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Updated: Jun 6, 2025
A Protein Microarray Assay for Serological Determination of Antigen-specific Antibody Responses Following Clostridium difficile Infection
Published on: June 15, 2018
Structural insight into recognition of Clostridioides difficile toxin A by novel neutralizing nanobodies targeting
Nikolai N Sluchanko1, Irina V Sokolova2, Irina A Favorskaya2
1Bach Institute of Biochemistry, Federal Research Centre "Fundamentals of Biotechnology", Russian Academy of Sciences, Moscow 119071, Russia; Gamaleya Research Center for Epidemiology and Microbiology, Russian Ministry of Public Health, Moscow 123098, Russia.
Abstract:
Clostridioides difficile causes a large proportion of nosocomial colon infections by producing toxins TcdA and TcdB as key virulence factors. TcdA and TcdB have analogous domain structures with a receptor-binding domain containing C-terminal combined repetitive oligopeptides (CROPs), an attractive target for the development of therapeutic antibodies. Here, we identify and characterize two potent neutralizing single-domain camelid anti-CROPsA antibodies, C4.2 and H5.2, with distinct mechanisms of action. Peptide mapping, high-resolution crystal structures and site-directed mutagenesis revealed that C4.2 and H5.2 nanobodies target the same C-terminal epitope centered on a 2667QTIN2670 motif, yet utilize different paratopes. Only for C4.2 is the complex geometry compatible with multisite binding using QTIN-like repeats throughout the CROPsA domain, as supported by Western blotting, ELISA, and SEC-MALS analysis. H5.2 binding is stronger and more selective for the C-terminal epitope than C4.2, although both nanobodies are sufficient to neutralize TcdA individually. The described epitope does not overlap with previously described epitopes of anti-CROPs antibodies and provides new modalities for disease treatment and diagnostics.
Insights
Two novel camelid antibodies, C4.2 and H5.2, effectively neutralize Clostridioides difficile toxin A by targeting a unique C-terminal epitope. These antibodies offer new therapeutic and diagnostic possibilities for C. difficile infections.
Area of Science:
- Immunology
- Microbiology
- Structural Biology
Background:
- Clostridioides difficile infections are a significant cause of hospital-acquired colon infections.
- Toxins TcdA and TcdB are key virulence factors, with their C-terminal combined repetitive oligopeptides (CROPs) domain being a potential therapeutic target.
Purpose of the Study:
- To identify and characterize novel neutralizing single-domain camelid antibodies against the CROPs domain of Clostridioides difficile toxin A.
- To elucidate the binding mechanisms and epitope specificities of these antibodies.
Main Methods:
- Peptide mapping, high-resolution crystal structure determination, and site-directed mutagenesis were employed.
- Binding affinities and neutralization capabilities were assessed using Western blotting, ELISA, and SEC-MALS analysis.
Main Results:
- Two potent neutralizing nanobodies, C4.2 and H5.2, targeting the C-terminal CROPsA epitope (QTIN motif) were identified.
- C4.2 exhibits multisite binding, while H5.2 shows stronger and more selective binding to the target epitope.
- Both antibodies individually neutralize TcdA and bind to a novel epitope distinct from previously described ones.
Conclusions:
- C4.2 and H5.2 represent promising candidates for developing new therapeutics and diagnostics against Clostridioides difficile.
- The distinct binding mechanisms of these antibodies provide new insights into toxin neutralization strategies.
