In vitro and in vivo characterization of DNA delivery using recombinant Lactococcus lactis expressing a mutated form of L. monocytogenes Internalin A - PubMed (original) (raw)

In vitro and in vivo characterization of DNA delivery using recombinant Lactococcus lactis expressing a mutated form of L. monocytogenes Internalin A

Marcela de Azevedo et al. BMC Microbiol. 2012.

Abstract

Background: The use of food-grade Lactic Acid Bacteria (LAB) as DNA delivery vehicles represents an attractive strategy to deliver DNA vaccines at the mucosal surfaces as they are generally regarded as safe (GRAS). We previously showed that either native Lactococcus lactis (LL) or recombinant invasive LL expressing Fibronectin Binding Protein A of Staphylococcus aureus (LL-FnBPA+) or Internalin A of Listeria monocytogenes (LL-InlA+), were able to deliver and trigger DNA expression by epithelial cells, either in vitro or in vivo. InlA does not bind to its receptor, the murine E-cadherin, thus limiting the use of LL-InlA+ in in vivo murine models. Moreover, FnBPA binds to its receptors, integrins, via fibronectin introducing another limiting factor. In order to avoid the limitations of LL-InlA+ and LL-FnBPA+, a new L. lactis strain was engineered to produce a previously described mutated form of InlA (LL-mInlA+) allowing the binding of mInlA on murine E-cadherin.

Results: After showing the expression of mInLA at the surface of LL-mInlA+ strain, in vitro gentamycin survival assay in Caco-2 cells showed that LL-mInlA+ is 1000 times more invasive than LL. LL-mInlA+ invasivity was also validated by fluorescence microscopy. LL and LL-mInlA+ were transformed with pValacBLG, a plasmid containing the cDNA of bovine β-Lactoglobulin (BLG), resulting in strains LL-BLG and LL-mInlA+BLG. The plasmid transfer in vitro using LL-mInlA+BLG was increased 10 times compared to LL-BLG. Moreover, the number of mice producing BLG in isolated enterocytes after oral administration of LL-mInlA+BLG in vivo was slightly higher than after oral administration of LL-BLG.

Conclusions: We confirmed in this study that the production of mInlA at the surface of L. lactis is a promising strategy for plasmid transfer in vitro and in vivo.

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Figures

Figure 1

Characterization of mInlA production at the surface of L. lactis. Black peak corresponds to the negative control, the wild type strain (LL) and the blue peak corresponds to L . lactis strain producing mInlA (LL-mInlA+).

Figure 2

Evaluation of the LL- mInlA+ invasiveness capacity in non- confluent Caco- 2 cells. Caco-2 cells were co-incubated with NZ9000 and LL-mInlA+ strains during 1 h and then treated with gentamicin for 2 h. Cells were lysed and the number of CFU internalized was measured by plating. **, survival rates were significantly different (One-way ANOVA, Bonferroni’s multiple comparison test, p < 0.05). Results are means standard deviations of three different experiments, each time done in triplicate.

Figure 3

LL- mInlA+ internalization in Caco- 2 cells analyzed by confocal microscopy. NZ9000 and L. lactis producing mutated internalin A (LL-mInlA+) were stained with CFSE dye (in green) and co-incubated with Caco-2 cells. Cell membranes were stained with DiI cell-labeling solution (in red) and the fluorescent samples were analyzed by confocal microscopy as described in the methods. 3A. Non-internalization of NZ9000 strain in Caco-2 cells. 3B. Intracellular localization of LL-mInlA+ in some cells. 3C. LL-mInlA+ bind to the membrane of cell clusters where mInlA receptor, E-cadherin, is exposed.

Figure 4

BLG production in Caco- 2 cells after co- incubation with LL- mInlA+ BLG or LL- BLG. Caco-2 cells were co-incubated with LL, LL-BLG or LL-mInlA+BLG during 3 h. BLG was assayed 72 h after co-incubation in cellular protein extracts (A) or medium (B). The results are expressed as mean ± SE values. Statistical significance between the groups was calculated using the One Way ANOVA followed by the “Bonferroni” post-test. Values of p < 0.05 were considered significant.

Figure 5

β- Lactoglobulin detection in mice isolated enterocytes after oral administration of noninvasive and invasive lactococci strains. Mice were orally administered 3 consecutive days with LL, LL-BLG or LL-mInlA+BLG. Seventy two hours after the last gavage, mice were sacrificed and BLG was assayed in protein extracts from isolated small intestine enterocytes. Results showed the sum of two independent experiments.

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In vitro and in vivo characterization of DNA delivery using recombinant Lactococcus lactis expressing a mutated form of L. monocytogenes Internalin A - PubMed (original) (raw)