Selection of Nanobodies that Block the Enzymatic and Cytotoxic Activities of the Binary Clostridium Difficile Toxin CDT

Abstract

The spore-forming gut bacterium Clostridium difficile is the leading cause of antibiotic-associated diarrhea in hospitalized patients. The major virulence factors are two large glucosylating cytotoxins. Hypervirulent strains (e.g. ribotype 027) with higher morbidity and mortality additionally produce the binary CDT toxin (Clostridium difficile transferase) that ADP-ribosylates actin and induces microtubule-based cell protrusions. Nanobodies are robust single domain antibodies derived from camelid heavy chain antibodies. Here we report the generation of functional nanobodies against the enzymatic CDTa and the heptameric receptor binding subunit CDTb. The nanobodies were obtained from a variable-domain repertoire library isolated from llamas immunized with recombinant CDTa or CDTb. Five CDTa-specific nanobodies blocked CDTa-mediated ADP-ribosylation of actin. Three CDTa-specific and two CDTb-specific nanobodies neutralized the cytotoxicity of CDTa+b. These nanobodies hold promise as new tools for research, diagnosis and therapy of C. difficile associated disease.

A nanobody that recognizes a 14-residue peptide epitope in the E2 ubiquitin-conjugating enzyme UBC6e modulates its activity

“…We have generated new VHHs as a tool to identify a region that allosterically regulates the enzymatic activity of UBC6e. VHHs have proven to be particularly useful as reagents for modulating the activity of enzymes [36][37][38] . VHHs have been identified that inhibit 37,39 or augment enzymatic activity 39, 40 , through direct active site binding 37,41 or via allosteric mechanisms 42,43 .…”

Mechanisms of Ricin Toxin Neutralization Revealed through Engineered Homodimeric and Heterodimeric Camelid Antibodies

“…Although a number of promising toxinneutralizing murine and humanized monoclonal antibodies (mAbs) are in the pipeline, the emergence of technologies surrounding the generation and expression of toxin-specific camelid heavy chain-only VH domains (VHHs) 3 has opened up new avenues for the rational design and delivery of antitoxin agents (5). VHHs with toxin-neutralizing activity have been described against botulinum neurotoxin (6), Clostridium difficile toxins TcdA and TcdB (7)(8)(9), Shiga toxins (10), ricin (11,12), and anthrax toxin (13). X-ray crystallography of toxin-VHH complexes has provided novel insights into the mechanisms of toxin neutralization (7,14) and single chain antibodies have been successfully delivered into the intracellular compartment of mammalian cells where they can effectively inactivate their targets (15).…”

Single Domain Antibodies and Bioengineering Drugs on Their Basis: New Opportunities for Diagnostics and Therapy

“…18, № 6 наноантитела. Эти наноантитела могут быть ис-пользованы в качестве инструментов для ис-следований, диагностики и терапии Clostridium difficile-ассоциированных заболеваний [114].…”