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.
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.
Innocentin
Silvia
S
Unité de Virologie et Immunologie Moléculaires, UR892 INRA, Domaine de Vilvert, F78352 Jouy en Josas Cedex, France.
Guimarães
Valeria
V
Miyoshi
Anderson
A
Azevedo
Vasco
V
Langella
Philippe
P
Chatel
Jean-Marc
JM
Lefèvre
François
F
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
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