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Surface display of an anti-DEC-205 single chain Fv fragment in Lactobacillus plantarum increases internalization and plasmid transfer to dendritic cells in vitro and in vivo.

Michon C, Christophe M, Kuczkowska K, Langella P, Eijsink VG, Mathiesen G, Chatel JM - Microb. Cell Fact. (2015)

Bottom Line: The results show that surface expression of aDec leads to increased internalization of L. plantarum and plasmid transfer in DCs and that efficiency depends on the type of anchor used.Interestingly, in vitro data indicates that cell wall anchoring is more effective, whereas in vivo data seem to indicate that anchoring to the cell membrane is preferable.It is likely that the more embedded localization of aDec in the latter case is favorable when cells are exposed to the harsh conditions of the gastro-intestinal tract.

View Article: PubMed Central - PubMed

Affiliation: INRA, UMR1319 MICALIS, Bat 440, R-2, 78352, Jouy-en-Josas, France. michon.christophe@yahoo.fr.

ABSTRACT

Background: Lactic acid bacteria (LAB) are promising vehicles for delivery of a variety of medicinal compounds, including antigens and cytokines. It has also been established that LAB are able to deliver cDNA to host cells. To increase the efficiency of LAB-driven DNA delivery we have constructed Lactobacillus plantarum strains targeting DEC-205, which is a receptor located at the surface of dendritic cells (DCs). The purpose was to increase uptake of bacterial cells, which could lead to improved cDNA delivery to immune cells.

Results: Anti-DEC-205 antibody (aDec) was displayed at the surface of L. plantarum using three different anchoring strategies: (1) covalent anchoring of aDec to the cell membrane (Lipobox domain, Lip); (2) covalent anchoring to the cell wall (LPXTG domain, CWA); (3) non-covalent anchoring to the cell wall (LysM domain, LysM). aDec was successfully expressed in all three strains, but surface location of the antibody could only be demonstrated for the two strains with cell wall anchors (CWA and LysM). Co-incubation of the engineered strains and DCs showed increased uptake when anchoring aDec using the CWA or LysM anchors. In a competition assay, free anti-DEC abolished the increased uptake, showing that the internalization is due to specific interactions between the DEC-205 receptor and aDec. To test plasmid transfer, a plasmid for expression of GFP under control of an eukaryotic promoter was transformed into the aDec expressing strains and GFP expression in DCs was indeed increased when using the strains producing cell-wall anchored aDec. Plasmid transfer to DCs in the gastro intestinal tract was also detected using a mouse model. Surprisingly, in mice the highest expression of GFP was observed for the strain in which aDec was coupled to the cell membrane.

Conclusion: The results show that surface expression of aDec leads to increased internalization of L. plantarum and plasmid transfer in DCs and that efficiency depends on the type of anchor used. Interestingly, in vitro data indicates that cell wall anchoring is more effective, whereas in vivo data seem to indicate that anchoring to the cell membrane is preferable. It is likely that the more embedded localization of aDec in the latter case is favorable when cells are exposed to the harsh conditions of the gastro-intestinal tract.

No MeSH data available.


Related in: MedlinePlus

Effect of surface expression of aDec on plasmid transfer to mouse DCs in vivo. Mice were orally administrated with Lp-WT/pValac-GFP, Lp-Lip-aDec/pValac-GFP, Lp-CWA-aDec/pValac-GFP or Lp-LysM-aDec/pValac-GFP during 4 days. At day 4 mice were sacrificed and DCs were extracted from the small intestine (a) or the colon (b). The percentage of DCs expressing GFP was measured by flow cytometry. Each point represents independent mice and the results are presented as mean ± SEM. Statistically significant differences are indicated as follows: *p < 0.05; **p < 0.01; ***p < 0.001.
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Fig4: Effect of surface expression of aDec on plasmid transfer to mouse DCs in vivo. Mice were orally administrated with Lp-WT/pValac-GFP, Lp-Lip-aDec/pValac-GFP, Lp-CWA-aDec/pValac-GFP or Lp-LysM-aDec/pValac-GFP during 4 days. At day 4 mice were sacrificed and DCs were extracted from the small intestine (a) or the colon (b). The percentage of DCs expressing GFP was measured by flow cytometry. Each point represents independent mice and the results are presented as mean ± SEM. Statistically significant differences are indicated as follows: *p < 0.05; **p < 0.01; ***p < 0.001.

Mentions: To investigate the potential of the recombinant strains for plasmid transfer in vivo the recombinant strains containing pValacGFP were administered to mice. After 4 days of oral administration of the four different strains, mice were euthanized and DCs were isolated from the intestine and the colon. Quantification of the fraction of fluorescent DCs by flow cytometry showed that, compared to mice fed with control strain, this fraction was higher in both the intestine (Figure 4a) and the colon (Figure 4b) for mice fed with Lp-Lip-Dec/pValac-GFP. A similar trend, but not significant, was observed for mice fed with Lp-LysM-aDec/pValac-GFP. Thus, expression of aDec at the membrane of L. plantarum enhances plasmid transfer in vivo, but the preferred anchor (Lip) differs from the anchors that were most efficient in vitro (CWA and LysM).Figure 4


Surface display of an anti-DEC-205 single chain Fv fragment in Lactobacillus plantarum increases internalization and plasmid transfer to dendritic cells in vitro and in vivo.

Michon C, Christophe M, Kuczkowska K, Langella P, Eijsink VG, Mathiesen G, Chatel JM - Microb. Cell Fact. (2015)

Effect of surface expression of aDec on plasmid transfer to mouse DCs in vivo. Mice were orally administrated with Lp-WT/pValac-GFP, Lp-Lip-aDec/pValac-GFP, Lp-CWA-aDec/pValac-GFP or Lp-LysM-aDec/pValac-GFP during 4 days. At day 4 mice were sacrificed and DCs were extracted from the small intestine (a) or the colon (b). The percentage of DCs expressing GFP was measured by flow cytometry. Each point represents independent mice and the results are presented as mean ± SEM. Statistically significant differences are indicated as follows: *p < 0.05; **p < 0.01; ***p < 0.001.
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4491208&req=5

Fig4: Effect of surface expression of aDec on plasmid transfer to mouse DCs in vivo. Mice were orally administrated with Lp-WT/pValac-GFP, Lp-Lip-aDec/pValac-GFP, Lp-CWA-aDec/pValac-GFP or Lp-LysM-aDec/pValac-GFP during 4 days. At day 4 mice were sacrificed and DCs were extracted from the small intestine (a) or the colon (b). The percentage of DCs expressing GFP was measured by flow cytometry. Each point represents independent mice and the results are presented as mean ± SEM. Statistically significant differences are indicated as follows: *p < 0.05; **p < 0.01; ***p < 0.001.
Mentions: To investigate the potential of the recombinant strains for plasmid transfer in vivo the recombinant strains containing pValacGFP were administered to mice. After 4 days of oral administration of the four different strains, mice were euthanized and DCs were isolated from the intestine and the colon. Quantification of the fraction of fluorescent DCs by flow cytometry showed that, compared to mice fed with control strain, this fraction was higher in both the intestine (Figure 4a) and the colon (Figure 4b) for mice fed with Lp-Lip-Dec/pValac-GFP. A similar trend, but not significant, was observed for mice fed with Lp-LysM-aDec/pValac-GFP. Thus, expression of aDec at the membrane of L. plantarum enhances plasmid transfer in vivo, but the preferred anchor (Lip) differs from the anchors that were most efficient in vitro (CWA and LysM).Figure 4

Bottom Line: The results show that surface expression of aDec leads to increased internalization of L. plantarum and plasmid transfer in DCs and that efficiency depends on the type of anchor used.Interestingly, in vitro data indicates that cell wall anchoring is more effective, whereas in vivo data seem to indicate that anchoring to the cell membrane is preferable.It is likely that the more embedded localization of aDec in the latter case is favorable when cells are exposed to the harsh conditions of the gastro-intestinal tract.

View Article: PubMed Central - PubMed

Affiliation: INRA, UMR1319 MICALIS, Bat 440, R-2, 78352, Jouy-en-Josas, France. michon.christophe@yahoo.fr.

ABSTRACT

Background: Lactic acid bacteria (LAB) are promising vehicles for delivery of a variety of medicinal compounds, including antigens and cytokines. It has also been established that LAB are able to deliver cDNA to host cells. To increase the efficiency of LAB-driven DNA delivery we have constructed Lactobacillus plantarum strains targeting DEC-205, which is a receptor located at the surface of dendritic cells (DCs). The purpose was to increase uptake of bacterial cells, which could lead to improved cDNA delivery to immune cells.

Results: Anti-DEC-205 antibody (aDec) was displayed at the surface of L. plantarum using three different anchoring strategies: (1) covalent anchoring of aDec to the cell membrane (Lipobox domain, Lip); (2) covalent anchoring to the cell wall (LPXTG domain, CWA); (3) non-covalent anchoring to the cell wall (LysM domain, LysM). aDec was successfully expressed in all three strains, but surface location of the antibody could only be demonstrated for the two strains with cell wall anchors (CWA and LysM). Co-incubation of the engineered strains and DCs showed increased uptake when anchoring aDec using the CWA or LysM anchors. In a competition assay, free anti-DEC abolished the increased uptake, showing that the internalization is due to specific interactions between the DEC-205 receptor and aDec. To test plasmid transfer, a plasmid for expression of GFP under control of an eukaryotic promoter was transformed into the aDec expressing strains and GFP expression in DCs was indeed increased when using the strains producing cell-wall anchored aDec. Plasmid transfer to DCs in the gastro intestinal tract was also detected using a mouse model. Surprisingly, in mice the highest expression of GFP was observed for the strain in which aDec was coupled to the cell membrane.

Conclusion: The results show that surface expression of aDec leads to increased internalization of L. plantarum and plasmid transfer in DCs and that efficiency depends on the type of anchor used. Interestingly, in vitro data indicates that cell wall anchoring is more effective, whereas in vivo data seem to indicate that anchoring to the cell membrane is preferable. It is likely that the more embedded localization of aDec in the latter case is favorable when cells are exposed to the harsh conditions of the gastro-intestinal tract.

No MeSH data available.


Related in: MedlinePlus