title: Use of native lactococci as vehicles for delivery of DNA into mammalian epithelial cells.
abstract: The use of the food-grade bacterium Lactococcus lactis as a DNA delivery vehicle at the mucosal level is an attractive DNA vaccination strategy. Previous experiments showed that recombinant L. lactis expressing the Listeria monocytogenes inlA gene can deliver a functional gene into mammalian cells. Here, we explored the potential use of noninvasive L. lactis strains as a DNA delivery vehicle. We constructed two Escherichia coli-L. lactis shuttle plasmids, pLIG:BLG1 and pLIG:BLG2, containing a eukaryotic expression cassette with the cDNA of bovine beta-lactoglobulin (BLG). The greatest BLG expression after transfection of Cos-7 cells was obtained with pLIG:BLG1, which was then used to transform L. lactis MG1363. The resulting L. lactis strain MG1363(pLIG:BLG1) was not able to express BLG. The potential of L. lactis as a DNA delivery vehicle was analyzed by detection of BLG in Caco-2 human colon carcinoma cells after 3 h of coincubation with (i) purified pLIG:BLG1, (ii) MG1363(pLIG:BLG1), (iii) a mix of MG1363(pLIG) and purified pLIG:BLG1, and (iv) MG1363. Both BLG cDNA and BLG expression were detected only in Caco-2 cells coincubated with MG1363(pLIG:BLG1). There was a decrease in the BLG cDNA level in Caco-2 cells between 24 and 48 h after coincubation. BLG expression by Caco-2 cells started at 24 h and increased between 24 and 72 h. BLG secretion by Caco-2 cells started 48 h after coincubation with MG1363(pLIG:BLG1). We conclude that lactococci can deliver BLG cDNA into mammalian epithelial cells, demonstrating their potential to deliver in vivo a DNA vaccine.
authors.affiliation: Unité de Virologie et Immunologie Moléculaire, INRA, Domaine de Vilvert, 78352 Jouy en Josas Cedex, France.
authors.forename: Valéria Dellaretti
authors.initials: VD
authors.lastname: Guimarães
authors.forename: Silvia
authors.initials: S
authors.lastname: Innocentin
authors.forename: François
authors.initials: F
authors.lastname: Lefèvre
authors.forename: Vasco
authors.initials: V
authors.lastname: Azevedo
authors.forename: Jean-Michel
authors.initials: JM
authors.lastname: Wal
authors.forename: Philippe
authors.initials: P
authors.lastname: Langella
authors.forename: Jean-Marc
authors.initials: JM
authors.lastname: Chatel
mesh_terms: D000818:Animals; D019556:COS Cells; D018938:Caco-2 Cells; D002417:Cattle; D002522:Chlorocebus aethiops; D003106:Colon / Q000166:cytology / Q000382:microbiology*; D018076:DNA, Complementary; D004847:Epithelial Cells / Q000382:microbiology*; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D006801:Humans; D013294:Lactococcus lactis / Q000235:genetics*; D007782:Lactoglobulins / Q000235:genetics / Q000378:metabolism; D010957:Plasmids
chemical_list: D018076:DNA, Complementary; D007782:Lactoglobulins