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      Mutations in EEA1 are associated with allergic bronchopulmonary aspergillosis and affect phagocytosis of Aspergillus fumigatus by human macrophages

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          Abstract

          Allergic bronchopulmonary aspergillosis (ABPA) in asthma is a severe, life-affecting disease that potentially affects over 4.8 million people globally. In the UK, ABPA is predominantly caused by the fungus Aspergillus fumigatus. Phagocytosis is important in clearance of this fungus, and Early Endosome Antigen 1 ( EEA1) has been demonstrated to be involved in phagocytosis of fungi. We sought to investigate the role of EEA1 mutations and phagocytosis in ABPA. We used exome sequencing to identify variants in EEA1 associated with ABPA. We then cultured monocyte-derived macrophages (MDMs) from 17 ABPA subjects with A. fumigatus conidia, and analyzed phagocytosis and phagolysosome acidification in relation to the presence of these variants. We found that variants in EEA1 were associated with ABPA and with the rate of phagocytosis of A. fumigatus conidia and the acidification of phagolysosomes. MDMs from ABPA subjects carrying the disease associated genotype showed increased acidification and phagocytosis compared to those from ABPA subjects carrying the non-associated genotypes or healthy controls.The identification of ABPA-associated variants in EEA that have functional effects on MDM phagocytosis and phagolysosome acidification of A. fumigatus conidia revolutionizes our understanding of susceptibility to this disease, which may in future benefit patients by earlier identification or improved treatments. We suggest that the increased phagocytosis and acidification observed demonstrates an over-active MDM profile in these patients, resulting in an exaggerated cellular response to the presence of A. fumigatus in the airways.

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          Most cited references32

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          A statistical framework for SNP calling, mutation discovery, association mapping and population genetical parameter estimation from sequencing data.

          Heng Li (2011)
          Most existing methods for DNA sequence analysis rely on accurate sequences or genotypes. However, in applications of the next-generation sequencing (NGS), accurate genotypes may not be easily obtained (e.g. multi-sample low-coverage sequencing or somatic mutation discovery). These applications press for the development of new methods for analyzing sequence data with uncertainty. We present a statistical framework for calling SNPs, discovering somatic mutations, inferring population genetical parameters and performing association tests directly based on sequencing data without explicit genotyping or linkage-based imputation. On real data, we demonstrate that our method achieves comparable accuracy to alternative methods for estimating site allele count, for inferring allele frequency spectrum and for association mapping. We also highlight the necessity of using symmetric datasets for finding somatic mutations and confirm that for discovering rare events, mismapping is frequently the leading source of errors. http://samtools.sourceforge.net. hengli@broadinstitute.org.
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            Aspergillus fumigatus and aspergillosis.

            J P Latgé (1999)
            Aspergillus fumigatus is one of the most ubiquitous of the airborne saprophytic fungi. Humans and animals constantly inhale numerous conidia of this fungus. The conidia are normally eliminated in the immunocompetent host by innate immune mechanisms, and aspergilloma and allergic bronchopulmonary aspergillosis, uncommon clinical syndromes, are the only infections observed in such hosts. Thus, A. fumigatus was considered for years to be a weak pathogen. With increases in the number of immunosuppressed patients, however, there has been a dramatic increase in severe and usually fatal invasive aspergillosis, now the most common mold infection worldwide. In this review, the focus is on the biology of A. fumigatus and the diseases it causes. Included are discussions of (i) genomic and molecular characterization of the organism, (ii) clinical and laboratory methods available for the diagnosis of aspergillosis in immunocompetent and immunocompromised hosts, (iii) identification of host and fungal factors that play a role in the establishment of the fungus in vivo, and (iv) problems associated with antifungal therapy.
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              EEA1 links PI(3)K function to Rab5 regulation of endosome fusion.

              GTPases and lipid kinases regulate membrane traffic along the endocytic pathway by mechanisms that are not completely understood. Fusion between early endosomes requires phosphatidylinositol-3-OH kinase (PI(3)K) activity as well as the small GTPase Rab5. Excess Rab5-GTP complex restores endosome fusion when PI(3)K is inhibited. Here we identify the early-endosomal autoantigen EEA1 which binds the PI(3)K product phosphatidylinositol-3-phosphate, as a new Rab5 effector that is required for endosome fusion. The association of EEA1 with the endosomal membrane requires Rab5-GTP and PI(3)K activity, and excess Rab5-GTP stabilizes the membrane association of EEA1 even when PI(3)K is inhibited. The identification of EEA1 as a direct Rab5 effector provides a molecular link between PI(3)K and Rab5, and its restricted distribution to early endosomes indicates that EEA1 may confer directionality to Rab5-dependent endocytic transport.
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                Author and article information

                Contributors
                Role: ConceptualizationRole: Formal analysisRole: Funding acquisitionRole: InvestigationRole: MethodologyRole: Writing – original draftRole: Writing – review & editing
                Role: MethodologyRole: Writing – review & editing
                Role: MethodologyRole: Writing – review & editing
                Role: ConceptualizationRole: Funding acquisitionRole: SupervisionRole: Writing – review & editing
                Role: ConceptualizationRole: Funding acquisitionRole: SupervisionRole: Writing – review & editing
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                16 March 2018
                2018
                : 13
                : 3
                : e0185706
                Affiliations
                [1 ] Manchester Fungal Infection Group (MFIG), Division of Infection, Immunity and Respiratory Medicine, School of Biological Sciences, Faculty of Biology, Medicine and Health, University of Manchester, Manchester Academic Health Science Centre, Manchester, United Kingdom
                [2 ] Department of Molecular and Applied Microbiology, Leibniz Institute for Natural Product Research and Infection Biology (HKI), Jena, Germany
                [3 ] Department of Microbiology and Molecular Biology, Institute of Microbiology, Friedrich Schiller University, Jena, Germany
                [4 ] The National Aspergillosis Centre, University Hospital of South Manchester NHS Foundation Trust, Manchester Academic Health Science Centre, Manchester, United Kingdom
                Woosuk University, REPUBLIC OF KOREA
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                [¤a]

                Current address: Clinical & Experimental Pharmacology Group, CRUK Manchester Institute, University of Manchester, Manchester, United Kingdom

                [¤b]

                Current address: BASF SE, Ludwigshafen, Ludwigshafen, Germany

                Author information
                http://orcid.org/0000-0003-0962-0268
                http://orcid.org/0000-0001-5626-2251
                http://orcid.org/0000-0002-1083-9286
                Article
                PONE-D-17-10371
                10.1371/journal.pone.0185706
                5856258
                29547649
                22cf59e1-6a82-4d68-9365-2041d6bfbfef
                © 2018 Overton 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.

                History
                : 20 March 2017
                : 18 September 2017
                Page count
                Figures: 4, Tables: 1, Pages: 15
                Funding
                This report is independent research supported by the Fungal Infection Trust ( www.fungalinfectiontrust.org/) (NLDO) and the National Institute for Health Research South Manchester Respiratory and Allergy Clinical Research Facility at University Hospital of South Manchester NHS Foundation Trust ( www.uhsm.nhs.uk/about/research/respiratory-hub/) (PB, DWD). PB was supported by the Medical Research Council ( www.mrc.ac.uk/) (PB, grant MR/M02010X/1). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The views expressed in this publication are those of the authors and not necessarily those of the NHS, the National Institute for Health Research or the Department of Health.
                Categories
                Research Article
                Biology and Life Sciences
                Cell Biology
                Cell Processes
                Phagocytosis
                Biology and Life Sciences
                Organisms
                Eukaryota
                Fungi
                Molds (Fungi)
                Aspergillus
                Aspergillus Fumigatus
                Biology and Life Sciences
                Microbiology
                Medical Microbiology
                Microbial Pathogens
                Fungal Pathogens
                Aspergillus Fumigatus
                Medicine and Health Sciences
                Pathology and Laboratory Medicine
                Pathogens
                Microbial Pathogens
                Fungal Pathogens
                Aspergillus Fumigatus
                Biology and Life Sciences
                Mycology
                Fungal Pathogens
                Aspergillus Fumigatus
                Biology and life sciences
                Genetics
                Gene expression
                Gene regulation
                Small interfering RNAs
                Biology and life sciences
                Biochemistry
                Nucleic acids
                RNA
                Non-coding RNA
                Small interfering RNAs
                Biology and Life Sciences
                Cell Biology
                Cellular Types
                Animal Cells
                Blood Cells
                White Blood Cells
                Macrophages
                Biology and Life Sciences
                Cell Biology
                Cellular Types
                Animal Cells
                Immune Cells
                White Blood Cells
                Macrophages
                Biology and Life Sciences
                Immunology
                Immune Cells
                White Blood Cells
                Macrophages
                Medicine and Health Sciences
                Immunology
                Immune Cells
                White Blood Cells
                Macrophages
                Medicine and Health Sciences
                Pulmonology
                Asthma
                Biology and Life Sciences
                Organisms
                Eukaryota
                Fungi
                Biology and Life Sciences
                Cell Biology
                Cellular Structures and Organelles
                Vesicles
                Endosomes
                Biology and Life Sciences
                Genetics
                Fungal Genetics
                Biology and Life Sciences
                Mycology
                Fungal Genetics
                Custom metadata
                The minimal underlying data set necessary for replication of the study are included in the paper and its Supporting Information files.

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