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To maintain this intracellular niche, the parasite secretes an array of dense granule proteins (GRAs) into the nascent parasitophorous vacuole. These GRAs are believed to play key roles in vacuolar remodeling, nutrient uptake, and immune evasion while the parasite is replicating within the host cell. Despite the central role of GRAs in the\n                    <jats:italic>Toxoplasma<\/jats:italic>\n                    life cycle, only a subset of these proteins have been identified, and many of their roles have not been fully elucidated. In this report, we utilize the promiscuous biotin ligase BirA* to biotinylate GRA proteins secreted into the vacuole and then identify those proteins by affinity purification and mass spectrometry. Using GRA-BirA* fusion proteins as bait, we have identified a large number of known and candidate GRAs and verified localization of 13 novel GRA proteins by endogenous gene tagging. We proceeded to functionally characterize three related GRAs from this group (GRA38, GRA39, and GRA40) by gene knockout. While \u0394\n                    <jats:italic>gra38<\/jats:italic>\n                    and \u0394\n                    <jats:italic>gra40<\/jats:italic>\n                    parasites showed no altered phenotype, disruption of\n                    <jats:italic>GRA39<\/jats:italic>\n                    results in slow-growing parasites that contain striking lipid deposits in the parasitophorous vacuole, suggesting a role in lipid regulation that is important for parasite growth. In addition, parasites lacking\n                    <jats:italic>GRA39<\/jats:italic>\n                    showed dramatically reduced virulence and a lower tissue cyst burden\n                    <jats:italic>in vivo<\/jats:italic>\n                    . Together, the findings from this work reveal a partial vacuolar proteome of\n                    <jats:named-content content-type=\"genus-species\">T. gondii<\/jats:named-content>\n                    and identify a novel GRA that plays a key role in parasite replication and pathogenesis.\n                  <\/jats:p>\n                  <jats:p>\n                    <jats:bold>IMPORTANCE<\/jats:bold>\n                    Most intracellular pathogens reside inside a membrane-bound vacuole within their host cell that is extensively modified by the pathogen to optimize intracellular growth and avoid host defenses. In\n                    <jats:italic>Toxoplasma<\/jats:italic>\n                    , this vacuole is modified by a host of secretory GRA proteins, many of which remain unidentified. Here we demonstrate that\n                    <jats:italic>in vivo<\/jats:italic>\n                    biotinylation of proximal and interacting proteins using the promiscuous biotin ligase BirA* is a powerful approach to rapidly identify vacuolar GRA proteins. We further demonstrate that one factor identified by this approach, GRA39, plays an important role in the ability of the parasite to replicate within its host cell and cause disease.\n                  <\/jats:p>","DOI":"10.1128\/mbio.00808-16","type":"journal-article","created":{"date-parts":[[2016,8,2]],"date-time":"2016-08-02T21:32:59Z","timestamp":1470173579000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":153,"title":["<i>In Vivo<\/i>\n                    Biotinylation of the\n                    <i>Toxoplasma<\/i>\n                    Parasitophorous Vacuole Reveals Novel Dense Granule Proteins Important for Parasite Growth and Pathogenesis"],"prefix":"10.1128","volume":"7","author":[{"given":"Santhosh M.","family":"Nadipuram","sequence":"first","affiliation":[{"name":"Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, Los Angeles, California, USA"},{"name":"Department of Pediatrics, David Geffen School of Medicine at the University of California, Los Angeles, Los Angeles, California, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Elliot W.","family":"Kim","sequence":"additional","affiliation":[{"name":"Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, Los Angeles, California, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ajay A.","family":"Vashisht","sequence":"additional","affiliation":[{"name":"Department of Biological Chemistry and Institute of Genomics and Proteomics, University of California, Los Angeles, Los Angeles, California, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Andrew H.","family":"Lin","sequence":"additional","affiliation":[{"name":"Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, Los Angeles, California, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hannah N.","family":"Bell","sequence":"additional","affiliation":[{"name":"Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, Los Angeles, California, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Isabelle","family":"Coppens","sequence":"additional","affiliation":[{"name":"Department of Molecular Microbiology and Immunology, The Johns Hopkins University Bloomberg School of Public Health, Baltimore, Maryland, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"James A.","family":"Wohlschlegel","sequence":"additional","affiliation":[{"name":"Department of Biological Chemistry and Institute of Genomics and Proteomics, University of California, Los Angeles, Los Angeles, California, USA"},{"name":"Molecular Biology Institute, University of California Los Angeles, Los Angeles, California, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Peter J.","family":"Bradley","sequence":"additional","affiliation":[{"name":"Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, Los Angeles, California, USA"},{"name":"Molecular Biology Institute, University of California Los Angeles, Los Angeles, California, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"235","reference":[{"key":"e_1_3_2_2_2","doi-asserted-by":"publisher","DOI":"10.1079\/AHR2005100"},{"key":"e_1_3_2_3_2","doi-asserted-by":"publisher","DOI":"10.1016\/S0140-6736(04)16412-X"},{"key":"e_1_3_2_4_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.ijpara.2008.03.007"},{"key":"e_1_3_2_5_2","first-page":"114","article-title":"Sequential protein secretion from three distinct organelles of Toxoplasma gondii accompanies invasion of human fibroblasts","volume":"73","author":"Carruthers VB","year":"1997","unstructured":"Carruthers VB, Sibley LD. 1997. 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