Retrograde transport is not required for cytosolic translocation of the B-subunit of Shiga toxin

Maria Daniela Garcia-Castillo (1, 2) , Thi Tran (3, 4, 5, 6, 7) , Alexandre Bobard (8) , Henri-François Renard (1, 2) , Stefan Rathjen (1, 2) , Estelle Dransart (1, 2) , Bahne Stechmann (1, 2) , Christophe Lamaze (1, 2) , Mike Lord (9) , Jean-Christophe Cintrat (10) , Jost Enninga (8) , Eric Tartour (3, 4, 5, 6, 7) , Ludger Johannes (1, 2)

1 CBMCT - UMR 3666 / U1143 - Chimie biologique des membranes et ciblage thérapeutique
2 Institut Curie [Paris]
3 PARCC - UMR-S U970 - Paris-Centre de Recherche Cardiovasculaire
4 HEGP - Hôpital Européen Georges Pompidou [APHP]
5 UPD5 - Université Paris Descartes - Paris 5
6 USPC - Université Sorbonne Paris Cité
7 AP-HP - Assistance publique - Hôpitaux de Paris (AP-HP)
8 Dynamique des Interactions Hôte-Pathogène - Dynamics of Host-Pathogen Interactions
9 University of Warwick [Coventry]
10 SCBM - Service de Chimie Bio-Organique et de Marquage

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.

Keywords: Endosome; Golgi; Immunotherapy; Infectious disease; Lactamase; Methyl-beta-cyclodextrin; Microdomain; Nanodomain; PPMP; Rab5; Rab6; Rab7; Raft; Retro compound; Sec22B; Shiga toxin; Bafilomycin A1; Brefeldin-A; Cancer; Cholesterol; Cytotoxic T-lymphocyte; Dendritic cell; Endoplasmic reticulum; Antigen cross-presentation; Endosomal escape

Domains: Life Sciences [q-bio]; Biochemistry, Molecular Biology; Biochemistry [q-bio.BM]; Life Sciences [q-bio]; Cellular Biology; Cell Behavior [q-bio.CB]

Maria Daniela Garcia-Castillo, Thi Tran, Alexandre Bobard, Henri-François Renard, Stefan Rathjen, et al.. Retrograde transport is not required for cytosolic translocation of the B-subunit of Shiga toxin. Journal of Cell Science, 2015, 128 (13), pp.2373 - 2387. ⟨10.1242/jcs.169383⟩. ⟨pasteur-01899449⟩