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Nanobody mediated inhibition of attachment of F18 Fimbriae expressing Escherichia coli.

Moonens K, De Kerpel M, Coddens A, Cox E, Pardon E, Remaut H, De Greve H - PLoS ONE (2014)

Bottom Line: Crystallization of the FedF lectin domain with the most potent inhibitory nanobodies revealed their mechanism of action.These either competed with the binding of the blood group antigen receptor on the FedF surface or induced a conformational change in which the CDR3 region of the nanobody displaces the D″-E loop adjacent to the binding site.This work demonstrates the feasibility of inhibiting the attachment of fimbriated pathogens by employing nanobodies directed against the adhesin domain.

View Article: PubMed Central - PubMed

Affiliation: Structural & Molecular Microbiology, Structural Biology Research Center, VIB, Brussels, Belgium; Structural Biology Brussels, Vrije Universiteit Brussel, Pleinlaan 2, 1050 Brussels, Belgium.

ABSTRACT
Post-weaning diarrhea and edema disease caused by F18 fimbriated E. coli are important diseases in newly weaned piglets and lead to severe production losses in farming industry. Protective treatments against these infections have thus far limited efficacy. In this study we generated nanobodies directed against the lectin domain of the F18 fimbrial adhesin FedF and showed in an in vitro adherence assay that four unique nanobodies inhibit the attachment of F18 fimbriated E. coli bacteria to piglet enterocytes. Crystallization of the FedF lectin domain with the most potent inhibitory nanobodies revealed their mechanism of action. These either competed with the binding of the blood group antigen receptor on the FedF surface or induced a conformational change in which the CDR3 region of the nanobody displaces the D″-E loop adjacent to the binding site. This D″-E loop was previously shown to be required for the interaction between F18 fimbriated bacteria and blood group antigen receptors in a membrane context. This work demonstrates the feasibility of inhibiting the attachment of fimbriated pathogens by employing nanobodies directed against the adhesin domain.

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Related in: MedlinePlus

Nanobodies that induce a conformational change in the D″-E loop do not inhibit the attachment of FedF towards the A6-1 carbohydrate.Consecutive injections of either FedF15–165 or FedF15–165-nanobody complexes over the sensor chip surface carrying an immobilized A6-1-human serum albumin glycoconjugate were performed. Nanobody NbFedF9, shown in the crystal structure to bind in the FedF carbohydrate binding site, completely blocks the FedF-A6-1 interaction. In contrary, nanobodies NbFedF6, NbFedF7 and NbFedF12 only slightly or not at all inhibit the binding of FedF on the A6-1 coated surface. The crystal structures show how NbFedF6 and NbFedF7 induce a conformational change in the D″-E loop but do not steric compete with A6-1 binding.
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pone-0114691-g006: Nanobodies that induce a conformational change in the D″-E loop do not inhibit the attachment of FedF towards the A6-1 carbohydrate.Consecutive injections of either FedF15–165 or FedF15–165-nanobody complexes over the sensor chip surface carrying an immobilized A6-1-human serum albumin glycoconjugate were performed. Nanobody NbFedF9, shown in the crystal structure to bind in the FedF carbohydrate binding site, completely blocks the FedF-A6-1 interaction. In contrary, nanobodies NbFedF6, NbFedF7 and NbFedF12 only slightly or not at all inhibit the binding of FedF on the A6-1 coated surface. The crystal structures show how NbFedF6 and NbFedF7 induce a conformational change in the D″-E loop but do not steric compete with A6-1 binding.

Mentions: Crystal structures of the complexes between FedF15–165 and both NbFedF6 and NbFedF7 were as well obtained to a resolution of respectively 2.5 Å and 1.7 Å (Fig. 4). In both elucidated complexes the nanobodies are occupying an overlapping epitope formed by strands D′ and D″ at the interface between the two β-sheets of FedF (Fig. 4). Nearly all interactions between NbFedF6 and FedF are mediated by the CDR3 loop. In the NbFedF7-FedF complex the contribution of CDR3 to the total binding affinity is even more pronounced as the other CDRs are not involved in any direct hydrogen bond formation at all. Direct hydrogen bond interactions that stabilize the NbFedF6-FedF15–165 complex are Arg45 (NbFedF6) and Ala96 (FedF), Ser54 (NbFedF6) and Gln84 (FedF), Phe100 (NbFedF6) and both Gly92/Asn81 (FedF), Tyr102 (NbFedF6) and Asn81 (FedF), Gln109 (NbFedF6) and both Gly98/Ala96 (FedF), Ala110 (NbFedF6) and Gly94 (FedF) (S4 Figure). In the NbFedF7-FedF complex Trp111 is inserted in a deep hydrophobic groove on the FedF surface with optimal shape complementary and as well forms a direct interaction with Ser79; other important interactions are Ser100 (NbFedF7) and Thr46 (FedF), Asn101 (NbFedF7) and both Asn81/Gly92 (FedF), Ser102 (NbFedF7) and Gln91 (FedF), Ala110 (NbFedF7) and Gly94 (FedF), Asn113 (NbFedF7) and Ser44 (FedF) (S4 Figure). Although a near identical epitope is targeted by NbFedF6 and NbFedF7 they differ significantly in the sequence of their CDR3 loop (S1 Figure) and the residues involved in the recognition of the epitope on the FedF surface. Both nanobody NbFedF6 and NbFedF7 are interacting distant from the A6-1 binding site (Fig. 5), thus contrary to the inhibitory complex between FedF- NbFedF9 in which NbFedF9 directly competed with the blood group antigen binding site. When superimposing the crystal structures of the FedF-A6-1 complex with the NbFedF6/7-FedF complex it shows how both nanobodies induce a conformational change in the D″-E loop (Fig. 5). The D″-E loop is displaced more outwards relative to the A6-1 binding site by the CDR3 loop of the nanobody and thereby pushed slightly upwards relative to the FedF surface. The conformation of none of the amino acid residues identified in our earlier study to interact with the A6-1 ligand is affected significantly [17]. To confirm that both nanobodies can still bind the ligand we performed an inhibition experiment using surface plasmon resonance. FedF was mixed with a fixed concentration of the different inhibitory nanobodies and injected over a chip on which a human serum albumine-A6-1 glycoconjugate was immobilized. As could be expected NbFedF9 completely abolished the binding of FedF with A6-1 (Fig 6). On the contrary when adding an excess of nanobodies NbFedF6 and NbFedF7, and as well nanobody NbFedF12, FedF was still able to fully or partially interact with the A6-1-HSA glycoconjugate (Fig. 6), and in addition these nanobodies are not altering the binding kinetics of the interaction. These results demonstrate the conformational change induced by the nanobodies NbFedF6 and NbFedF7 cannot completely explain their inhibitory capacity. This is despite their full binding inhibition in a biological context, when FedF is binding membrane-embedded sphingolipids. The D″-E loop harbors two positively charged lysine residues that we identified previously to add non-specific binding affinity in proximity to the membrane [17]. By targeting this loop and changing its conformation we could thus block the affinity of F18 fimbriated bacteria towards membrane imbedded glycosphingolipid receptors. Either this inhibitory effect stems from the disruption of the conformation of the critical D″-E loop, or another possibility that cannot be excluded is that NbFedF6 and NbFedF7 upon binding impart steric hindrance with the nearby phospholipid bilayer.


Nanobody mediated inhibition of attachment of F18 Fimbriae expressing Escherichia coli.

Moonens K, De Kerpel M, Coddens A, Cox E, Pardon E, Remaut H, De Greve H - PLoS ONE (2014)

Nanobodies that induce a conformational change in the D″-E loop do not inhibit the attachment of FedF towards the A6-1 carbohydrate.Consecutive injections of either FedF15–165 or FedF15–165-nanobody complexes over the sensor chip surface carrying an immobilized A6-1-human serum albumin glycoconjugate were performed. Nanobody NbFedF9, shown in the crystal structure to bind in the FedF carbohydrate binding site, completely blocks the FedF-A6-1 interaction. In contrary, nanobodies NbFedF6, NbFedF7 and NbFedF12 only slightly or not at all inhibit the binding of FedF on the A6-1 coated surface. The crystal structures show how NbFedF6 and NbFedF7 induce a conformational change in the D″-E loop but do not steric compete with A6-1 binding.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4263667&req=5

pone-0114691-g006: Nanobodies that induce a conformational change in the D″-E loop do not inhibit the attachment of FedF towards the A6-1 carbohydrate.Consecutive injections of either FedF15–165 or FedF15–165-nanobody complexes over the sensor chip surface carrying an immobilized A6-1-human serum albumin glycoconjugate were performed. Nanobody NbFedF9, shown in the crystal structure to bind in the FedF carbohydrate binding site, completely blocks the FedF-A6-1 interaction. In contrary, nanobodies NbFedF6, NbFedF7 and NbFedF12 only slightly or not at all inhibit the binding of FedF on the A6-1 coated surface. The crystal structures show how NbFedF6 and NbFedF7 induce a conformational change in the D″-E loop but do not steric compete with A6-1 binding.
Mentions: Crystal structures of the complexes between FedF15–165 and both NbFedF6 and NbFedF7 were as well obtained to a resolution of respectively 2.5 Å and 1.7 Å (Fig. 4). In both elucidated complexes the nanobodies are occupying an overlapping epitope formed by strands D′ and D″ at the interface between the two β-sheets of FedF (Fig. 4). Nearly all interactions between NbFedF6 and FedF are mediated by the CDR3 loop. In the NbFedF7-FedF complex the contribution of CDR3 to the total binding affinity is even more pronounced as the other CDRs are not involved in any direct hydrogen bond formation at all. Direct hydrogen bond interactions that stabilize the NbFedF6-FedF15–165 complex are Arg45 (NbFedF6) and Ala96 (FedF), Ser54 (NbFedF6) and Gln84 (FedF), Phe100 (NbFedF6) and both Gly92/Asn81 (FedF), Tyr102 (NbFedF6) and Asn81 (FedF), Gln109 (NbFedF6) and both Gly98/Ala96 (FedF), Ala110 (NbFedF6) and Gly94 (FedF) (S4 Figure). In the NbFedF7-FedF complex Trp111 is inserted in a deep hydrophobic groove on the FedF surface with optimal shape complementary and as well forms a direct interaction with Ser79; other important interactions are Ser100 (NbFedF7) and Thr46 (FedF), Asn101 (NbFedF7) and both Asn81/Gly92 (FedF), Ser102 (NbFedF7) and Gln91 (FedF), Ala110 (NbFedF7) and Gly94 (FedF), Asn113 (NbFedF7) and Ser44 (FedF) (S4 Figure). Although a near identical epitope is targeted by NbFedF6 and NbFedF7 they differ significantly in the sequence of their CDR3 loop (S1 Figure) and the residues involved in the recognition of the epitope on the FedF surface. Both nanobody NbFedF6 and NbFedF7 are interacting distant from the A6-1 binding site (Fig. 5), thus contrary to the inhibitory complex between FedF- NbFedF9 in which NbFedF9 directly competed with the blood group antigen binding site. When superimposing the crystal structures of the FedF-A6-1 complex with the NbFedF6/7-FedF complex it shows how both nanobodies induce a conformational change in the D″-E loop (Fig. 5). The D″-E loop is displaced more outwards relative to the A6-1 binding site by the CDR3 loop of the nanobody and thereby pushed slightly upwards relative to the FedF surface. The conformation of none of the amino acid residues identified in our earlier study to interact with the A6-1 ligand is affected significantly [17]. To confirm that both nanobodies can still bind the ligand we performed an inhibition experiment using surface plasmon resonance. FedF was mixed with a fixed concentration of the different inhibitory nanobodies and injected over a chip on which a human serum albumine-A6-1 glycoconjugate was immobilized. As could be expected NbFedF9 completely abolished the binding of FedF with A6-1 (Fig 6). On the contrary when adding an excess of nanobodies NbFedF6 and NbFedF7, and as well nanobody NbFedF12, FedF was still able to fully or partially interact with the A6-1-HSA glycoconjugate (Fig. 6), and in addition these nanobodies are not altering the binding kinetics of the interaction. These results demonstrate the conformational change induced by the nanobodies NbFedF6 and NbFedF7 cannot completely explain their inhibitory capacity. This is despite their full binding inhibition in a biological context, when FedF is binding membrane-embedded sphingolipids. The D″-E loop harbors two positively charged lysine residues that we identified previously to add non-specific binding affinity in proximity to the membrane [17]. By targeting this loop and changing its conformation we could thus block the affinity of F18 fimbriated bacteria towards membrane imbedded glycosphingolipid receptors. Either this inhibitory effect stems from the disruption of the conformation of the critical D″-E loop, or another possibility that cannot be excluded is that NbFedF6 and NbFedF7 upon binding impart steric hindrance with the nearby phospholipid bilayer.

Bottom Line: Crystallization of the FedF lectin domain with the most potent inhibitory nanobodies revealed their mechanism of action.These either competed with the binding of the blood group antigen receptor on the FedF surface or induced a conformational change in which the CDR3 region of the nanobody displaces the D″-E loop adjacent to the binding site.This work demonstrates the feasibility of inhibiting the attachment of fimbriated pathogens by employing nanobodies directed against the adhesin domain.

View Article: PubMed Central - PubMed

Affiliation: Structural & Molecular Microbiology, Structural Biology Research Center, VIB, Brussels, Belgium; Structural Biology Brussels, Vrije Universiteit Brussel, Pleinlaan 2, 1050 Brussels, Belgium.

ABSTRACT
Post-weaning diarrhea and edema disease caused by F18 fimbriated E. coli are important diseases in newly weaned piglets and lead to severe production losses in farming industry. Protective treatments against these infections have thus far limited efficacy. In this study we generated nanobodies directed against the lectin domain of the F18 fimbrial adhesin FedF and showed in an in vitro adherence assay that four unique nanobodies inhibit the attachment of F18 fimbriated E. coli bacteria to piglet enterocytes. Crystallization of the FedF lectin domain with the most potent inhibitory nanobodies revealed their mechanism of action. These either competed with the binding of the blood group antigen receptor on the FedF surface or induced a conformational change in which the CDR3 region of the nanobody displaces the D″-E loop adjacent to the binding site. This D″-E loop was previously shown to be required for the interaction between F18 fimbriated bacteria and blood group antigen receptors in a membrane context. This work demonstrates the feasibility of inhibiting the attachment of fimbriated pathogens by employing nanobodies directed against the adhesin domain.

Show MeSH
Related in: MedlinePlus