title: Phenotypes Associated with Knockouts of Eight Dense Granule Gene Loci (GRA2-9) in Virulent Toxoplasma gondii.
abstract: Toxoplasma gondii actively invades host cells and establishes a parasitophorous vacuole (PV) that accumulates many proteins secreted by the dense granules (GRA proteins). To date, at least 23 GRA proteins have been reported, though the function(s) of most of these proteins still remains unknown. We targeted gene knockouts at ten GRA gene loci (GRA1-10) to investigate the cellular roles and essentiality of these classical GRA proteins during acute infection in the virulent type I RH strain. While eight of these genes (GRA2-9) were successfully knocked out, targeted knockouts at the GRA1 and GRA10 loci were not obtained, suggesting these GRA proteins may be essential. As expected, the Δgra2 and Δgra6 knockouts failed to form an intravacuolar network (IVN). Surprisingly, Δgra7 exhibited hyper-formation of the IVN in both normal and lipid-free growth conditions. No morphological alterations were identified in parasite or PV structures in the Δgra3, Δgra4, Δgra5, Δgra8, or Δgra9 knockouts. With the exception of the Δgra3 and Δgra8 knockouts, all of the GRA knockouts exhibited defects in their infection rate in vitro. While the single GRA knockouts did not exhibit reduced replication rates in vitro, replication rate defects were observed in three double GRA knockout strains (Δgra4Δgra6, Δgra3Δgra5 and Δgra3Δgra7). However, the virulence of single or double GRA knockout strains in CD1 mice was not affected. Collectively, our results suggest that while the eight individual GRA proteins investigated in this study (GRA2-9) are not essential, several GRA proteins may provide redundant and potentially important functions during acute infection.
authors.affiliation: Department of Microbiology and Immunology, The Geisel School of Medicine at Dartmouth, Lebanon, NH, United States of America.
authors.forename: Leah M
authors.identifier: http://orcid.org/0000-0003-3834-4549
authors.initials: LM
authors.lastname: Rommereim
authors.affiliation: Laboratoire Adaptation et Pathogénie des Micro-organismes, Université Grenoble Alpes, Université Joseph Fourier, Grenoble, France.|Centre National de la Recherche Scientifique, Unité Mixte de Recherche 5163, Grenoble, France.
authors.forename: Valeria
authors.initials: V
authors.lastname: Bellini
authors.affiliation: Department of Microbiology and Immunology, The Geisel School of Medicine at Dartmouth, Lebanon, NH, United States of America.
authors.forename: Barbara A
authors.initials: BA
authors.lastname: Fox
authors.affiliation: Laboratoire Adaptation et Pathogénie des Micro-organismes, Université Grenoble Alpes, Université Joseph Fourier, Grenoble, France.|Centre National de la Recherche Scientifique, Unité Mixte de Recherche 5163, Grenoble, France.
authors.forename: Graciane
authors.initials: G
authors.lastname: Pètre
authors.affiliation: Laboratoire Adaptation et Pathogénie des Micro-organismes, Université Grenoble Alpes, Université Joseph Fourier, Grenoble, France.|Centre National de la Recherche Scientifique, Unité Mixte de Recherche 5163, Grenoble, France.
authors.forename: Camille
authors.initials: C
authors.lastname: Rak
authors.affiliation: Laboratoire Adaptation et Pathogénie des Micro-organismes, Université Grenoble Alpes, Université Joseph Fourier, Grenoble, France.|Centre National de la Recherche Scientifique, Unité Mixte de Recherche 5163, Grenoble, France.|Station de Cytométrie en Images en Microbiologie (SCIMI platform), Grenoble, France.
authors.forename: Bastien
authors.initials: B
authors.lastname: Touquet
authors.affiliation: Laboratoire Adaptation et Pathogénie des Micro-organismes, Université Grenoble Alpes, Université Joseph Fourier, Grenoble, France.|Centre National de la Recherche Scientifique, Unité Mixte de Recherche 5163, Grenoble, France.
authors.forename: Delphine
authors.initials: D
authors.lastname: Aldebert
authors.affiliation: Université Montpellier 2, Place Eugène Bataillon, Montpellier, France.|Centre National de al Recherché Scientifique, Unité Mixte de Recherché 5235, Montpellier, France.
authors.forename: Jean-François
authors.initials: JF
authors.lastname: Dubremetz
authors.affiliation: Laboratoire Adaptation et Pathogénie des Micro-organismes, Université Grenoble Alpes, Université Joseph Fourier, Grenoble, France.|Centre National de la Recherche Scientifique, Unité Mixte de Recherche 5163, Grenoble, France.
authors.forename: Marie-France
authors.initials: MF
authors.lastname: Cesbron-Delauw
authors.affiliation: Laboratoire Adaptation et Pathogénie des Micro-organismes, Université Grenoble Alpes, Université Joseph Fourier, Grenoble, France.|Centre National de la Recherche Scientifique, Unité Mixte de Recherche 5163, Grenoble, France.
authors.forename: Corinne
authors.initials: C
authors.lastname: Mercier
authors.affiliation: Department of Microbiology and Immunology, The Geisel School of Medicine at Dartmouth, Lebanon, NH, United States of America.
authors.forename: David J
authors.initials: DJ
authors.lastname: Bzik
mesh_terms: D000818:Animals; D017353:Gene Deletion; D055786:Gene Knockout Techniques; D023061:Gene Order; D018390:Gene Targeting; D006790:Host-Parasite Interactions; D051379:Mice; D010641:Phenotype; D010957:Plasmids / Q000235:genetics; D015800:Protozoan Proteins / Q000235:genetics*; D040641:Quantitative Trait Loci; D014122:Toxoplasma / Q000472:pathogenicity / Q000502:physiology* / Q000648:ultrastructure; D014123:Toxoplasmosis / Q000469:parasitology; D014774:Virulence / Q000235:genetics
chemical_list: D015800:Protozoan Proteins