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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 internalization of L. plantarum by DCs. a Monocyte derived hDCs were co-incubated with Lp-WT, Lp-Lip-aDec, Lp-CWA-aDec or Lp-LysM-aDec with or without addition of a free anti human DEC205 antibody. After incubation, the hDCs were washed and non-internalized bacteria were killed by treatment with gentamicin. The numbers of internalized bacteria were counted as CFU. Same amount of DCs was used in each experiment. b BMDCs were co-incubated with Lp-WT, Lp-Lip-aDec, Lp-CWA-aDec or Lp-LysM-aDec. After incubation, the BMDCs were washed and non-internalized bacteria were killed by treatment with gentamicin. The numbers of internalized bacteria were counted as CFU. Each point represents independent wells and results are presented as mean ± SEM. The results presented are from one experiment representative of three performed independently. Statistically significant differences are indicated as follows: *p < 0.05; **p < 0.01; ***p < 0.001.
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Fig2: Effect of surface expression of aDec on internalization of L. plantarum by DCs. a Monocyte derived hDCs were co-incubated with Lp-WT, Lp-Lip-aDec, Lp-CWA-aDec or Lp-LysM-aDec with or without addition of a free anti human DEC205 antibody. After incubation, the hDCs were washed and non-internalized bacteria were killed by treatment with gentamicin. The numbers of internalized bacteria were counted as CFU. Same amount of DCs was used in each experiment. b BMDCs were co-incubated with Lp-WT, Lp-Lip-aDec, Lp-CWA-aDec or Lp-LysM-aDec. After incubation, the BMDCs were washed and non-internalized bacteria were killed by treatment with gentamicin. The numbers of internalized bacteria were counted as CFU. Each point represents independent wells and results are presented as mean ± SEM. The results presented are from one experiment representative of three performed independently. Statistically significant differences are indicated as follows: *p < 0.05; **p < 0.01; ***p < 0.001.

Mentions: To test interactions of the recombinant strains with DCs derived from humans (hDCs) or mice bone marrow dendritic cells (BMDCs), we first performed internalization assays. Figure 2 shows that the number of internalized bacteria was significantly higher after co-incubation with Lp-CWA-aDec and Lp-LysM-aDec compared to Lp-WT or Lp-Lip-aDec. To investigate the specificity of the interactions between the displayed anti-mouse ScFv and human DCs, a competition experiment was carried in which the hDCs were pre-treated with a monoclonal antibody against human DEC-205. The results (Figure 2a) clearly show that increased internalization caused by surface display of aDec is abolished in the presence of the competing free antibody, which proofs that increased internalization is due to a specific interaction between the recombinant strains and DEC-205 on the DCs.Figure 2


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 internalization of L. plantarum by DCs. a Monocyte derived hDCs were co-incubated with Lp-WT, Lp-Lip-aDec, Lp-CWA-aDec or Lp-LysM-aDec with or without addition of a free anti human DEC205 antibody. After incubation, the hDCs were washed and non-internalized bacteria were killed by treatment with gentamicin. The numbers of internalized bacteria were counted as CFU. Same amount of DCs was used in each experiment. b BMDCs were co-incubated with Lp-WT, Lp-Lip-aDec, Lp-CWA-aDec or Lp-LysM-aDec. After incubation, the BMDCs were washed and non-internalized bacteria were killed by treatment with gentamicin. The numbers of internalized bacteria were counted as CFU. Each point represents independent wells and results are presented as mean ± SEM. The results presented are from one experiment representative of three performed independently. 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

Fig2: Effect of surface expression of aDec on internalization of L. plantarum by DCs. a Monocyte derived hDCs were co-incubated with Lp-WT, Lp-Lip-aDec, Lp-CWA-aDec or Lp-LysM-aDec with or without addition of a free anti human DEC205 antibody. After incubation, the hDCs were washed and non-internalized bacteria were killed by treatment with gentamicin. The numbers of internalized bacteria were counted as CFU. Same amount of DCs was used in each experiment. b BMDCs were co-incubated with Lp-WT, Lp-Lip-aDec, Lp-CWA-aDec or Lp-LysM-aDec. After incubation, the BMDCs were washed and non-internalized bacteria were killed by treatment with gentamicin. The numbers of internalized bacteria were counted as CFU. Each point represents independent wells and results are presented as mean ± SEM. The results presented are from one experiment representative of three performed independently. Statistically significant differences are indicated as follows: *p < 0.05; **p < 0.01; ***p < 0.001.
Mentions: To test interactions of the recombinant strains with DCs derived from humans (hDCs) or mice bone marrow dendritic cells (BMDCs), we first performed internalization assays. Figure 2 shows that the number of internalized bacteria was significantly higher after co-incubation with Lp-CWA-aDec and Lp-LysM-aDec compared to Lp-WT or Lp-Lip-aDec. To investigate the specificity of the interactions between the displayed anti-mouse ScFv and human DCs, a competition experiment was carried in which the hDCs were pre-treated with a monoclonal antibody against human DEC-205. The results (Figure 2a) clearly show that increased internalization caused by surface display of aDec is abolished in the presence of the competing free antibody, which proofs that increased internalization is due to a specific interaction between the recombinant strains and DEC-205 on the DCs.Figure 2

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