Article Title: The structure of bacterial toxin CNFY reveals requirements for secretion, host cell recognition and endosomal release
Figure Lengend Snippet: Synthesis, secretion, host cell binding and effects of C-terminal deletion variants of CNF Y . A C-terminally 3xFlag-tagged CNF Y and different C-terminally deleted toxin variants were expressed in Y. pseudotuberculosis YP147 (Δ cnfY ) from plasmids under control of their own promoter and were detected in whole cell extracts using an anti-Flag antibody. B HEp-2 cells remained untreated or were incubated with full-length CNF Y or the C-terminally deleted toxin variants for 4 h. Cells were lysed and the deamidation of RhoA was analyzed by the mobility shift of the modified Rho GTPase on SDS PAGE. C HEp-2 cells were incubated with 500 nM full-length CNF Y or the C-terminal deleted toxin variants for 24 h. The cell nuclei were strained with DAPI (blue) and the actin cytoskeleton was stained using FITC-phalloidin (green). The formation of large, multinuclear cells was observed by fluorescence microscopy and the formation of actin stress fibers and membrane actin folding were only observed with full-length-CNF Y -treated cells. The white scale bar is 40 µm. Cells incubated with extracts of YP147 (Δ cnfY ) harboring the empty expression vector were used as negative controls. D HEp-2 cells were incubated with 500 nM full-length CNF Y or the C-terminally deleted toxin variants fused to beta-lactamase (TEM) for 4 h. Cleavage of the reporter dye CCF4-AM was used to visualize toxin delivery. After cell entry CCF4-AM is rapidly converted into the negatively charged form CCF4, which is retained in the cytosol and emits a green fluorescence signal (520 nm). In the presence of translocated beta-lactamase fusion proteins, CCF4-AM is cleaved, and disruption of FRET results in blue fluorescence (447 nm). White bar: 20 µm. E 3xFlag-tagged CNF1, CNF Y and C-terminally deleted toxin variants were added to HEp-2 cells for 4 h. The cells were thoroughly washed, pelleted, lysed and the toxin variants bound to the cells were identified by western blotting using an anti-Flag antibody. F To test secretion of the CNF Y variants, CNF Y and different C-terminally deleted variants fused to beta-lactamase (TEM) were expressed in Y. pseudotuberculosis YP147 (Δ cnfY ). Beta-lactamase activity in the culture supernatant was subsequently measured using nitrocefin as substrate.
Article Snippet: HEp-2 cells (ATCC CCL-23) were grown at 37°C, 5% CO2 in RPMI (Gibco) supplemented with 7.5% newborn calf serum (NCS; Sigma).
Techniques: Binding Assay, Incubation, Mobility Shift, Modification, SDS Page, Staining, Fluorescence, Microscopy, Expressing, Plasmid Preparation, Transmission Electron Microscopy, Western Blot, Activity Assay