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

journal contribution
posted on 2024-06-17, 03:53 authored by CY Kuo, S Sinha, Jalal A Jazayeri, CW Pouton
We have generated a recombinant stable, suicidal Listeria monocytogenes strain (rsΔ2) capable of delivering antigens as protein or DNA into nondividing intestinal epithelial cells. The rsΔ2 strain was generated by inserting a cell wall hydrolysin gene, "ply118" together with its associated holin gene from a Listeria-specific phage, into the attenuated L. monocytonegenes genome of strain Δ2. The hol118/ply118 gene was placed under the control of the Listeria promoter PactA, inducing bacteria to undergo autolysis in eukaryotic cells. The rsΔ2 strain had normal growth rate in rich bacterial growth medium, but its replication in eukaryotic cells was limited, and its autolysis was used to deliver its contents to the cytoplasm of eukaryotic cells. The delivery potential of rsΔ2 was explored using engineered shuttle vectors designed to facilitate expression of a transgene, either in rsΔ2 (driven by Phly) or in the mammalian cell (driven by P CMV), or both (using our engineered dual Listeria and mammalian expression vector, pDuLX). The luciferase reporter was used to demonstrate that pDuLX vector allowed delivery of both protein and DNA to dividing Caco-2 human epithelial cells. As expected, nondividing fully differentiated Caco-2 monolayers were resistant to transfection with Lipofectamine, which can be explained by lack of access to the cell nucleus. We demonstrated that when Caco-2 monolayers were treated with rsΔ2, the bacteria were able to deliver a significant quantity of luciferase protein. By implication the bacteria were also able to deliver DNA, but expression driven by the eukaryotic promoter in host Caco-2 cells was not observed. When the rsΔ2 strain was taken up by Caco-2 cells, there was little or no bacterial growth, whereas the control Δ2 strain was viable and grew by approximately three log cycles within the Caco-2 cells. A small mass of protein or DNA was delivered by the Δ2 strain perhaps because some bacteria died, but despite the level of growth the mass of protein delivered to dividing Caco-2 cells by the Δ2 strain was considerably less than that delivered by the rsΔ2 strain. We concluded that the Listeria delivery system has prospects for oral vaccination using antigens synthesized by the bacterium itself. © 2009 American Chemical Society.

Funding

Category 2 - Other Public Sector Grants Category

History

Volume

6

Issue

4

Start Page

1052

End Page

1061

Number of Pages

10

eISSN

1543-8392

ISSN

1543-8384

Publisher

American Chemical Society (ACS)

Language

en

Peer Reviewed

  • Yes

Open Access

  • No

Acceptance Date

2009-03-24

Era Eligible

  • Yes

Medium

Print

Journal

Molecular Pharmaceutics

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