Limits...
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.

Show MeSH

Related in: MedlinePlus

NbFedF9 inhibits the binding of F18 fimbriated E. coli to piglet enterocytes by occupying the carbohydrate binding site on the FedF surface.Left: structure of the complex between the inhibitory NbFedF9 (green) and FedF15–165 (β-strands, α-helices and loops are colored respectively cyan, red and grey), which shows NbFedF9 interacting at the side of the FedF fold. The three complementary determining regions (CDRs) of NbFedF9 are colored respectively in orange, purple and yellow. Right: overlay of the NbFedF9-FedF15–165 structure with the previously elucidated structure of the co-complex between FedF15-165 and the blood group A type 1 hexasaccharide [17]. Both the carbohydrate ligand and NbFedF9 compete for the same binding site on the FedF fold. Blood group A type 1 hexasaccharide is depicted in stick model with carbon, oxygen and nitrogen atoms colored respectively purple, red and blue.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4263667&req=5

pone-0114691-g003: NbFedF9 inhibits the binding of F18 fimbriated E. coli to piglet enterocytes by occupying the carbohydrate binding site on the FedF surface.Left: structure of the complex between the inhibitory NbFedF9 (green) and FedF15–165 (β-strands, α-helices and loops are colored respectively cyan, red and grey), which shows NbFedF9 interacting at the side of the FedF fold. The three complementary determining regions (CDRs) of NbFedF9 are colored respectively in orange, purple and yellow. Right: overlay of the NbFedF9-FedF15–165 structure with the previously elucidated structure of the co-complex between FedF15-165 and the blood group A type 1 hexasaccharide [17]. Both the carbohydrate ligand and NbFedF9 compete for the same binding site on the FedF fold. Blood group A type 1 hexasaccharide is depicted in stick model with carbon, oxygen and nitrogen atoms colored respectively purple, red and blue.

Mentions: Crystallization trials of the NbFedF9-FedF15–165 co-complex were set up in order to obtain structural information about the inhibitory mechanism of NbFedF9. These efforts resulted in the elucidation of the FedF15–165- NbFedF9 structure to a resolution of 1.5 angstrom using molecular replacement with the earlier determined apo-FedF15–165 structure (PDB identifier 4B4P). NbFedF9 interacts along the side of the FedF immunoglobulin-like fold (Fig. 3 left) by exclusively hydrogen bond formation, either directly by the interaction between residues of both NbFedF9 and FedF15–165 or indirectly with intermediary water molecules (S2 Figure). All but one of the interactions between NbFedF9 and FedF15–165 are governed by CDR3, which is more expanded in nanobodies compared to their VHVL antibody counterpart. Important direct interactions that stabilize the complex are hydrogen bonds between the side chains of His88 (FedF) and Tyr114 (NbFedF9), Arg117 (FedF) and Glu101 (NbFedF9), Glu122 (FedF) and Arg112 (NbFedF9), Glu96 (FedF) and Arg108 (NbFedF9) (S2 Figure). As well two direct interactions involving only main chain atoms are formed, more precisely between the amide group of Ile94 (FedF) and the carboxyl group of Arg108 (NbFedF9) and between the carboxyl group of Gly92 (FedF) and the amide group of Ser110 (NbFedF9) (S2 Figure). When the structure of the previously elucidated co-complex between FedF15–165 and the blood group A type 1 hexasaccharide (A6-1)[17] is overlaid on the NbFedF9-FedF15–165 complex it shows clearly how the binding sites for the glycan A6-1 and NbFedF9 on the surface of FedF are overlapping (Fig. 3 right; S3 Figure). Amino acid residues His88 and Arg117 on the FedF surface have been shown to be crucial in mediating the attachment of F18-fimbriated bacteria towards enterocytes in a mutational study [17]. In the FedF- NbFedF9 crystal structure these residues are involved in the formation of hydrogen bonds with NbFedF9 and thus unable to interact with the A6-1 ligand (Fig. 3; S3 Figure). All together the presented co-complex structure demonstrates how NbFedF9 inhibits the attachment of F18 fimbriated E. coli to villi by directly competing with the carbohydrate binding site on the surface of FedF.


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)

NbFedF9 inhibits the binding of F18 fimbriated E. coli to piglet enterocytes by occupying the carbohydrate binding site on the FedF surface.Left: structure of the complex between the inhibitory NbFedF9 (green) and FedF15–165 (β-strands, α-helices and loops are colored respectively cyan, red and grey), which shows NbFedF9 interacting at the side of the FedF fold. The three complementary determining regions (CDRs) of NbFedF9 are colored respectively in orange, purple and yellow. Right: overlay of the NbFedF9-FedF15–165 structure with the previously elucidated structure of the co-complex between FedF15-165 and the blood group A type 1 hexasaccharide [17]. Both the carbohydrate ligand and NbFedF9 compete for the same binding site on the FedF fold. Blood group A type 1 hexasaccharide is depicted in stick model with carbon, oxygen and nitrogen atoms colored respectively purple, red and blue.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0114691-g003: NbFedF9 inhibits the binding of F18 fimbriated E. coli to piglet enterocytes by occupying the carbohydrate binding site on the FedF surface.Left: structure of the complex between the inhibitory NbFedF9 (green) and FedF15–165 (β-strands, α-helices and loops are colored respectively cyan, red and grey), which shows NbFedF9 interacting at the side of the FedF fold. The three complementary determining regions (CDRs) of NbFedF9 are colored respectively in orange, purple and yellow. Right: overlay of the NbFedF9-FedF15–165 structure with the previously elucidated structure of the co-complex between FedF15-165 and the blood group A type 1 hexasaccharide [17]. Both the carbohydrate ligand and NbFedF9 compete for the same binding site on the FedF fold. Blood group A type 1 hexasaccharide is depicted in stick model with carbon, oxygen and nitrogen atoms colored respectively purple, red and blue.
Mentions: Crystallization trials of the NbFedF9-FedF15–165 co-complex were set up in order to obtain structural information about the inhibitory mechanism of NbFedF9. These efforts resulted in the elucidation of the FedF15–165- NbFedF9 structure to a resolution of 1.5 angstrom using molecular replacement with the earlier determined apo-FedF15–165 structure (PDB identifier 4B4P). NbFedF9 interacts along the side of the FedF immunoglobulin-like fold (Fig. 3 left) by exclusively hydrogen bond formation, either directly by the interaction between residues of both NbFedF9 and FedF15–165 or indirectly with intermediary water molecules (S2 Figure). All but one of the interactions between NbFedF9 and FedF15–165 are governed by CDR3, which is more expanded in nanobodies compared to their VHVL antibody counterpart. Important direct interactions that stabilize the complex are hydrogen bonds between the side chains of His88 (FedF) and Tyr114 (NbFedF9), Arg117 (FedF) and Glu101 (NbFedF9), Glu122 (FedF) and Arg112 (NbFedF9), Glu96 (FedF) and Arg108 (NbFedF9) (S2 Figure). As well two direct interactions involving only main chain atoms are formed, more precisely between the amide group of Ile94 (FedF) and the carboxyl group of Arg108 (NbFedF9) and between the carboxyl group of Gly92 (FedF) and the amide group of Ser110 (NbFedF9) (S2 Figure). When the structure of the previously elucidated co-complex between FedF15–165 and the blood group A type 1 hexasaccharide (A6-1)[17] is overlaid on the NbFedF9-FedF15–165 complex it shows clearly how the binding sites for the glycan A6-1 and NbFedF9 on the surface of FedF are overlapping (Fig. 3 right; S3 Figure). Amino acid residues His88 and Arg117 on the FedF surface have been shown to be crucial in mediating the attachment of F18-fimbriated bacteria towards enterocytes in a mutational study [17]. In the FedF- NbFedF9 crystal structure these residues are involved in the formation of hydrogen bonds with NbFedF9 and thus unable to interact with the A6-1 ligand (Fig. 3; S3 Figure). All together the presented co-complex structure demonstrates how NbFedF9 inhibits the attachment of F18 fimbriated E. coli to villi by directly competing with the carbohydrate binding site on the surface of FedF.

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