Use of native lactococci as vehicles for delivery of DNA into mammalian epithelial cells

Abstract

The use of the food-grade bacterium Lactococcus lactis as a DNA delivery vehicle at the mucosal level is an attractive DNA vaccination strategy. Previous experiments showed that recombinant L. lactis expressing the Listeria monocytogenes inlA gene can deliver a functional gene into mammalian cells. Here, we explored the potential use of noninvasive L. lactis strains as a DNA delivery vehicle. We constructed two Escherichia coli-L. lactis shuttle plasmids, pLIG:BLG1 and pLIG:BLG2, containing a eukaryotic expression cassette with the cDNA of bovine beta-lactoglobulin (BLG). The greatest BLG expression after transfection of Cos-7 cells was obtained with pLIG:BLG1, which was then used to transform L. lactis MG1363. The resulting L. lactis strain MG1363(pLIG:BLG1) was not able to express BLG. The potential of L. lactis as a DNA delivery vehicle was analyzed by detection of BLG in Caco-2 human colon carcinoma cells after 3 h of coincubation with (i) purified pLIG:BLG1, (ii) MG1363(pLIG:BLG1), (iii) a mix of MG1363(pLIG) and purified pLIG:BLG1, and (iv) MG1363. Both BLG cDNA and BLG expression were detected only in Caco-2 cells coincubated with MG1363(pLIG:BLG1). There was a decrease in the BLG cDNA level in Caco-2 cells between 24 and 48 h after coincubation. BLG expression by Caco-2 cells started at 24 h and increased between 24 and 72 h. BLG secretion by Caco-2 cells started 48 h after coincubation with MG1363(pLIG:BLG1). We conclude that lactococci can deliver BLG cDNA into mammalian epithelial cells, demonstrating their potential to deliver in vivo a DNA vaccine.

FIG. 1.

Structures of pLIG:BLG1 and pLIG:BLG2. Arrows indicate the directions of transcription of BLG cDNA, the ampicillin resistance gene (Amp), and the erythromycin resistance gene (Ery). Boxes indicate P_cmv, the cytomegalovirus eukaryotic promoter; RepA, the origin of replication of L. lactis; and ColE1, the origin of replication of E. coli. The thin part of the vector is derived from plasmid pIL253, and the thick part is derived from pcDNA3BLG5. The BamHI and BglII restriction sites used to obtain the two plasmids and the HindIII restriction site are shown.

FIG. 2.

BLG expression in Cos-7 cells after transfection. Cos-7 cells were transfected with plasmids pcDNA3BLG5, pLIG:BLG1, and pLIG:BLG2. Two days later, the transfection medium was collected and cells were harvested. BLG was assayed in the culture medium and in cell extracts. Results are expressed in micrograms per milliliter of culture medium (A) and of cell extracts (B). The results correspond to the averages of two independent assays. Error bars correspond to standard deviations. Statistical significance was set at a P value of <0.05.

FIG. 3.

BLG expression in MG1363(pLIG1) and MG1363(pLIG:BLG1). (A) Detection of β-actin and BLG-specific mRNA by RT-PCR in total RNA from saturated growth of MG1363(pLIG1) and MG1363(pLIG:BLG1). Lanes 1 and 2, β-actin mRNA detection by RT-PCR from MG1363(pLIG1) and MG1363(pLIG:BLG1), respectively; lanes 3 and 4, BLG mRNA detection by RT-PCR from MG1363(pLIG1) and MG1363(pLIG:BLG1), respectively. (B) BLG assay of L. lactis strains grown to saturation.

FIG. 4.

Plasmid transfer detection in Caco-2 cells determined by amplification by PCR from total DNA purified 24, 48, and 72 h after gentamicin treatment of Caco-2 cells cocultured with MG1363(pLIG:BLG1), MG1363(pLIG1), and purified plasmid pLIG:BLG1. (A) Specific amplification by PCR using oligonucleotides primed at the N terminus and the C terminus of pcDNA3. (B) Specific amplification by PCR using oligonucleotides primed at the N terminus and the C terminus of BLG cDNA. Control (C) amplifications were made with purified plasmid pcDNA3BLG5.

FIG. 5.

BLG expression in Caco-2 cells. Caco-2 cells were cocultivated with purified plasmid pLIG:BLG1, MG1363(pLIG1), purified plasmid pLIG:BLG1 mixed with MG1363(pLIG1), and MG1363(pLIG:BLG1). Twenty-four, 48, and 72 h after treatment with gentamicin, the cells were harvested and culture supernatants were collected. (A) BLG assayed in the cellular extract. (B) BLG assayed in the culture supernatant. The results correspond to the averages of three independent assays. Error bars correspond to standard deviations.

FIG. 6.

Detection of BLG mRNA in Caco-2 cells. Caco-2 cells were cocultivated with purified plasmid pLIG:BLG1 (lanes 1 and 4), MG1363(pLIG1) (lanes 2 and 5), and MG1363(pLIG:BLG1) (lanes 3 and 6). Forty-eight hours after treatment with gentamicin, the cells were harvested and total RNA was collected and used for RT-PCR experiments with β-actin- and BLG-specific oligonucleotides. M, nucleic acid marker.