Development of a bispecific Nanobody anti-F17 fimbria as a potential therapeutic tool.

Pathogenic strains of Escherichia coli F17+ are associated with various intestinal and extra-intestinal pathologies, including diarrhea, and result in significant animal mortality. These infections rely on the expression of virulence factors, such as F17 fimbriae, for adhesion. F17 fimbriae form a protective layer on the surface of E. coli bacteria, consisting of a major structural subunit, F17A, and a minor functional subunit, F17G. Because of the evolution of bacterial resistance, conventional antibiotic treatments have limited efficacy. Therefore, there is a pressing need to develop novel therapeutic tools. In this study, we cloned and produced the F17G protein. We then immunized a camel with the purified F17G protein and constructed a VHH library consisting of 2 × 109 clones. The library was then screened against F17G protein using phage display technology. Through this process, we identified an anti-F17G nanobody that was subsequently linked, via a linker, to an anti-F17A nanobody, resulting in the creation of an effective bispecific nanobody. Comprehensive characterization of this bispecific nanobody demonstrated excellent production, specific binding capacity to both recombinant forms of the two F17 antigens and the E. coli F17+ strain, remarkable stability in camel serum, and superior resistance to pepsin protease. The successful generation of this bispecific nanobody with excellent production, specific binding capacity and stability highlights its potential as a valuable tool for fighting infections caused by pathogenic E. coli F17+ strain.

Nanobody-Based Sandwich Immunoassay for Pathogenic Escherichia coli F17 Strain Detection.

Rapid and specific detection of pathogenic bacteria in fecal samples is of critical importance for the diagnosis of neonatal diarrhea in veterinary clinics. Nanobodies are a promising tool for the treatment and diagnosis of infectious diseases due to their unique recognition properties. In this study, we report the design of a nanobody-based magnetofluorescent immunoassay for the sensitive detection of pathogenic Escherichia coli F17-positive strains (E. coli F17). For this, a camel was immunized with purified F17A protein from F17 fimbriae and a nanobody library was constructed by phage display. Two specific anti-F17A nanobodies (Nbs) were selected to design the bioassay. The first one (Nb1) was conjugated to magnetic beads (MBs) to form a complex capable of efficiently capturing the target bacteria. A second horseradish peroxidase (HRP)-conjugated nanobody (Nb4) was used for detection by oxidizing o-phenylenediamine (OPD) to fluorescent 2,3-diaminophenazine (DAP). Our results show that the immunoassay recognizes E. coli F17 with high specificity and sensitivity, with a detection limit of 1.8 CFU/mL in only 90 min. Furthermore, we showed that the immunoassay can be applied to fecal samples without pretreatment and remains stable for at least one month when stored at 4 °C.

Sandwich-Based Immunosensor for Dual-Mode Detection of Pathogenic F17-Positive Escherichia coli Strains.

Bacterial diseases cause tremendous economic losses due to high morbidity and mortality in livestock animals. F17A protein, the major subunit of F17 fimbriae, is one of the most prevalent and crucial virulence factors among the pathogenic Escherichia coli (E. coli) isolated from diarrheic and septicemic animals of various species. Purification and detection of this protein is regarded as an interesting field of investigation due to its important role as a therapeutic target, such as vaccines, and as a diagnostic tool. In this context, polyclonal rabbit antibodies recognizing F17A protein (anti-F17A antibody) were developed and used for its detection. In fact, sandwich biosensor using anti-F17A/gold nanoparticles conjugates as capture probe and anti-F17A antibody labelled with horseradish peroxidase as signal amplification probe was developed for electrochemical and fluorescent detection of purified F17A protein and live F17-positive E. coli bacteria. Good specificity and sensitivity for detection of F17-positive E. coli strains were obtained. The dynamic range for the biosensor varies from 1 × 102 to 1 × 109 CFU·mL-1 (R2 = 0.998) and the detection limit (LOD) and the IC50 value were estimated to be 37 CFU·mL-1 and 75 CFU·mL-1, respectively.