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Applied and Environmental Microbiology, July 2009, p. 4870-4878, Vol. 75, No. 14
0099-2240/09/$08.00+0     doi:10.1128/AEM.00825-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

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

Silvia Innocentin,¹ Valeria Guimarães,¹ Anderson Miyoshi,² Vasco Azevedo,² Philippe Langella,³ Jean-Marc Chatel,^3* and François Lefèvre¹

Unité de Virologie et Immunologie Moléculaires, UR892 INRA, Domaine de Vilvert, F78352 Jouy en Josas Cedex, France,¹ Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais (UFMG-ICB), Belo Horizonte—MG, Brazil,² Unité d'Ecologie et Physiologie du Système Digestif, UR910 INRA, Domaine de Vilvert, F78352 Jouy en Josas Cedex, France³

Received 10 April 2009/ Accepted 22 May 2009

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.

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* Corresponding author. Mailing address: Unité d'Ecologie et Physiologie du Système Digestif, INRA, Domaine de Vilvert, F78352 Jouy en Josas Cedex, France. Phone: 33 01 34 65 24 68. Fax: 33 01 34 65 24 62. E-mail: jean-marc.chatel{at}jouy.inra.fr

Published ahead of print on 29 May 2009.

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Applied and Environmental Microbiology, July 2009, p. 4870-4878, Vol. 75, No. 14
0099-2240/09/$08.00+0     doi:10.1128/AEM.00825-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.