title: Lactococcus lactis expressing either Staphylococcus aureus fibronectin-binding protein A or Listeria monocytogenes internalin A can efficiently internalize and deliver DNA in human epithelial cells.
abstract: Lactococci are noninvasive bacteria frequently used as protein delivery vectors and, more recently, as in vitro and in vivo DNA delivery vehicles. We previously showed that a functional eukaryotic enhanced green fluorescent protein (eGFP) expression plasmid vector was delivered in epithelial cells by Lactococcus lactis producing Listeria monocytogenes internalin A (L. lactis InlA(+)), but this strategy is limited in vivo to transgenic mice and guinea pigs. In this study, we compare the internalization ability of L. lactis InlA(+) and L. lactis producing either the fibronectin-binding protein A of Staphylococcus aureus (L. lactis FnBPA(+)) or its fibronectin binding domains C and D (L. lactis CD(+)). L. lactis FnBPA(+) and L. lactis InlA(+) showed comparable internalization rates in Caco-2 cells, while the internalization rate observed with L. lactis CD(+) was lower. As visualized by conventional and confocal fluorescence microscopy, large clusters of L. lactis FnBPA(+), L. lactis CD(+), and L. lactis InlA(+) were present in the cytoplasm of Caco-2 cells after internalization. Moreover, the internalization rates of Lactobacillus acidophilus NCFM and of an NCFM mutant strain with the gene coding for the fibronectin-binding protein (fbpA) inactivated were also evaluated in Caco-2 cells. Similar low internalization rates were observed for both wild-type L. acidophilus NCFM and the fbpA mutant, suggesting that commensal fibronectin binding proteins have a role in adhesion but not in invasion. L. lactis FnBPA(+), L. lactis CD(+), and L. lactis InlA(+) were then used to deliver a eukaryotic eGFP expression plasmid in Caco-2 cells: flow cytometry analysis showed that the highest percentage of green fluorescent Caco-2 cells was observed after coculture with either L. lactis FnBPA(+) or L. lactis InlA(+). Analysis of the in vivo efficiency of these invasive recombinant strains is currently in progress to validate their potential as DNA vaccine delivery vehicles.
authors.affiliation: Unité de Virologie et Immunologie Moléculaires, UR892 INRA, Domaine de Vilvert, F78352 Jouy en Josas Cedex, France.
authors.forename: Silvia
authors.initials: S
authors.lastname: Innocentin
authors.forename: Valeria
authors.initials: V
authors.lastname: Guimarães
authors.forename: Anderson
authors.initials: A
authors.lastname: Miyoshi
authors.forename: Vasco
authors.initials: V
authors.lastname: Azevedo
authors.forename: Philippe
authors.initials: P
authors.lastname: Langella
authors.forename: Jean-Marc
authors.initials: JM
authors.lastname: Chatel
authors.forename: François
authors.initials: F
authors.lastname: Lefèvre
mesh_terms: D018829:Adhesins, Bacterial / Q000096:biosynthesis* / Q000235:genetics; D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000235:genetics; D018938:Caco-2 Cells; D004247:DNA / Q000235:genetics / Q000378:metabolism*; D004847:Epithelial Cells / Q000382:microbiology*; D005434:Flow Cytometry; D018014:Gene Transfer Techniques; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D013294:Lactococcus lactis / Q000235:genetics* / Q000378:metabolism; D008089:Listeria monocytogenes / Q000235:genetics; D013211:Staphylococcus aureus / Q000235:genetics; D014170:Transformation, Genetic
chemical_list: D018829:Adhesins, Bacterial; D001426:Bacterial Proteins; C485184:enhanced green fluorescent protein; C078635:fibronectin-binding proteins, bacterial; C069761:internalin protein, Bacteria; D049452:Green Fluorescent Proteins; D004247:DNA