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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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Inhibitory nanobodies recognize FedF15–165 with low nanomolar affinity.Microscale thermophoresis (MST) was employed to determine the in solution affinity between Nb-FedF6, Nb-FedF7, Nb-FedF9 and Nb-FedF12 with FedF15–165. (A) Typical MST measurement showing the interaction between Nb-FedF6 and FedF15–165. Data points are indicated by black diamonds, the fit by the NT Analysis software is shown as a red line. (B) Overview on the determined dissociation constants (KD) for the indicated Nb-FedF15–165 interactions.
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pone-0114691-g002: Inhibitory nanobodies recognize FedF15–165 with low nanomolar affinity.Microscale thermophoresis (MST) was employed to determine the in solution affinity between Nb-FedF6, Nb-FedF7, Nb-FedF9 and Nb-FedF12 with FedF15–165. (A) Typical MST measurement showing the interaction between Nb-FedF6 and FedF15–165. Data points are indicated by black diamonds, the fit by the NT Analysis software is shown as a red line. (B) Overview on the determined dissociation constants (KD) for the indicated Nb-FedF15–165 interactions.

Mentions: A purified truncate (residues 15–165) of the FedF tipadhesin (FedF15–165) corresponding to the N-terminal lectin domain, where the binding capability to blood group type 1 antigens was shown to reside [17], was used to generate specific nanobodies [35]. Despite the smaller size of nanobodies compared to conventional antibodies they confer high affinity and antigen specificity, high stability and solubility, and are easy and inexpensive to produce [36]. A llama was immunized with FedF15–165 and specific nanobodies were selected by panning the immune library, derived from the llama peripheral blood lymphocytes, in two consecutive rounds using the phage display technology [21]. In total twelve nanobodies specific for FedF15–165 were obtained and further used to screen for their inhibitory capacity in an in vitro adherence assay on piglet enterocytes. The wild type E. coli strain 107/86 expressing F18 fimbriae [14] was incubated with each of the twelve different nanobodies and then added to the villi of piglets. The amount of bacteria adhering to the villi lining were counted, the results are summarized in Fig. 1 and showed that three categories of nanobodies could be distinguished. The adherence of the first group of nanobodies (NbFedF2, NbFedF3, NbFedF4, NbFedF8 and NbFedF11) remains unchanged compared to the control sample without added nanobody. In a second category, binding was reduced compared to wild type binding but still residual binding to the piglets enterocytes remained (NbFedF1, NbFedF5 and NbFedF10). A third group of nanobodies leads to the (near) complete loss of attachment of the wild type strain 107/86 to piglet villi (NbFedF6, NbFedF7, NbFedF9 and NbFedF12). Sequence alignment of these four inhibitory nanobodies reveals great sequence variability between them in all three complementary determining regions (S1 Figure), hinting that varying epitopes are recognized by these nanobodies. Microscale thermophoresis was used to determine the in solution affinity between FedF15–165 and the four inhibitory nanobodies (Fig. 2). Nanobodies NbFedF6, NbFedF7, NbFedF9 and NbFedF12 recognize FedF with low nanomolar affinity (Kd's of 3.57, 5.25, 1.58 and 29.02 nM, respectively), which is up to a thousand fold higher affinity in comparison with the interaction between FedF and the natural occurring glycan ligand blood group A type 1 hexasaccharide (2.9 µM) [17]. In the next sections we further characterized the three best inhibiting nanobodies (NbFedF6, NbFedF7 and NbFedF9) by X-ray crystallography.


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)

Inhibitory nanobodies recognize FedF15–165 with low nanomolar affinity.Microscale thermophoresis (MST) was employed to determine the in solution affinity between Nb-FedF6, Nb-FedF7, Nb-FedF9 and Nb-FedF12 with FedF15–165. (A) Typical MST measurement showing the interaction between Nb-FedF6 and FedF15–165. Data points are indicated by black diamonds, the fit by the NT Analysis software is shown as a red line. (B) Overview on the determined dissociation constants (KD) for the indicated Nb-FedF15–165 interactions.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0114691-g002: Inhibitory nanobodies recognize FedF15–165 with low nanomolar affinity.Microscale thermophoresis (MST) was employed to determine the in solution affinity between Nb-FedF6, Nb-FedF7, Nb-FedF9 and Nb-FedF12 with FedF15–165. (A) Typical MST measurement showing the interaction between Nb-FedF6 and FedF15–165. Data points are indicated by black diamonds, the fit by the NT Analysis software is shown as a red line. (B) Overview on the determined dissociation constants (KD) for the indicated Nb-FedF15–165 interactions.
Mentions: A purified truncate (residues 15–165) of the FedF tipadhesin (FedF15–165) corresponding to the N-terminal lectin domain, where the binding capability to blood group type 1 antigens was shown to reside [17], was used to generate specific nanobodies [35]. Despite the smaller size of nanobodies compared to conventional antibodies they confer high affinity and antigen specificity, high stability and solubility, and are easy and inexpensive to produce [36]. A llama was immunized with FedF15–165 and specific nanobodies were selected by panning the immune library, derived from the llama peripheral blood lymphocytes, in two consecutive rounds using the phage display technology [21]. In total twelve nanobodies specific for FedF15–165 were obtained and further used to screen for their inhibitory capacity in an in vitro adherence assay on piglet enterocytes. The wild type E. coli strain 107/86 expressing F18 fimbriae [14] was incubated with each of the twelve different nanobodies and then added to the villi of piglets. The amount of bacteria adhering to the villi lining were counted, the results are summarized in Fig. 1 and showed that three categories of nanobodies could be distinguished. The adherence of the first group of nanobodies (NbFedF2, NbFedF3, NbFedF4, NbFedF8 and NbFedF11) remains unchanged compared to the control sample without added nanobody. In a second category, binding was reduced compared to wild type binding but still residual binding to the piglets enterocytes remained (NbFedF1, NbFedF5 and NbFedF10). A third group of nanobodies leads to the (near) complete loss of attachment of the wild type strain 107/86 to piglet villi (NbFedF6, NbFedF7, NbFedF9 and NbFedF12). Sequence alignment of these four inhibitory nanobodies reveals great sequence variability between them in all three complementary determining regions (S1 Figure), hinting that varying epitopes are recognized by these nanobodies. Microscale thermophoresis was used to determine the in solution affinity between FedF15–165 and the four inhibitory nanobodies (Fig. 2). Nanobodies NbFedF6, NbFedF7, NbFedF9 and NbFedF12 recognize FedF with low nanomolar affinity (Kd's of 3.57, 5.25, 1.58 and 29.02 nM, respectively), which is up to a thousand fold higher affinity in comparison with the interaction between FedF and the natural occurring glycan ligand blood group A type 1 hexasaccharide (2.9 µM) [17]. In the next sections we further characterized the three best inhibiting nanobodies (NbFedF6, NbFedF7 and NbFedF9) by X-ray crystallography.

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