Anti-InlA single-domain antibodies that inhibit the cell invasion of Listeria monocytogenes

Abstract

Listeriosis, caused by infection with Listeria monocytogenes, is a severe disease with a high mortality rate. The L. monocytogenes virulence factor, internalin family protein InlA, which binds to the host receptor E-cadherin, is necessary to invade host cells. Here, we isolated two single-domain antibodies (V_HHs) that bind to InlA with picomolar affinities from an alpaca immune library using the phage display method. These InlA-specific V_HHs inhibited the binding of InlA to the extracellular domains of E-cadherin in vitro as shown by biophysical interaction analysis. Furthermore, we determined that the V_HHs inhibited the invasion of L. monocytogenes into host cells in culture. High-resolution X-ray structure analyses of the complexes of V_HHs with InlA revealed that the V_HHs bind to the same binding site as E-cadherin against InlA. We conclude that these V_HHs have the potential for use as drugs to treat listeriosis.

Keywords: InlA; Listeria monocytogenes; isothermal titration calorimetry (ITC); listeriosis; protein-protein interaction; single-domain V(H)H antibody; surface plasmon resonance (SPR).

Figure 1

Selection of anti-InlA V_HH antibodies.A, phage ELISA analysis of single clones from phage display library. ELISA assays were performed twice on each clone (first and second). The asterisks mark clones selected for sequence analysis (total 28 clones). B, SPR analysis of the interaction between InlA and vhh10. C, SPR analysis of the interaction between InlA and vhh24. D, SPR analysis of the interaction between InlA and D3-L11. The color lines are the raw data and the black lines are the fitting curves in SPR. E, ITC analysis of the interaction between InlA and vhh10. F, ITC analysis of the interaction between InlA and vhh24. G, ITC analysis of the interaction between InlA and D3-L11. ITC, isothermal titration calorimetry; SPR, surface plasmon resonance.

Figure 2

Anti-InlA V_HHs inhibit the interaction between InlA and EC12. ITC profiles of the interaction of InlA and EC12 (A) in the absence of V_HH, (B) in the presence of vhh10, and (C) in the presence of vhh24. EC, extracellular cadherin; ITC, isothermal titration calorimetry.

Figure 3

Anti-InlA V_HHs inhibit invasion of L. monocytogenes into human cells in a dose-dependent manner.A, percentages of bacterial cells adhered to Caco-2 cells in the presence of vhh10 and vhh24 at indicated concentrations. B, percentage of bacterial cells inside Caco-2 cells in the presence of vhh10 and vhh24 at indicated concentrations. C, percentages of V_HH pretreated bacterial cells adhered to Caco-2 cells. D, percentages of V_HH pretreated bacterial cells inside Caco-2 cells. Anti-lysozyme V_HH antibody D3-L11 was used as a control. All values are expressed as mean ± S.D. One-way ANOVA, followed by Dunnett's test was used to evaluate differences compared to control. ∗∗∗∗ p < 0.0001, ∗∗∗ p < 0.001.

Figure 4

Three dimensional structures of InlA in complex with the EC12 domains of E-cadherin.A, InlA (gray) in complex with EC12 (green). B, structure of InlA with EC12 contact interface in green. C, interactions in the InlA-EC12 complex. InlA and EC12 are drawn as cartoon displays in gray and green, respectively. Ca²⁺ is depicted as blue spheres. Structures were created with Pymol software (Version 2.3.2, Schrödinger, LLC). EC, extracellular cadherin.

Figure 5

Interactions in the InlA-V_HHs complex.A, InlA (gray) in complex with vhh10 (magenta). B, structure of InlA with vhh10 contact interface in magenta color. C, InlA (gray) in complex with vhh24 (orange). D, structure of InlA with vhh24 contact interface in orange. E, overlay of complexes. The LRR region of InlA is shown in beige color. All of the structures were created with Pymol software (Version 2.3.2, Schrödinger, LLC).

Figure 6

Three dimensional structures of InlA in complex with the V_HHs.A, interactions at CDR-H1, CDR-H2, and CDR-H3 in the InlA-vhh10 complex. InlA, vhh10, and CDR are drawn as cartoon displays in gray, magenta, and light purple, respectively. B, interactions at CDR-H1, CDR-H2, and CDR-H3 in the InlA-vhh24 complex. InlA, vhh24, and CDR are drawn as cartoon displays in gray, orange, and light purple, respectively. Structures were created with Pymol software (Version 2.3.2, Schrödinger, LLC). CDR, complementarity-determining region.