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.The mucosal administration of bacterial carriers to deliver antigens and plasmid DNA constitutes a promising vaccination strategy. Pathogenic bacteria that have the capacity to invade cells, such as
Listeria,
Salmonella, and
Shigella strains, have been used to deliver DNA constructs into mammalian cells (
23). Nevertheless, the risk associated with possible reversion to a virulent phenotype of these pathogens is a major concern (
5).
Lactococcus lactis, the food-grade, gram-positive, noninvasive model bacterium, has been intensively used to deliver antigens and cytokines at the mucosal level (
30). We previously showed (i) that native
L. lactis can deliver a eukaryotic expression cassette coding for the bovine β-lactoglobulin (BLG), one of the major cow''s milk allergens, into mammalian epithelial Caco-2 cells, and (ii) that these cells were able to express and secrete BLG protein in its native conformation (
10). Recently, we demonstrated the ability of native noninvasive
L. lactis to deliver a fully functional plasmid to murine intestinal cells in vivo (
2).The internalization of the bacterial carrier is a fundamental step to achieve efficient DNA delivery in eukaryotic cells (
7). In order to increase DNA delivery by lactic acid bacteria (LAB), invasin genes were expressed in
L. lactis. Due to the safety profile of LAB, recombinant lactococci expressing invasin genes from intracellular bacteria are attractive as potential DNA delivery vectors compared to the attenuated pathogens presently used.In this field, we previously demonstrated that
L. lactis bacteria expressing the main
Listeria monocytogenes invasin, internalin A (
L. lactis InlA
+), were able to invade eukaryotic cells and efficiently deliver a functional green fluorescent protein (GFP) expression plasmid into epithelial/endothelial cells (
9). Even though attractive, the experimental use of lactococci expressing InlA in a mouse model has a major bottleneck: InlA, which binds to human E-cadherin (
15), does not interact with murine E-cadherin. Consequently, in vivo experimental studies using lactococci expressing InlA as DNA delivery vehicles are limited to transgenic mice expressing human E-cadherin or to guinea pigs (
13).Fibronectin binding protein A (FnBPA) of
Staphylococcus aureus is another bacterial invasin that is involved in intracellular spreading of
S. aureus in the host (
27). It is a multifunctional adhesion protein having both fibrinogen and fibronectin binding capacities (
24). Its N-terminal part, also called domain A, is responsible for fibrinogen (
29) and elastin (
20) binding, whereas its C-terminal part, including domains B, C, and D, binds to fibronectin (
25). FnBPA is known to mediate adhesion to host tissue and bacterial uptake into nonphagocytic host cells (
27). Its expression by
L. lactis was previously shown to be sufficient to confer the ability to invade nonphagocytic cells in vitro and in vivo, while the expression of domains C and D confers invasivity only in vitro (
19).In this study, we show that
L. lactis bacteria expressing full-length FnBPA of
S. aureus (
L. lactis FnBPA
+) or a truncated form encompassing only its C and D domains (
L. lactis CD
+) are internalized as efficiently as
L. lactis InlA
+ in the human intestinal cell line Caco-2. We also provide, for the first time, direct microscopic evidence of the intracellular location of the internalized lactococci, showing that the bacteria are heterogeneously distributed in the cell monolayer and that their number per cell can reach a surprisingly high level. However, we demonstrate that FbpA, a fibronectin binding protein from the commensal
Lactobacillus acidophilus NCFM, does not mediate bacterial internalization: no difference in invasivity was observed between the wild-type (wt) strain and the mutant with
fbpA inactivated. This result indicates that, although widely distributed among bacteria, fibronectin binding proteins are not universal mediators of bacterial internalization, even at low levels. Finally, we also demonstrate that, similarly to
L. lactis InlA
+,
L. lactis FnBPA
+ and
L. lactis CD
+ can efficiently deliver a eukaryotic GFP expression plasmid in Caco-2 cells and trigger GFP expression in these cells. Consequently,
L. lactis FnBPA
+ can be used for further DNA delivery experiments in vivo.
相似文献