Nanobody mediated inhibition of attachment of F18 fimbriae expressing Escherichia coli

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

Binding of the F18 fimbrial adhesin FedF to piglet intestinal epithelium involves specific receptor recognition and non-specific electrostatic attraction with the phospholipid membrane

Post-weaning diarrhoea and oedema disease are serious infectious diseases of piglets caused by pathogenic E. coli strains, including enterotoxigenic E. coli (ETEC) and Shiga toxin-producing E. coli (STEC). These strains account for substantial economical losses in the pig industry (Bertschinger et al., 1994; E. coli in Domestic Animals and Humans (Gyles, C. L. ed) pp. 193, CAB, Wallingford, Oxon, UK). The first and crucial step during most of these pathogenic infections is the recognition and adhesion of these pathogens to a specific host tissue. Adhesins mediating such an interaction are often presented to the host cell receptors by pili or fimbriae. We examined FedF, the adhesive tipsubunit of F18 fimbriae expressed by STEC which infect recently weaned piglets. Recently, the carbohydrates interacting with the FedF adhesin were identified by Coddens et al. (2009, J. Biol. Chem. 284, 9713). The authors showed that FedF is interacting with ABH blood group type 1 determinants

Secretion and functional display of fusion proteins through the curli biogenesis pathway.

Curli are functional amyloids expressed as fibres on the surface of Enterobacteriaceae. Contrary to the protein misfolding events associated with pathogenic amyloidosis, curli are the result of a dedicated biosynthetic pathway. A specialized transporter in the outer membrane, CsgG, operates in conjunction with the two accessory proteins CsgE and CsgF to secrete curlin subunits to the extracellular surface, where they nucleate into cross-beta strand fibres. Here we investigate the substrate tolerance of the CsgG transporter and the capability of heterologous sequences to be built into curli fibres. Non-native polypeptides ranging up to at least 260 residues were exported when fused to the curli subunit CsgA. Secretion efficiency depended on the folding properties of the passenger sequences, with substrates exceeding an approximately 2 nm transverse diameter blocking passage through the transport channel. Secretion of smaller passengers was compatible with prior DsbA-mediated disulphide bridge formation in the fusion partner, indicating that CsgG is capable of translocating non-linear polypeptide stretches. Using fusions we further demonstrate the exported or secreted heterologous passenger proteins can attain their native, active fold, establishing curli biogenesis pathway as a platform for the secretion and surface display of small heterologous proteins.Journal ArticleSCOPUS: ar.jFLWINSCOPUS: ar.jinfo:eu-repo/semantics/publishe

Structural and thermodynamic characterization of pre- and postpolymerization states in the F4 fimbrial subunit FaeG.

Enterotoxigenic Escherichia coli expressing F4 fimbriae are the major cause of porcine colibacillosis and are responsible for significant death and morbidity in neonatal and postweaned piglets. Via the chaperone-usher pathway, F4 fimbriae are assembled into thin, flexible polymers mainly composed of the single-domain adhesin FaeG. The F4 fimbrial system has been labeled eccentric because the F4 pilins show some features distinct from the features of pilins of other chaperone-usher-assembled structures. In particular, FaeG is much larger than other pilins (27 versus approximately 17 kDa), grafting an additional carbohydrate binding domain on the common immunoglobulin-like core. Structural data of FaeG during different stages of the F4 fimbrial biogenesis process, combined with differential scanning calorimetry measurements, confirm the general principles of the donor strand complementation/exchange mechanisms taking place during pilus biogenesis via the chaperone-usher pathway.info:eu-repo/semantics/publishe

Blocking the attachment of F18 positive <i>E. coli</i> to piglet enterocytes by nanobodies.

<p>Nanobodies directed against the N-terminal domain of the FedF tipadhesin (FedF_15–165) were assayed in an <i>in vitro</i> adherence test of wild type F18-positive <i>E. coli</i> strain 107/86 to piglet intestinal enterocytes. As a negative control PBS buffer was added instead of nanobody. Bacterial cells adhering to villi were counted under a microscope and plotted as a percentage of wild type binding.</p

Nanobodies that induce a conformational change in the D″-E loop do not inhibit the attachment of FedF towards the A6-1 carbohydrate.

<p>Consecutive injections of either FedF_15–165 or FedF_15–165-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.</p

Inhibitory nanobodies recognize FedF_15–165 with low nanomolar affinity.

<p>Microscale thermophoresis (MST) was employed to determine the in solution affinity between Nb-FedF6, Nb-FedF7, Nb-FedF9 and Nb-FedF12 with FedF_15–165. (<b>A</b>) Typical MST measurement showing the interaction between Nb-FedF6 and FedF_15–165. Data points are indicated by black diamonds, the fit by the NT Analysis software is shown as a red line. (<b>B</b>) Overview on the determined dissociation constants (K_D) for the indicated Nb-FedF_15–165 interactions.</p

A unique hetero-hexadecameric architecture displayed by the Escherichia coli O157 PaaA2-ParE2 antitoxin-toxin complex

Many bacterial pathogens modulate their metabolic activity, virulence and pathogenicity through so-called "toxin-antitoxin" (TA) modules. The genome of the human pathogen Escherichia coli O157 contains two three-component TA modules related to the known parDE module. Here, we show that the toxin EcParE2 maps in a branch of the RelE/ParE toxin superfamily that is distinct from the branches that contain verified gyrase and ribosome inhibitors. The structure of EcParE2 closely resembles that of Caulobacter crescentus ParE but shows a distinct pattern of conserved surface residues, in agreement with its apparent inability to interact with GyrA. The antitoxin EcPaaA2 is characterized by two α-helices (H1 and H2) that serve as molecular recognition elements to wrap itself around EcParE2. Both EcPaaA2 H1 and H2 are required to sustain a high-affinity interaction with EcParE2 and for the inhibition of EcParE2-mediated killing in vivo. Furthermore, evidence demonstrates that EcPaaA2 H2, but not H1, determines specificity for EcParE2. The initially formed EcPaaA2-EcParE2 heterodimer then assembles into a hetero-hexadecamer, which is stable in solution and is formed in a highly cooperative manner. Together these findings provide novel data on quaternary structure, TA interactions and activity of a hitherto poorly characterized family of TA modules.SCOPUS: ar.jinfo:eu-repo/semantics/publishe