Ligand-Displaying Cells and Minicells for Programmable Delivery of Toxic Payloads via Type IV Secretion Systems.

Unlabelled: Bacterial type IV secretion systems (T4SSs) are highly versatile macromolecular translocators and offer great potential for deployment as delivery systems for therapeutic intervention. One major T4SS subfamily, the conjugation machines, are well-adapted for delivery of DNA cargoes of interest to other bacteria or eukaryotic cells, but generally exhibit modest transfer frequencies and lack specificity for target cells. Here, we tested the efficacy of a surface-displayed nanobody/antigen (Nb/Ag) pairing system to enhance the conjugative transfer of IncN (pKM101), IncF (F/pOX38), or IncP (RP4) plasmids, or of mobilizable plasmids including those encoding CRISPR/Cas9 systems (pCrispr), to targeted recipient cells. donors displaying Nb's transferred plasmids to and recipients displaying the cognate Ag's at significantly higher frequencies than to recipients lacking Ag's. Nb/Ag pairing functionally substituted for the surface adhesin activities of F-encoded TraN and pKM101-encoded Pep, although not conjugative pili or VirB5-like adhesins. Nb/Ag pairing further elevated the killing effects accompanying delivery of pCrispr plasmids to and transconjugants bearing CRISPR/Cas9 target sequences. Finally, we determined that anucleate minicells, which are clinically safer delivery vectors than intact cells, transferred self-transmissible and mobilizable plasmids to and cells. Minicell-mediated mobilization of pCrispr plasmids to recipients elicited significant killing of transconjugants, although Nb/Ag pairing did not enhance conjugation frequencies or killing. Together, our findings establish the potential for deployment of bacteria or minicells as Programmed Delivery Systems (PDSs) for suppression of targeted bacterial species in infection settings.

Importance: The rapid emergence of drug-resistant bacteria and current low rate of antibiotic discovery emphasize an urgent need for alternative antibacterial strategies. We engineered to conjugatively transfer plasmids to specific and recipient cells through surface display of cognate nanobody/antigen (Nb/Ag) pairs. We further engineered mobilizable plasmids to carry CRISPR/Cas9 systems (pCrispr) for selective killing of recipient cells harboring CRISPR/Cas9 target sequences. In the assembled Programmed Delivery System (PDS), Nb-displaying donors with different conjugation systems and mobilizable pCrispr plasmids suppressed growth of Ag-displaying recipient cells to significantly greater extents than unpaired recipients. We also showed that anucleate minicells armed with conjugation machines and pCrispr plasmids were highly effective in killing of recipients. Together, our findings suggest that bacteria or minicells armed with PDSs may prove highly effective as an adjunct or alternative to antibiotics for antimicrobial intervention.