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Specific antibody-receptor interactions trigger InlAB-independent uptake of Listeria monocytogenes into tumor cell lines.

Heisig M, Frentzen A, Bergmann B, Galmbacher K, Gentschev I, Hotz C, Schoen C, Stritzker J, Fensterle J, Rapp UR, Goebel W - BMC Microbiol. (2011)

Bottom Line: Here, we describe the construction of a novel, internalin A and B (InlAB)-deficient Listeria monocytogenes strain (Lm-spa+), which expresses protein A of Staphylococcus aureus (SPA) and anchors SPA in the correct orientation on the bacterial cell surface.Binding of Trastuzumab (Herceptin®) or Cetuximab (Erbitux®) to Lm-spa+, two clinically approved monoclonal antibodies directed against HER2/neu and EGFR/HER1, respectively, triggers InlAB-independent internalization into non-phagocytic cancer cell lines overexpressing the respective receptors.Binding of receptor-specific antibodies to SPA-expressing L. monocytogenes may represent a promising approach to target L. monocytogenes to host cells expressing specific receptors triggering internalization.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institut für Medizinische Strahlenkunde und Zellforschung (MSZ), Universität Würzburg, Versbacher Strasse 8, Würzburg, 97078, Deutschland. martin.heisig@yale.edu

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Expression of S. aureus protein A (SPA) on the cell surface of L. monocytogenes strain ΔtrpS,aroA,inlA/B,int::Phly-spa × pFlo-trpS (Lm-spa+). (a) Western blot analysis with polyclonal goat-anti-Protein A antibody of protein extracts from ΔtrpS,aroA,inlA/B × pFlo-trpS (Lm-spa-, lanes 1 and 2) and Lm-spa+ (lanes 3 and 4); lanes 1 and 3: cell surface protein extracts; lanes 2 and 4: internal protein extracts. The arrow indicates the position of SPA in the SDS-PAGE. (b) Immunofluorescence micrographs showing specific binding of antibody Fc-part to SPA on the surface of Lm-spa+. Lm-spa+ were incubated with polyclonal anti-OVA antibody and stained with OVA-FITC protein (vii-ix). Lm-spa- stained with antibody and OVA-FITC (i-iii) and Lm-spa+ stained without antibody but with OVA-FITC protein (iv-vi) were used as negative controls. Phase contrast pictures are shown in the left column; FITC-stained images in middle column; picture overlays in the right column. (c) Flow cytometry quantifying the specific Fc-mediated antibody binding to SPA on the surface of L. monocytogenes strains. Mid-logarithmic grown bacteria were stained with polyclonal FITC-conjugated rabbit-anti-goat immunoglobulin G (H+L). Grey area indicates strain Lm-spa-, while the white area indicates strain Lm-spa+. (d) Western blot analysis was used for indirect quantitation of protein A on the surface of Lm-spa+. 5 × 108 bacteria were incubated simultaneously with antibody directed against native albumin and an excess of albumin. After incubation bacteria were washed and the amount of albumin bound to the bacteria via antibody was quantified by Western blot analysis with a primary antibody directed against denatured albumin. In the right lane 10 ng of pure serum albumin was applied as control.
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Figure 1: Expression of S. aureus protein A (SPA) on the cell surface of L. monocytogenes strain ΔtrpS,aroA,inlA/B,int::Phly-spa × pFlo-trpS (Lm-spa+). (a) Western blot analysis with polyclonal goat-anti-Protein A antibody of protein extracts from ΔtrpS,aroA,inlA/B × pFlo-trpS (Lm-spa-, lanes 1 and 2) and Lm-spa+ (lanes 3 and 4); lanes 1 and 3: cell surface protein extracts; lanes 2 and 4: internal protein extracts. The arrow indicates the position of SPA in the SDS-PAGE. (b) Immunofluorescence micrographs showing specific binding of antibody Fc-part to SPA on the surface of Lm-spa+. Lm-spa+ were incubated with polyclonal anti-OVA antibody and stained with OVA-FITC protein (vii-ix). Lm-spa- stained with antibody and OVA-FITC (i-iii) and Lm-spa+ stained without antibody but with OVA-FITC protein (iv-vi) were used as negative controls. Phase contrast pictures are shown in the left column; FITC-stained images in middle column; picture overlays in the right column. (c) Flow cytometry quantifying the specific Fc-mediated antibody binding to SPA on the surface of L. monocytogenes strains. Mid-logarithmic grown bacteria were stained with polyclonal FITC-conjugated rabbit-anti-goat immunoglobulin G (H+L). Grey area indicates strain Lm-spa-, while the white area indicates strain Lm-spa+. (d) Western blot analysis was used for indirect quantitation of protein A on the surface of Lm-spa+. 5 × 108 bacteria were incubated simultaneously with antibody directed against native albumin and an excess of albumin. After incubation bacteria were washed and the amount of albumin bound to the bacteria via antibody was quantified by Western blot analysis with a primary antibody directed against denatured albumin. In the right lane 10 ng of pure serum albumin was applied as control.

Mentions: Expression of SPA by the constructed Lm strains was analyzed by Western blotting using polyclonal protein A antibody. Bacterial cell surface and cytoplasmic protein fractions were examined after growth of Lm-spa+ in BHI containing 1% amberlite XAD-4. Addition of XAD-4 to the culture medium enhances the activity of the virulence gene activator PrfA and hence leads to an enhanced transcription of the spa gene which is under the control of the PrfA-dependent hly promoter [25]. SPA was readily detected in the cell surface protein fraction of Lm-spa+ and to a lower extent in the internal protein extract fraction. (Figure 1A). As expected, no SPA was present in the parental strain ΔtrpS,aroA,inlA/B × pFlo-trpS, termed Lm-spa- (Figure 1A).


Specific antibody-receptor interactions trigger InlAB-independent uptake of Listeria monocytogenes into tumor cell lines.

Heisig M, Frentzen A, Bergmann B, Galmbacher K, Gentschev I, Hotz C, Schoen C, Stritzker J, Fensterle J, Rapp UR, Goebel W - BMC Microbiol. (2011)

Expression of S. aureus protein A (SPA) on the cell surface of L. monocytogenes strain ΔtrpS,aroA,inlA/B,int::Phly-spa × pFlo-trpS (Lm-spa+). (a) Western blot analysis with polyclonal goat-anti-Protein A antibody of protein extracts from ΔtrpS,aroA,inlA/B × pFlo-trpS (Lm-spa-, lanes 1 and 2) and Lm-spa+ (lanes 3 and 4); lanes 1 and 3: cell surface protein extracts; lanes 2 and 4: internal protein extracts. The arrow indicates the position of SPA in the SDS-PAGE. (b) Immunofluorescence micrographs showing specific binding of antibody Fc-part to SPA on the surface of Lm-spa+. Lm-spa+ were incubated with polyclonal anti-OVA antibody and stained with OVA-FITC protein (vii-ix). Lm-spa- stained with antibody and OVA-FITC (i-iii) and Lm-spa+ stained without antibody but with OVA-FITC protein (iv-vi) were used as negative controls. Phase contrast pictures are shown in the left column; FITC-stained images in middle column; picture overlays in the right column. (c) Flow cytometry quantifying the specific Fc-mediated antibody binding to SPA on the surface of L. monocytogenes strains. Mid-logarithmic grown bacteria were stained with polyclonal FITC-conjugated rabbit-anti-goat immunoglobulin G (H+L). Grey area indicates strain Lm-spa-, while the white area indicates strain Lm-spa+. (d) Western blot analysis was used for indirect quantitation of protein A on the surface of Lm-spa+. 5 × 108 bacteria were incubated simultaneously with antibody directed against native albumin and an excess of albumin. After incubation bacteria were washed and the amount of albumin bound to the bacteria via antibody was quantified by Western blot analysis with a primary antibody directed against denatured albumin. In the right lane 10 ng of pure serum albumin was applied as control.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3142209&req=5

Figure 1: Expression of S. aureus protein A (SPA) on the cell surface of L. monocytogenes strain ΔtrpS,aroA,inlA/B,int::Phly-spa × pFlo-trpS (Lm-spa+). (a) Western blot analysis with polyclonal goat-anti-Protein A antibody of protein extracts from ΔtrpS,aroA,inlA/B × pFlo-trpS (Lm-spa-, lanes 1 and 2) and Lm-spa+ (lanes 3 and 4); lanes 1 and 3: cell surface protein extracts; lanes 2 and 4: internal protein extracts. The arrow indicates the position of SPA in the SDS-PAGE. (b) Immunofluorescence micrographs showing specific binding of antibody Fc-part to SPA on the surface of Lm-spa+. Lm-spa+ were incubated with polyclonal anti-OVA antibody and stained with OVA-FITC protein (vii-ix). Lm-spa- stained with antibody and OVA-FITC (i-iii) and Lm-spa+ stained without antibody but with OVA-FITC protein (iv-vi) were used as negative controls. Phase contrast pictures are shown in the left column; FITC-stained images in middle column; picture overlays in the right column. (c) Flow cytometry quantifying the specific Fc-mediated antibody binding to SPA on the surface of L. monocytogenes strains. Mid-logarithmic grown bacteria were stained with polyclonal FITC-conjugated rabbit-anti-goat immunoglobulin G (H+L). Grey area indicates strain Lm-spa-, while the white area indicates strain Lm-spa+. (d) Western blot analysis was used for indirect quantitation of protein A on the surface of Lm-spa+. 5 × 108 bacteria were incubated simultaneously with antibody directed against native albumin and an excess of albumin. After incubation bacteria were washed and the amount of albumin bound to the bacteria via antibody was quantified by Western blot analysis with a primary antibody directed against denatured albumin. In the right lane 10 ng of pure serum albumin was applied as control.
Mentions: Expression of SPA by the constructed Lm strains was analyzed by Western blotting using polyclonal protein A antibody. Bacterial cell surface and cytoplasmic protein fractions were examined after growth of Lm-spa+ in BHI containing 1% amberlite XAD-4. Addition of XAD-4 to the culture medium enhances the activity of the virulence gene activator PrfA and hence leads to an enhanced transcription of the spa gene which is under the control of the PrfA-dependent hly promoter [25]. SPA was readily detected in the cell surface protein fraction of Lm-spa+ and to a lower extent in the internal protein extract fraction. (Figure 1A). As expected, no SPA was present in the parental strain ΔtrpS,aroA,inlA/B × pFlo-trpS, termed Lm-spa- (Figure 1A).

Bottom Line: Here, we describe the construction of a novel, internalin A and B (InlAB)-deficient Listeria monocytogenes strain (Lm-spa+), which expresses protein A of Staphylococcus aureus (SPA) and anchors SPA in the correct orientation on the bacterial cell surface.Binding of Trastuzumab (Herceptin®) or Cetuximab (Erbitux®) to Lm-spa+, two clinically approved monoclonal antibodies directed against HER2/neu and EGFR/HER1, respectively, triggers InlAB-independent internalization into non-phagocytic cancer cell lines overexpressing the respective receptors.Binding of receptor-specific antibodies to SPA-expressing L. monocytogenes may represent a promising approach to target L. monocytogenes to host cells expressing specific receptors triggering internalization.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institut für Medizinische Strahlenkunde und Zellforschung (MSZ), Universität Würzburg, Versbacher Strasse 8, Würzburg, 97078, Deutschland. martin.heisig@yale.edu

Show MeSH
Related in: MedlinePlus