A stably engineered, suicidal strain of listeria monocytogenes delivers protein and/or DNA to fully differentiated intestinal epithelial monolayers

We have generated a recombinant stable, suicidal Listeria monocytogenes strain (rs'2)capable of delivering antigens as protein or DNA into nondividing intestinal epithelial cells. The rs'2strain was generated by inserting a cell wall hydrolysin gene, 'ply118' together with its associatedholin gene from a Listeria-specific phage, into the attenuated L. monocytonegenes genome of strain'2. The hol118/ply118 gene was placed under the control of the Listeria promoter PactA, inducingbacteria to undergo autolysis in eukaryotic cells. The rs'2 strain had normal growth rate in richbacterial growth medium, but its replication in eukaryotic cells was limited, and its autolysis wasused to deliver its contents to the cytoplasm of eukaryotic cells. The delivery potential of rs'2 wasexplored using engineered shuttle vectors designed to facilitate expression of a transgene, either inrs'2 (driven by Phly) or in the mammalian cell (driven by PCMV), or both (using our engineered dualListeria and mammalian expression vector, pDuLX). The luciferase reporter was used to demonstratethat pDuLX vector allowed delivery of both protein and DNA to dividing Caco-2 human epithelialcells. As expected, nondividing fully differentiated Caco-2 monolayers were resistant to transfectionwith Lipofectamine, which can be explained by lack of access to the cell nucleus. We demonstratedthat when Caco-2 monolayers were treated with rs'2, the bacteria were able to deliver a significantquantity of luciferase protein. By implication the bacteria were also able to deliver DNA, but expressiondriven by the eukaryotic promoter in host Caco-2 cells was not observed. When the rs'2 strain wastaken up by Caco-2 cells, there was little or no bacterial growth, whereas the control '2 strain wasviable and grew by approximately three log cycles within the Caco-2 cells. A small mass of proteinor DNA wasdelivered by the '2 strain perhaps because some bacteria died, but despite the levelof growth the mass of protein delivered to dividing Caco-2 cells by the '2 strain was considerablyless than that delivered by the rs'2 strain. We concluded that the Listeria delivery system hasprospects for oral vaccination using antigens synthesized by the bacterium itself.