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

• DOI: 10.1128/aem.00825-09
• Publication Date: 2009-05-30T01:00:16Z
• Authors: Silvia Innocentin; Valeria Guimarães; Anderson Miyoshi; Vasco Azevedo; Philippe Langella; Jean-Marc Chatel; François Lefèvre
• Keywords: [SDV.EE]Life Sciences [q-bio]/Ecology, environment; Staphylococcus aureus; 0303 health sciences; Green Fluorescent Proteins; Gene Transfer Techniques; Epithelial Cells; DNA; Flow Cytometry; Listeria monocytogenes; 3. Good health; Lactococcus lactis; 03 medical and health sciences; Transformation, Genetic; Bacterial Proteins; Genes, Reporter; [SDV.EE]Life Sciences [q-bio]/Ecology; Humans; Caco-2 Cells; Adhesins, Bacterial; environment

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.