Xiaorong Lin", "Jianwu Pei", "Lei Li", "Thomas A. Ficht", "Paul de Figueiredo"], "doi"=>"https://dx.doi.org/10.1371/journal.ppat.1002078.g003", "stats"=>{"downloads"=>0, "page_views"=>2, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Cryptococcus_neoformans_intracellular_trafficking_in_Drosophila_S2_/435488", "title"=>"<i>Cryptococcus neoformans</i> intracellular trafficking in <i>Drosophila</i> S2.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2011-06-16 01:31:28"}
  • {"files"=>["https://ndownloader.figshare.com/files/765867"], "description"=>"<p>Host cell phagocytosis of Cn cells is class III PI3-kinase (C3_PI3K) and actin-dependent. This process is blocked by the PI3-kinase inhibitors 3-MA, LY294002 and the actin polymerization inhibitor cytochalasin D (CytD) (1). Internalized Cn resides in a Cn containing vacuole (CnCv). Early CnCvs interact with components of the endocytic pathway and sequentially obtain early endosome (EEA1), late endosome (M6PR) and lysosome (LAMP-1 and cathepsin D) markers (2). Infection by Cn may also activate the host cell autophagy pathway. Atg proteins such as Atg5, Atg9a and LC3 are recruited to the vicinity of CnCVs. Activated host cell Atg proteins promote the maturation of early CnCVs to form intermediate CnCVs (2). These compartments support Cn replication (3). Finally, late CnCVs may fuse with the plasma membrane, via an undefined mechanism, which results in the escape of replicative Cn cells from infected host cells (4). Extrusion in the absence of Cn replication and/or CnCv maturation may also occur (5).</p>", "links"=>[], "tags"=>["depicting", "cn", "subversion", "atg", "proteins", "intracellular", "replication"], "article_id"=>436228, "categories"=>["Infectious Diseases"], "users"=>["Qing-Ming Qin", "Jijing Luo", "Xiaorong Lin", "Jianwu Pei", "Lei Li", "Thomas A. Ficht", "Paul de Figueiredo"], "doi"=>"https://dx.doi.org/10.1371/journal.ppat.1002078.g009", "stats"=>{"downloads"=>1, "page_views"=>5, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Proposed_model_depicting_Cn_subversion_of_Atg_proteins_for_intracellular_replication_and_dissemination_/436228", "title"=>"Proposed model depicting Cn subversion of Atg proteins for intracellular replication and dissemination.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2011-06-16 01:43:48"}
  • {"files"=>["https://ndownloader.figshare.com/files/765634"], "description"=>"<p>Murine RAW264.7 macrophages were transfected with the indicated siRNAs (10 nM). Two days post-transfection, the host cells were infected with Cn (H99) for 3 hrs. The infected cells were extensively washed to remove the extracellular population of Cn cells, and fresh medium supplemented with 20 µg/ml fluconazole was added. At the indicated time points post infection, the supernatant and lysate from infected cells were collected separately. The materials were then plated, and the number of CFUs per well was determined. The resultant CFUs were used to calculate the relative phagocytosis (<b>A</b>), Cn escape (<b>B</b>) and intracellular replication (<b>C</b>) as described in the <a href=\"http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002078#s4\" target=\"_blank\"><i>Materials and Methods</i></a>. Data represent the means ± SD from at least four independent experiments with triplicate wells examined for each treatment. *, **, *** indicates significance at p<0.05, 0.01 and 0.001, respectively.</p>", "links"=>[], "tags"=>["atg", "proteins", "mediate"], "article_id"=>436001, "categories"=>["Infectious Diseases"], "users"=>["Qing-Ming Qin", "Jijing Luo", "Xiaorong Lin", "Jianwu Pei", "Lei Li", "Thomas A. Ficht", "Paul de Figueiredo"], "doi"=>"https://dx.doi.org/10.1371/journal.ppat.1002078.g007", "stats"=>{"downloads"=>1, "page_views"=>6, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Host_Atg_proteins_mediate_Cryptococcus_neoformans_infection_of_host_cells_/436001", "title"=>"Host Atg proteins mediate <i>Cryptococcus neoformans</i> infection of host cells.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2011-06-16 01:40:01"}

PMC Usage Stats | Further Information

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Relative Metric

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