Targeting Cattle-Borne Zoonoses and Cattle Pathogens Using a Novel Trypanosomatid-Based Delivery System
Distribution of expressed Bd37 assayed via its fusion to T. brucei BiP and detected using a BiP-specific antibody. Cells were fixed with paraformaldehyde and then either 0.1% Triton-X100 permeabilised, or not, to distinguish intracellular from surface exposed protein following immunofluorescence. In wild-type cells, only weak staining was observed (A–C) in the absence of permeabilisation whereas permeabilisation (D–F) allows visualisation of endogenous, intracellular T. theileri BiP protein, which cross-reacts with the T. brucei BiP antibody. In contrast, the N-BiP-Bd37 fusion protein generates intense staining at the flagellar-pocket in intact cells (G–I), plus an intense intracellular staining in permeabilised cells comprised of endogenous BiP and N-BiP-Bd37 protein in the ER (J–L). Expression of the N-BiP-Bd37-GPI fusion protein generates a strong signal on intact cells indicating the presence of cell-surface protein (M–O); permeabilised cells also show strong straining representing the combined endogenous BiP and the expressed fusion protein (P–R). Camera exposures with respect to the BiP fluorescence and image handling were precisely consistent between cells under each condition to allow comparison of the relative intensity of signal. DAPI images reveal the kinetoplast and nuclear position, these often overlying one another. Scale Bar = 10 µm.