title: Production of Fibronectin Binding Protein A at the surface of Lactococcus lactis increases plasmid transfer in vitro and in vivo.
abstract: Lactococci are noninvasive lactic acid bacteria frequently used as protein delivery vectors and, more recently, as DNA delivery vehicles. We previously showed that Lactococcus lactis (LL) expressing the Fibronectin-Binding Protein A of Staphylococcus aureus (LL-FnBPA+) showed higher internalization rates in vitro in Caco-2 cells than the native (wt) lactococci and were able to deliver a eukaryotic Green Fluorescent Protein (GFP) expression plasmid in 1% of human Caco-2 cells. Here, using the bovine beta-lactoglobulin (BLG), one of the major cow's milk allergen, and GFP we characterized the potential of LL-FnBPA+ as an in vivo DNA vaccine delivery vehicle. We first showed that the invasive strain LL-FnBPA+ carrying the plasmid pValac:BLG (LL-FnBPA+ BLG) was more invasive than LL-BLG and showed the same invasivity as LL-FnBPA+. Then we demonstrated that the Caco-2 cells, co-incubated with LL-FnBPA+ BLG produced up to 30 times more BLG than the Caco-2 cells co-incubated with the non invasive LL-BLG. Using two different gene reporters, BLG and GFP, and two different methods of detection, EIA and fluorescence microscopy, we showed in vivo that: i) in order to be effective, LL-FnBPA+ required a pre-coating with Fetal Calf Serum before oral administration; ii) plasmid transfer occurred in enterocytes without regard to the strains used (invasive or not); iii) the use of LL-FnBPA+ increased the number of mice producing BLG, but not the level of BLG produced. We thus confirmed the good potential of invasive recombinant lactic acid bacteria as DNA delivery vector in vivo.
authors.affiliation: INRA, UMR1319 Micalis, Jouy-en-Josas, France.
authors.forename: Daniela
authors.initials: D
authors.lastname: Pontes
authors.forename: Silvia
authors.initials: S
authors.lastname: Innocentin
authors.forename: Silvina
authors.initials: S
authors.lastname: Del Carmen
authors.forename: Juliana Franco
authors.initials: JF
authors.lastname: Almeida
authors.forename: Jean-Guy
authors.initials: JG
authors.lastname: Leblanc
authors.forename: Alejandra
authors.initials: A
authors.lastname: de Moreno de Leblanc
authors.forename: Sébastien
authors.initials: S
authors.lastname: Blugeon
authors.forename: Claire
authors.initials: C
authors.lastname: Cherbuy
authors.forename: François
authors.initials: F
authors.lastname: Lefèvre
authors.forename: Vasco
authors.initials: V
authors.lastname: Azevedo
authors.forename: Anderson
authors.initials: A
authors.lastname: Miyoshi
authors.forename: Philippe
authors.initials: P
authors.lastname: Langella
authors.forename: Jean-Marc
authors.initials: JM
authors.lastname: Chatel
mesh_terms: D018829:Adhesins, Bacterial / Q000096:biosynthesis* / Q000235:genetics; D000818:Animals; D001692:Biological Transport; D018938:Caco-2 Cells; D002417:Cattle; D018076:DNA, Complementary / Q000235:genetics; D020895:Enterocytes / Q000378:metabolism / Q000382:microbiology; D005260:Female; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D013294:Lactococcus lactis / Q000235:genetics / Q000378:metabolism*; D007782:Lactoglobulins / Q000235:genetics; D051379:Mice; D010957:Plasmids / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000235:genetics; D013045:Species Specificity; D013211:Staphylococcus aureus / Q000235:genetics; D019444:Vaccines, DNA / Q000378:metabolism
chemical_list: D018829:Adhesins, Bacterial; D018076:DNA, Complementary; D007782:Lactoglobulins; D019444:Vaccines, DNA; C078635:fibronectin-binding proteins, bacterial; D049452:Green Fluorescent Proteins