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. Author manuscript; available in PMC: 2013 Jul 1.
Published in final edited form as: Nat Commun. 2013;4:1590. doi: 10.1038/ncomms2594

Fig. 3. T3SS-dependent antigen delivery by minicellsin-vitro.

Fig. 3

(a) Schematic of the SopE-OVA construct used in these studies. (b) Western blot analysis of minicells obtained from wild type or T3SS-defective (ΔinvA) S. Typhimurium strains expressing the SopE-OVA construct and used in the experiment shown in (c). When indicated, the SopE-OVA construct was co-expressed with the SopE chaperone InvB and the T3SS protein translocases and their chaperones to improve protein secretion and/or translocation. Equal amount of total protein was loaded in each sample. (c) Analysis of antigen delivery by minicells to antigen-presenting cells. RMA cells (C57BL/6 mouse hybridomas) were pulsed for 3 hs with minicells isolated from wild type or T3SS-defective (ΔinvA) S. Typhimurium strains. After pulsing, RMA cells were fixed, and used as APCs in a B3Z T-cell activation assay as described in experimental procedures. Values represent the levels of antigen presentation based on the β-galactosidase activity detected in the B3Z-T cell hybridoma reporter and are normalized relative to the values of the OVA peptide positive control, which was considered 100 %. The values are the mean ± standard deviation of three independent experiments. (d and e) Minicells can deliver antigen to dendritic cells ex vivo. (d) Western blot analysis of minicells obtained from wild type or T3SS-defective (ΔinvA) S. Typhimurium strains expressing the SopE-OVA construct and used in the experiment shown in (e). Equal amount of total protein was loaded in each sample. (e) Bone marrow-derived dendritic cells were pulsed for 3 hs with minicells isolated from the indicated S. Typhimurium strains carrying a plasmid expressing SopE-OVA and the indicated SPI-1 T3SS-associated proteins. After pulsing, dendritic cells were fixed and used as APCs in a B3Z T-cell activation assay as described in Supplementary Materials. Values represent the levels of antigen presentation based on the β-galactosidase activity detected in the B3Z-T cell hybridoma reporter and they are normalized relative to the values of the OVA peptide positive control, which was considered 100 %. The values are the mean ± standard deviation of three independent experiments.