Extended Data Fig. 6: High-resolution imaging of PVC-mediated binding and killing of human cells.
From: Programmable protein delivery with a bacterial contractile injection system
a, A549 cells exhibit F-actin condensation after exposure to EGFR-specific PVCs (harbouring Pvc13–E01-DARPin), indicating the endogenous PVC toxin payloads (which are known to target the actin cytoskeleton24) were successfully delivered. Scale bars, 100 μm (upper row) and 20 μm (lower row). b, SEM images of A549 cells treated with the same PVC designs as in (a). Scale bars, 100 μm (upper row) and 20 μm (lower row). c, EGFR-specific PVCs can be directly visualized binding to A549 cells. Scale bars, 1 μm (left panels) and 100 nm (right panels). d, Contracted PVCs are visible on the cell surface, suggesting PVC-mediated protein delivery in human cells is mediated by sheath contraction. Scale bar, 100 nm. e, Locations of additional nonbinding mutations within the AlphaFold predicted structure of Pvc13–Ad5-knob. We screened two additional mutants—S408E and L485K—each containing single amino acid substitutions shown previously to inhibit the binding of Ad5 to target cells51. f, New Ad5 knob mutants are unable to retarget PVCs toward HEK 293FT cells. PVCs equipped with Pvc13–Ad5-knob (S408E or L485K) were loaded with Cre and administered to HEK 293FT cells harbouring a loxP-GFP plasmid (as in Fig. 2a); only PVCs equipped with tail fibres containing wild-type Ad5 knob domains produced efficient GFP expression. Values are mean ± s.d. with n = 3 biological replicates. Scale bar, 150 μm. g, Titration of PVCs harbouring Pvc13–Ad5-knob over HEK 293FT cells. Cells were exposed to a dilution series of Pvc13–Ad5-knob particles loaded with either ZFD-L or ZFD-R (the same particles as those in Fig. 2c) and PVC-mediated base substitution was measured with deep sequencing. PVC efficiency saturated at about 1,000 ng/µL. Values are mean with n = 2 biological replicates.
