Design of 8-mer Peptides that BlockClostridioides difficileToxin A in Intestinal Cells

Abstract

Clostridioides difficile (C. diff.) is a bacterium that causes severe diarrhea and inflammation of the colon. The pathogenicity of C. diff. infection is derived from two major toxins, toxins A (TcdA) and B (TcdB). Peptide inhibitors that can be delivered to the gut to inactivate these toxins are an attractive therapeutic strategy. In this work, we present a new approach that combines a peptide binding design algorithm (PepBD), molecular-level simulations, rapid screening of candidate peptides for toxin binding, a primary human cell-based assay, and surface plasmon resonance (SPR) measurements to develop peptide inhibitors that block the glucosyltransferase activity of TcdA by targeting its glucosyltransferase domain (GTD). Using PepBD and explicit-solvent molecular dynamics simulations, we identified seven candidate peptides, SA1-SA7. These peptides were selected for specific TcdA GTD binding through a custom solid-phase peptide screening system, which eliminated the weaker inhibitors SA5-SA7. The efficacies of SA1-SA4 were then tested using a trans-epithelial electrical resistance (TEER) assay on monolayers of the human gut epithelial culture model. One peptide, SA1, was found to block TcdA toxicity in primary-derived human jejunum (small intestinal) and colon (large intestinal) epithelial cells. SA1 bound TcdA with a KD of 56.1 +/- 61617; 29.8 nM as measured by surface plasmon resonance (SPR).

Programming Probiotics: Diet-responsive gene expression and colonization control in engineeredS. boulardii

“…Upon expression of the fusion protein, the protein of interest localizes to the cell surface through disulfide bonds that form between Aga1p and Aga2p (48). To investigate the utility of inducible systems for enabling surface display in Sb , we engineered SbGal⁺ to display a short peptide that binds to and inhibits the activity of Toxin A from Clostridioides difficile (SA1) (49) . C. difficile infections cause colon inflammation and diarrhea and are the most frequently reported nosocomial infection in the United States (50).…”

Design of parallel 𝛽‐sheet nanofibrils using Monte Carlo search, coarse‐grained simulations, and experimental testing

“…More details regarding the PepAD algorithm and $$$ {\varGamma}_{\mathrm{score}} $$$ can be found in our previous work (Xiao, Robang, et al, 2022). The development of the PepAD algorithm has been inspired by our previous work on designing peptides that bind to biomolecular targets using a Pep tide B inding D esign algorithm (Sarma et al, 2022; Sarma et al, 2023; Xiao, Kilgore, et al, 2022; Xiao, Sarma, et al, 2022).…”

Development of MHC Class I Blocking Peptides to Target Metabolic Dysfunction-Associated Steatohepatitis CD8+T Cell Activation

“…Here, we use the Peptide Binding Design algorithm (PepBD), a Monte-Carlo search algorithm in peptide sequence and conformation space, to discover peptides that bind with higher affinity and selectivity to a biomolecular target than a known "reference ligand." The performance of the best peptide binders predicted by PepBD is evaluated in-silico by performing atomistic molecular dynamics (MD) simulations and calculating the binding free energy (∆G binding ) of the peptide:protein complex using the implicit-solvent molecular mechanics/generalized Born surface area (MM/GBSA) approach with the variable internal dielectric constant model [10][11][12][13][14][15]. Using the PepBD algorithm, we developed blocking peptides that competitively inhibit the interaction between Ncf2 peptide and H2-K b to prevent CD8 + T cell activation and that might ultimately serve as an effective anti-MASH therapeutic strategy.…”