On the Hunt for Peptide Inhibitors of Clostridium difficile Toxins

ROONEY, B.L.; LEBLANC, JEAN GUY; THOMAS, N.; LEBLANC, J.

Congreso; American Society for Microbiology 115th General Meeting; 2015

Introduction: C. difficle infection is a leading cause of nosocomial antibiotic-associated diarrhea. Though treatment is available, recurrent disease becomes a large burden to healthcare systems. Since pathogenesis is associated with two toxins, TcdA and TcdB, this study aimed to identify peptides that could neutralize the effects of these toxins.Methods: Three M13-phage display libraries (Ph.D.-7, 12, C7C) expressing peptides were tested against TcdA and TcdB toxins that were labeled at their terminus with a hexameric (His6)-tag and purified from a Bacillus megaterium expression system. Briefly, each bacteriophage library expressing the various peptides was incubated in the presence of either His6-tagged TcdA or TcdB that was immobilized onto nickel-nitrilotriacetic acid magnetic beads. Following several washes, the remaining phage bound to toxin were eluted with native toxin. After several biopanning steps with increasing amounts of native toxin, high affinity toxin-binding peptides were isolated. To evaluate whether the toxin-binding peptides could prevent the cytopathic effects (CPE) of TcdA and TcdB on cultured FSK and HT29 cells, a cell culture cytotoxicity neutralization assay (CCCNA) was performed. The assay was performed using toxins derived from wild-type C. difficile strain 630 or hypervirulent strain Nap1 or their toxin-deletion mutants (tcdA+B- or tcdA-B+). Similar CCCNA experiments were performed using synthetic decameric peptide libraries mimicking the TcdA or TcdB receptor-binding domain sequence composition. Results: Phage display experiments demonstrated that thousands of toxin-binding peptides could be isolated against TcdA and TcdB. However, to date, no peptide has been isolated that could neutralize the CPE of TcdA or TcdB on FSK or HT29 cells. In contrast, a synthetic peptide was able to prevent entry of TcdB at concentrations found in clinical specimens.Conclusion: This study suggests that toxin-binding properties of peptides may be insufficient to neutralize the CPE of C. difficile toxins, but peptides mimicking the amino acid composition of the receptor-binding domain may be a fruitful avenue of research. Further investigations are underway to identify peptides able to prevent CPE derived from TcdA and methods for peptide delivery.