title: Retrograde transport is not required for cytosolic translocation of the B-subunit of Shiga toxin.
abstract: Antigen-presenting cells have the remarkable capacity to transfer exogenous antigens to the cytosol for processing by proteasomes and subsequent presentation on major histocompatibility complex class-I (MHC-I) molecules, a process termed cross-presentation. This is the target of biomedical approaches that aim to trigger a therapeutic immune response. The receptor-binding B-subunit of Shiga toxin (STxB) has been developed as an antigen delivery tool for such immunotherapy applications. In this study, we have analyzed pathways and trafficking factors that are involved in this process. A covalent conjugate between STxB and saporin was generated to quantitatively sample the membrane translocation step to the cytosol in differentiated monocyte-derived THP-1 cells. We have found that retrograde trafficking to the Golgi complex was not required for STxB-saporin translocation to the cytosol or for STxB-dependent antigen cross-presentation. Depletion of endosomal Rab7 inhibited, and lowering membrane cholesterol levels favored STxB-saporin translocation. Interestingly, experiments with reducible and non-reducible linker-arm-STxB conjugates led to the conclusion that after translocation, STxB remains associated with the cytosolic membrane leaflet. In summary, we report new facets of the endosomal escape process bearing relevance to antigen cross-presentation.
authors.affiliation: Institut Curie, PSL Research University, Endocytic Trafficking and Therapeutic Delivery Group, 26 rue d'Ulm, Paris Cedex 05 75248, France CNRS UMR3666, Paris 75005, France INSERM U1143, Paris 75005, France.
authors.forename: Maria Daniela
authors.initials: MD
authors.lastname: Garcia-Castillo
authors.affiliation: INSERM U970, PARCC Université Paris Descartes Sorbonne Paris Cité, Paris 75006, France Hôpital Européen Georges-Pompidou, AP-HP, Service d'Immunologie Biologique, Paris Cedex 15 75908, France.
authors.forename: Thi
authors.initials: T
authors.lastname: Tran
authors.affiliation: Dynamique des Interactions Hôte Pathogène, Institut Pasteur, Paris Cedex 15 75724, France.
authors.forename: Alexandre
authors.initials: A
authors.lastname: Bobard
authors.affiliation: Institut Curie, PSL Research University, Endocytic Trafficking and Therapeutic Delivery Group, 26 rue d'Ulm, Paris Cedex 05 75248, France CNRS UMR3666, Paris 75005, France INSERM U1143, Paris 75005, France.
authors.forename: Henri-François
authors.initials: HF
authors.lastname: Renard
authors.affiliation: Institut Curie, PSL Research University, Endocytic Trafficking and Therapeutic Delivery Group, 26 rue d'Ulm, Paris Cedex 05 75248, France CNRS UMR3666, Paris 75005, France INSERM U1143, Paris 75005, France.
authors.forename: Stefan J
authors.initials: SJ
authors.lastname: Rathjen
authors.affiliation: Institut Curie, PSL Research University, Endocytic Trafficking and Therapeutic Delivery Group, 26 rue d'Ulm, Paris Cedex 05 75248, France CNRS UMR3666, Paris 75005, France INSERM U1143, Paris 75005, France.
authors.forename: Estelle
authors.initials: E
authors.lastname: Dransart
authors.affiliation: Institut Curie, PSL Research University, Endocytic Trafficking and Therapeutic Delivery Group, 26 rue d'Ulm, Paris Cedex 05 75248, France CNRS UMR3666, Paris 75005, France INSERM U1143, Paris 75005, France.
authors.forename: Bahne
authors.initials: B
authors.lastname: Stechmann
authors.affiliation: CNRS UMR3666, Paris 75005, France INSERM U1143, Paris 75005, France Institut Curie - Centre de Recherche, Membrane Dynamics and Mechanics of Intracellular Signaling Group, 26 rue d'Ulm, Paris Cedex 05 75248, France.
authors.forename: Christophe
authors.initials: C
authors.lastname: Lamaze
authors.affiliation: Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, UK.
authors.forename: Mike
authors.initials: M
authors.lastname: Lord
authors.affiliation: CEA, iBiTec-S/SCBM, CEA-Saclay, LabEx LERMIT, Gif-sur-Yvette F-91191, France.
authors.forename: Jean-Christophe
authors.initials: JC
authors.lastname: Cintrat
authors.affiliation: Dynamique des Interactions Hôte Pathogène, Institut Pasteur, Paris Cedex 15 75724, France.
authors.forename: Jost
authors.initials: J
authors.lastname: Enninga
authors.affiliation: INSERM U970, PARCC Université Paris Descartes Sorbonne Paris Cité, Paris 75006, France Hôpital Européen Georges-Pompidou, AP-HP, Service d'Immunologie Biologique, Paris Cedex 15 75908, France.
authors.forename: Eric
authors.initials: E
authors.lastname: Tartour
authors.affiliation: Institut Curie, PSL Research University, Endocytic Trafficking and Therapeutic Delivery Group, 26 rue d'Ulm, Paris Cedex 05 75248, France CNRS UMR3666, Paris 75005, France INSERM U1143, Paris 75005, France ludger.johannes@curie.fr.
authors.forename: Ludger
authors.initials: L
authors.lastname: Johannes
mesh_terms: D001692:Biological Transport; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology; D002451:Cell Compartmentation; D003587:Cytomegalovirus / Q000502:physiology; D003600:Cytosol / Q000378:metabolism*; D004705:Endocytosis; D011992:Endosomes / Q000378:metabolism; D000939:Epitopes / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D006367:HeLa Cells; D006801:Humans; D014176:Protein Biosynthesis; D054789:Ribosome Inactivating Proteins, Type 1 / Q000378:metabolism; D000076985:Saporins; D022621:Shiga Toxin / Q000378:metabolism*; D020691:rab GTP-Binding Proteins / Q000378:metabolism; D000090802:rab7 GTP-Binding Proteins
chemical_list: D000939:Epitopes; D054789:Ribosome Inactivating Proteins, Type 1; D000090802:rab7 GTP-Binding Proteins; C000716463:rab7 GTP-binding proteins, human; D022621:Shiga Toxin; D000076985:Saporins; D020691:rab GTP-Binding Proteins
keywords: Antigen cross-presentation; Bafilomycin A1; Brefeldin-A; Cancer; Cholesterol; Cytotoxic T-lymphocyte; Dendritic cell; Endoplasmic reticulum; Endosomal escape; Endosome; Golgi; Immunotherapy; Infectious disease; Lactamase; Methyl-beta-cyclodextrin; Microdomain; Nanodomain; PPMP; Rab5; Rab6; Rab7; Raft; Retro compound; Sec22B; Shiga toxin