Fig. 2: Reprogramming PVCs for injection of human cells.
From: Programmable protein delivery with a bacterial contractile injection system
a, AlphaFold-guided engineering of Pvc13 (tail fibre) results in PVCs exhibiting efficient delivery activity in human cells. Top, the putative target recognition ___domain of Pvc13 (amino acids 403–476) was deleted (producing Pvc13 (truncated)) and replaced with either the target-binding ___domain from human adenovirus 5 (producing Pvc13–Ad5-knob) or a DARPin specific for human EGFR (producing Pvc13–E01-DARPin). Pvc13 variants containing non-targeting binding domains (Pvc13–Ad5-knob(Δ491/492) and Pvc13–A4-DARPin) were also included as negative controls. Bottom, PVC particles were loaded with wild-type (WT) toxin payloads or with a novel payload (Cre), and PVC-driven protein delivery was measured as cytotoxicity or GFP expression, respectively. Bottom row, novel PVC designs can be observed binding to human cells (in this case U2OS to facilitate immunofluorescence imaging). Binding of target cells required the presence of Pvc13–Ad5-knob or Pvc13–E01-DARPin; PVCs harbouring wild-type Pvc13, truncated Pvc13 or a non-targeting fusion protein did not cluster on the cell surface. Scale bars, 300 μm. b, Human-targeting PVC designs can deliver SpCas9 protein into HEK 293FT cells, enabling PVC-mediated gene editing in human cells. Indel formation required the presence of an unmutated Ad5 knob ___domain in Pvc13, indicating that this activity required the action of the PVC. Conditions are listed in the format PVC(Pvc13 design)–payload. c, Human-targeting PVC designs can deliver ZFDs into HEK 293FT cells, enabling base editing in human cells. On-target G-to-A base substitution was observed only when each ZFD half (ZFD-L and ZFD-R) was delivered by a properly targeted PVC; non-targeting PVCs produced negligible activity. d, Human-targeting PVC designs can kill leukaemia cells (Jurkat). Cytotoxicity was produced only when PVCs were retargeted towards a T cell receptor known to be expressed by Jurkat cells (CD4), but not when retargeted towards a myeloid receptor not found on Jurkat cells (CD11b). Data are mean ± s.d. with n = 3 biological replicates; one-way ANOVA with Bonferroni post hoc test.
