Ligand-displaying
Escherichia coli
cells and minicells for programmable delivery of toxic payloads via type IV secretion systems
Authors: Li Yang Grace , Kishida Kouhei , Ogawa-Kishida Natsumi , Christie Peter J. 
Published: September 2023

ABSTRACT

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.
Escherichia coli
donors displaying Nbs transferred plasmids to
E. coli
and
Pseudomonas aeruginosa
recipients displaying the cognate Ags at significantly higher frequencies than recipients lacking Ags. 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 the delivery of pCrispr plasmids to
E. coli
and
P. aeruginosa
transconjugants bearing CRISPR/Cas9 target sequences. Finally, we determined that anucleate
E. coli
minicells, which are clinically safer delivery vectors than intact cells, transferred self-transmissible and mobilizable plasmids to
E. coli
and
P. aeruginosa
cells. Minicell-mediated mobilization of pCrispr plasmids to
E. coli
recipients elicited significant killing of transconjugants, although Nb/Ag pairing did not enhance conjugation frequencies or killing. Together, our findings establish the potential for the deployment of bacteria or minicells as programmed delivery systems for the suppression of targeted bacterial species in infection settings.

IMPORTANCE

The rapid emergence of drug-resistant bacteria and current low rate of antibiotic discovery emphasize the urgent need for alternative antibacterial strategies. We engineered
Escherichia coli
to conjugatively transfer plasmids to specific
E. coli
and
Pseudomonas aeruginosa
recipient cells through the surface display of cognate nanobody/antigen (Nb/Ag) pairs. We further engineered mobilizable plasmids to carry CRISPR/Cas9 systems (pCrispr) for the selective killing of recipient cells harboring CRISPR/Cas9 target sequences. In the assembled programmed delivery system (PDS), Nb-displaying
E. coli
donors with different conjugation systems and mobilizable pCrispr plasmids suppressed the 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
E. coli
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.