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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

Characterization of aDec-expressing L. plantarum strains. a Schematic representation of the three anchors used. b Expression cassettes in which the gene fragment encoding the desired protein is translationally fused to the inducible PsppA promoter [47]. The cassette was PCR-generated using the primers listed in Table 2 and inserted into previously described anchoring vectors [31, 36] digested with the restriction enzymes indicated in the Figure, as summarized in Table 3. All constructs include a N-terminal signal sequence (SP) for secretion and a HA-tag for immune detection. Three different anchoring domains were used, as described in the main text. Note that in the CWA construct, the anchoring domain is located C-terminally, meaning that the HA-tag will protrude from the cells after secretion and subsequent anchoring. c Growth curves for Lp-WT (control strain) and aDec-expressing strains; protein production was induced by addition of peptide pheromone at OD600 ~0.3. d Western blot analysis of cell-free protein extracts from aDec-expressing strains harvested 2 h after induction, using a mouse anti-HA primary antibody and a HRP-conjugated goat anti-mouse secondary antibody. e Flow cytometry analysis of the presence of aDec at the surface of Lp-WT (in black) compared to Lp-Lip-aDec, Lp-CWA-aDec or Lp-LysM-aDec (all in red).
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Fig1: Characterization of aDec-expressing L. plantarum strains. a Schematic representation of the three anchors used. b Expression cassettes in which the gene fragment encoding the desired protein is translationally fused to the inducible PsppA promoter [47]. The cassette was PCR-generated using the primers listed in Table 2 and inserted into previously described anchoring vectors [31, 36] digested with the restriction enzymes indicated in the Figure, as summarized in Table 3. All constructs include a N-terminal signal sequence (SP) for secretion and a HA-tag for immune detection. Three different anchoring domains were used, as described in the main text. Note that in the CWA construct, the anchoring domain is located C-terminally, meaning that the HA-tag will protrude from the cells after secretion and subsequent anchoring. c Growth curves for Lp-WT (control strain) and aDec-expressing strains; protein production was induced by addition of peptide pheromone at OD600 ~0.3. d Western blot analysis of cell-free protein extracts from aDec-expressing strains harvested 2 h after induction, using a mouse anti-HA primary antibody and a HRP-conjugated goat anti-mouse secondary antibody. e Flow cytometry analysis of the presence of aDec at the surface of Lp-WT (in black) compared to Lp-Lip-aDec, Lp-CWA-aDec or Lp-LysM-aDec (all in red).

Mentions: A DNA fragment encoding the anti-mouse Dec205 ScFv (aDec) preceded by a HA-tag for immune detection was cloned into plasmids previously developed for production and surface localization of proteins in L. plantarum (32, 26) using three different types of anchors as outlined in Figure 1a, b. The anchors are: a Lipobox membrane anchor for covalent coupling of aDec to a membrane component, a cell wall anchor based on sortase-catalyzed covalent coupling to the peptidoglycan and a cell wall anchor based on non-covalent interactions between a LysM domain and the cell wall. The three resulting plasmids, pLip-aDec, pCWA-aDec and pLysM-aDec (Figure 1b) were transformed into L. plantarum. In addition, we used Lp-WT, containing a control plasmid (pEV) [31] that does not encode for the anchors or aDec, as a negative control. The growth rate of strains producing aDec was substantially lower compared to control strain (Figure 1c), in particular for the strain harboring the lipoprotein anchor. Still, all strains showed reasonable growth and bacteria harvested 2 h after induction, where all transformants have a similar OD600 (Figure 1c) and similar cell counts, were used for further studies.Figure 1


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)

Characterization of aDec-expressing L. plantarum strains. a Schematic representation of the three anchors used. b Expression cassettes in which the gene fragment encoding the desired protein is translationally fused to the inducible PsppA promoter [47]. The cassette was PCR-generated using the primers listed in Table 2 and inserted into previously described anchoring vectors [31, 36] digested with the restriction enzymes indicated in the Figure, as summarized in Table 3. All constructs include a N-terminal signal sequence (SP) for secretion and a HA-tag for immune detection. Three different anchoring domains were used, as described in the main text. Note that in the CWA construct, the anchoring domain is located C-terminally, meaning that the HA-tag will protrude from the cells after secretion and subsequent anchoring. c Growth curves for Lp-WT (control strain) and aDec-expressing strains; protein production was induced by addition of peptide pheromone at OD600 ~0.3. d Western blot analysis of cell-free protein extracts from aDec-expressing strains harvested 2 h after induction, using a mouse anti-HA primary antibody and a HRP-conjugated goat anti-mouse secondary antibody. e Flow cytometry analysis of the presence of aDec at the surface of Lp-WT (in black) compared to Lp-Lip-aDec, Lp-CWA-aDec or Lp-LysM-aDec (all in red).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4491208&req=5

Fig1: Characterization of aDec-expressing L. plantarum strains. a Schematic representation of the three anchors used. b Expression cassettes in which the gene fragment encoding the desired protein is translationally fused to the inducible PsppA promoter [47]. The cassette was PCR-generated using the primers listed in Table 2 and inserted into previously described anchoring vectors [31, 36] digested with the restriction enzymes indicated in the Figure, as summarized in Table 3. All constructs include a N-terminal signal sequence (SP) for secretion and a HA-tag for immune detection. Three different anchoring domains were used, as described in the main text. Note that in the CWA construct, the anchoring domain is located C-terminally, meaning that the HA-tag will protrude from the cells after secretion and subsequent anchoring. c Growth curves for Lp-WT (control strain) and aDec-expressing strains; protein production was induced by addition of peptide pheromone at OD600 ~0.3. d Western blot analysis of cell-free protein extracts from aDec-expressing strains harvested 2 h after induction, using a mouse anti-HA primary antibody and a HRP-conjugated goat anti-mouse secondary antibody. e Flow cytometry analysis of the presence of aDec at the surface of Lp-WT (in black) compared to Lp-Lip-aDec, Lp-CWA-aDec or Lp-LysM-aDec (all in red).
Mentions: A DNA fragment encoding the anti-mouse Dec205 ScFv (aDec) preceded by a HA-tag for immune detection was cloned into plasmids previously developed for production and surface localization of proteins in L. plantarum (32, 26) using three different types of anchors as outlined in Figure 1a, b. The anchors are: a Lipobox membrane anchor for covalent coupling of aDec to a membrane component, a cell wall anchor based on sortase-catalyzed covalent coupling to the peptidoglycan and a cell wall anchor based on non-covalent interactions between a LysM domain and the cell wall. The three resulting plasmids, pLip-aDec, pCWA-aDec and pLysM-aDec (Figure 1b) were transformed into L. plantarum. In addition, we used Lp-WT, containing a control plasmid (pEV) [31] that does not encode for the anchors or aDec, as a negative control. The growth rate of strains producing aDec was substantially lower compared to control strain (Figure 1c), in particular for the strain harboring the lipoprotein anchor. Still, all strains showed reasonable growth and bacteria harvested 2 h after induction, where all transformants have a similar OD600 (Figure 1c) and similar cell counts, were used for further studies.Figure 1

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