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

Accuracy of transcranial magnetic stimulation and a Bayesian latent class model for diagnosis of spinal cord dysfunction in horses

Background: Spinal cord dysfunction/compression and ataxia are common in horses. Presumptive diagnosis is most commonly based on neurological examination and cervical radiography, but the interest into the diagnostic value of transcranial magnetic stimulation (TMS) with recording of magnetic motor evoked potentials has increased. The problem for the evaluation of diagnostic tests for spinal cord dysfunction is the absence of a gold standard in the living animal. Objectives: To compare diagnostic accuracy of TMS, cervical radiography, and neurological examination. Animals: One hundred seventy-four horses admitted at the clinic for neurological examination. Methods: Retrospective comparison of neurological examination, cervical radiography, and different TMS criteria, using Bayesian latent class modeling to account for the absence of a gold standard. Results: The Bayesian estimate of the prevalence (95% CI) of spinal cord dysfunction was 58.1 (48.3%-68.3%). Sensitivity and specificity of neurological examination were 97.6 (91.4%-99.9%) and 74.7 (61.0%-96.3%), for radiography they were 43.0 (32.3%-54.6%) and 77.3 (67.1%-86.1%), respectively. Transcranial magnetic stimulation reached a sensitivity and specificity of 87.5 (68.2%-99.2%) and 97.4 (90.4%-99.9%). For TMS, the highest accuracy was obtained using the minimum latency time for the pelvic limbs (Youden's index = 0.85). In all evaluated models, cervical radiography performed poorest. Clinical Relevance: Transcranial magnetic stimulation-magnetic motor evoked potential (TMS-MMEP) was the best test to diagnose spinal cord disease, the neurological examination was the second best, but the accuracy of cervical radiography was low. Selecting animals based on neurological examination (highest sensitivity) and confirming disease by TMS-MMEP (highest specificity) would currently be the optimal diagnostic strategy

Determination of magnetic motor evoked potential latency time cutoff values for detection of spinal cord dysfunction in horses

Background: Transcranial magnetic stimulation (TMS) and recording of magnetic motor evoked potentials (MMEP) can detect neurological dysfunction in horses but cutoff values based on confirmed spinal cord dysfunction are lacking. Objectives: To determine latency time cutoff for neurological dysfunction. Animals Five control horses and 17 horses with proprioceptive ataxia. Methods: Case-control study with receiver operating characteristic curve analysis, based on diagnostic imaging, TMS, and histopathological findings. Horses were included if all 3 examinations were performed. Results: Diagnostic imaging and histopathology did not show abnormalities in the control group but confirmed spinal cord compression in 14 of 17 ataxic horses. In the remaining 3 horses, histopathological lesions were mild to severe, but diagnostic imaging did not confirm spinal cord compression. In control horses, latency time values of thoracic and pelvic limbs were significantly lower than in ataxic horses (20 +/- 1 vs 34 +/- 16 milliseconds, P = .05; and 39 +/- 1 vs 78 +/- 26 milliseconds, P = .004). Optimal cutoff values to detect spinal cord dysfunction were 22 milliseconds (sensitivity [95% CI interval], 88% [73%-100%]; specificity, 100% [100%-100%]) in thoracic and 40 milliseconds (sensitivity, 94% [83%-100%]; specificity, 100% [100%-100%]) in pelvic limbs. To detect spinal cord dysfunction caused by compression, the optimal cutoff for thoracic limbs remained 22 milliseconds, while it increased to 43 milliseconds in pelvic limbs (sensitivity, 100% [100%-100%]; specificity, 100% [100%-100%] for thoracic and pelvic limbs). Conclusions and Clinical Importance: Magnetic motor evoked potential analysis using these cutoff values is a promising diagnostic tool for spinal cord dysfunction diagnosis in horses

Diversity in kinetics correlated with structure in nano body-stabilized LacY.

Funder: research foundation-flandersThe structure of lactose permease, stabilized in a periplasmic open conformation by two Gly to Trp replacements (LacYww) and complexed with a nanobody directed against this conformation, provides the highest resolution structure of the symporter. The nanobody binds in a different manner than two other nanobodies made against the same mutant, which also bind to the same general region on the periplasmic side. This region of the protein may represent an immune hotspot. The CDR3 loop of the nanobody is held by hydrogen bonds in a conformation that partially blocks access to the substrate-binding site. As a result, kon and koff for galactoside binding to either LacY or the double mutant complexed with the nanobody are lower than for the other two LacY/nanobody complexes though the Kd values are similar, reflecting the fact that the nanobodies rigidify structures along the pathway. While the wild-type LacY/nanobody complex clearly stabilizes a similar 'extracellular open' conformation in solution, judged by binding kinetics, the complex with wild-type LacY did not yet crystallize, suggesting the nanobody does not bind strongly enough to shift the equilibrium to stabilize a periplasmic side-open conformation suitable for crystallization. However, the similarity of the galactoside binding kinetics for the nanobody-bound complexes with wild type LacY and with LacYWW indicates that they have similar structures, showing that the reported co-structures reliably show nanobody interactions with LacY

In vitro reconstitution of dynamically interacting integral membrane subunits of energy-coupling factor transporters

Energy-coupling factor (ECF) transporters mediate import of micronutrients in prokaryotes. They consist of an integral membrane S-component (that binds substrate) and ECF module (that powers transport by ATP hydrolysis). It has been proposed that different S-components compete for docking onto the same ECF module, but a minimal liposome-reconstituted system, required to substantiate this idea, is lacking. Here, we co-reconstituted ECF transporters for folate (ECF-FolT2) and pantothenate (ECF-PanT) into proteoliposomes, and assayed for crosstalk during active transport. The kinetics of transport showed that exchange of S-components is part of the transport mechanism. Competition experiments suggest much slower substrate association with FolT2 than with PanT. Comparison of a crystal structure of ECF-PanT with previously determined structures of ECF-FolT2 revealed larger conformational changes upon binding of folate than pantothenate, which could explain the kinetic differences. Our work shows that a minimal in vitro system with two reconstituted transporters recapitulates intricate kinetics behaviour observed in vivo

The Molecular Mechanism of Transport by the Mitochondrial ADP/ATP Carrier.

Mitochondrial ADP/ATP carriers transport ADP into the mitochondrial matrix for ATP synthesis, and ATP out to fuel the cell, by cycling between cytoplasmic-open and matrix-open states. The structure of the cytoplasmic-open state is known, but it has proved difficult to understand the transport mechanism in the absence of a structure in the matrix-open state. Here, we describe the structure of the matrix-open state locked by bongkrekic acid bound in the ADP/ATP-binding site at the bottom of the central cavity. The cytoplasmic side of the carrier is closed by conserved hydrophobic residues, and a salt bridge network, braced by tyrosines. Glycine and small amino acid residues allow close-packing of helices on the matrix side. Uniquely, the carrier switches between states by rotation of its three domains about a fulcrum provided by the substrate-binding site. Because these features are highly conserved, this mechanism is likely to apply to the whole mitochondrial carrier family. VIDEO ABSTRACT