{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,1,15]],"date-time":"2026-01-15T18:33:36Z","timestamp":1768502016023,"version":"3.49.0"},"posted":{"date-parts":[[2023,1,12]]},"group-title":"Bioengineering","reference-count":58,"publisher":"openRxiv","content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2023,1,12]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:p>\n                  <jats:italic>Clostridioides difficile<\/jats:italic>\n                  (\n                  <jats:italic>C. diff<\/jats:italic>\n                  .) is a bacterium that causes severe diarrhea and inflammation of the colon. The pathogenicity of\n                  <jats:italic>C. diff<\/jats:italic>\n                  . 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\n                  <jats:italic>pep<\/jats:italic>\n                  tide\n                  <jats:italic>b<\/jats:italic>\n                  inding\n                  <jats:italic>d<\/jats:italic>\n                  esign 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 K\n                  <jats:sub>D<\/jats:sub>\n                  of 56.1 \u00b1 29.8 nM as measured by surface plasmon resonance (SPR).\n                <\/jats:p>\n                <jats:sec>\n                  <jats:title>Significance Statement<\/jats:title>\n                  <jats:p>\n                    Infections by\n                    <jats:italic>Clostridioides difficile<\/jats:italic>\n                    , a bacterium that targets the large intestine (colon), impact a significant number of people worldwide. Bacterial colonization is mediated by two exotoxins: toxins A and B. Short peptides that can inhibit the biocatalytic activity of these toxins represent a promising strategy to prevent and treat\n                    <jats:italic>C. diff<\/jats:italic>\n                    . infection. We describe an approach that combines a\n                    <jats:italic>Peptide B<\/jats:italic>\n                    inding\n                    <jats:italic>D<\/jats:italic>\n                    esign (PepBD) algorithm, molecular-level simulations, a rapid screening assay to evaluate peptide:toxin binding, a primary human cell-based assay, and surface plasmon resonance (SPR) measurements to develop peptide inhibitors that block Toxin A in small intestinal and colon epithelial cells. Importantly, our designed peptide, SA1, bound toxin A with nanomolar affinity and blocked toxicity in colon cells.\n                  <\/jats:p>\n                <\/jats:sec>","DOI":"10.1101\/2023.01.10.523493","type":"posted-content","created":{"date-parts":[[2023,1,12]],"date-time":"2023-01-12T20:45:18Z","timestamp":1673556318000},"source":"Crossref","is-referenced-by-count":0,"title":["Design of 8-mer Peptides that Block\n                  <i>Clostridioides difficile<\/i>\n                  Toxin A in Intestinal Cells"],"prefix":"10.64898","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-4642-7689","authenticated-orcid":false,"given":"Sudeep","family":"Sarma","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8140-0959","authenticated-orcid":false,"given":"Carly M.","family":"Catella","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Ellyce T. San","family":"Pedro","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3462-3049","authenticated-orcid":false,"given":"Xingqing","family":"Xiao","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4450-509X","authenticated-orcid":false,"given":"Deniz","family":"Durmusoglu","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5633-434X","authenticated-orcid":false,"given":"Stefano","family":"Menegatti","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6165-1972","authenticated-orcid":false,"given":"Nathan","family":"Crook","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2746-1224","authenticated-orcid":false,"given":"Scott T.","family":"Magness","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7425-587X","authenticated-orcid":false,"given":"Carol K.","family":"Hall","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"54368","reference":[{"key":"2023011409050561000_2023.01.10.523493v1.1","doi-asserted-by":"crossref","first-page":"286","DOI":"10.1056\/NEJMc1506004","article-title":"Clostridium difficile Infection","volume":"373","year":"2015","journal-title":"N. 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