{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,1]],"date-time":"2026-10-01T01:43:41Z","timestamp":1790819021919,"version":"4.1.0"},"update-to":[{"DOI":"10.1371\/journal.ppat.1010323","type":"new_version","label":"New version","source":"publisher","updated":{"date-parts":[[2022,3,2]],"date-time":"2022-03-02T00:00:00Z","timestamp":1646179200000}}],"reference-count":47,"publisher":"Public Library of Science (PLoS)","issue":"2","license":[{"start":{"date-parts":[[2022,2,17]],"date-time":"2022-02-17T00:00:00Z","timestamp":1645056000000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"publisher","award":["T32 DK007673"],"award-info":[{"award-number":["T32 DK007673"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"publisher","award":["AI957555"],"award-info":[{"award-number":["AI957555"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000738","name":"U.S. Department of Veterans Affairs","doi-asserted-by":"publisher","award":["BX002943"],"award-info":[{"award-number":["BX002943"]}],"id":[{"id":"10.13039\/100000738","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["www.plospathogens.org"],"crossmark-restriction":false},"short-container-title":["PLoS Pathog"],"abstract":"<jats:p>\n                    <jats:italic>Clostridioides difficile<\/jats:italic>\n                    infection (CDI) is the leading cause of nosocomial diarrhea and pseudomembranous colitis in the USA. In addition to these symptoms, patients with CDI can develop severe inflammation and tissue damage, resulting in life-threatening toxic megacolon. CDI is mediated by two large homologous protein toxins, TcdA and TcdB, that bind and hijack receptors to enter host cells where they use glucosyltransferase (GT) enzymes to inactivate Rho family GTPases. GT-dependent intoxication elicits cytopathic changes, cytokine production, and apoptosis. At higher concentrations TcdB induces GT-independent necrosis in cells and tissue by stimulating production of reactive oxygen species via recruitment of the NADPH oxidase complex. Although GT-independent necrosis has been observed\n                    <jats:italic>in vitro<\/jats:italic>\n                    , the relevance of this mechanism during CDI has remained an outstanding question in the field. In this study we generated novel\n                    <jats:italic>C<\/jats:italic>\n                    .\n                    <jats:italic>difficile<\/jats:italic>\n                    toxin mutants in the hypervirulent BI\/NAP1\/PCR-ribotype 027 R20291 strain to test the hypothesis that GT-independent epithelial damage occurs during CDI. Using the mouse model of CDI, we observed that epithelial damage occurs through a GT-independent process that does not involve immune cell influx. The GT-activity of either toxin was sufficient to cause severe edema and inflammation, yet GT activity of both toxins was necessary to produce severe watery diarrhea. These results demonstrate that both TcdA and TcdB contribute to disease pathogenesis when present. Further, while inactivating GT activity of\n                    <jats:italic>C<\/jats:italic>\n                    .\n                    <jats:italic>difficile<\/jats:italic>\n                    toxins may suppress diarrhea and deleterious GT-dependent immune responses, the potential of severe GT-independent epithelial damage merits consideration when developing toxin-based therapeutics against CDI.\n                  <\/jats:p>","DOI":"10.1371\/journal.ppat.1010323","type":"journal-article","created":{"date-parts":[[2022,2,17]],"date-time":"2022-02-17T13:50:55Z","timestamp":1645105855000},"page":"e1010323","update-policy":"https:\/\/doi.org\/10.1371\/journal.ppat.corrections_policy","source":"Crossref","is-referenced-by-count":34,"title":["Glucosyltransferase-dependent and independent effects of Clostridioides difficile toxins during infection"],"prefix":"10.1371","volume":"18","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-0920-2285","authenticated-orcid":true,"given":"F. Christopher","family":"Peritore-Galve","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6011-557X","authenticated-orcid":true,"given":"John A.","family":"Shupe","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Rory J.","family":"Cave","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kevin O.","family":"Childress","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"M. Kay","family":"Washington","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sarah A.","family":"Kuehne","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2273-8121","authenticated-orcid":true,"given":"D. Borden","family":"Lacy","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"340","published-online":{"date-parts":[[2022,2,17]]},"reference":[{"key":"ppat.1010323.ref001","doi-asserted-by":"crossref","DOI":"10.15620\/cdc:82532","author":"Centers for Disease Control and Prevention","year":"2019","journal-title":"Antibiotic resistance threats in the United States"},{"key":"ppat.1010323.ref002","doi-asserted-by":"crossref","first-page":"825","DOI":"10.1056\/NEJMoa1408913","article-title":"Burden of Clostridium difficile infection in the United States","volume":"372","author":"FC Lessa","year":"2015","journal-title":"N. Engl. J. Med"},{"key":"ppat.1010323.ref003","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1038\/nrdp.2016.20","article-title":"Clostridium difficile infection","volume":"2","author":"WK Smits","year":"2016","journal-title":"Nat. Rev. Dis. 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Liver Physiol"},{"key":"ppat.1010323.ref033","doi-asserted-by":"crossref","first-page":"1257","DOI":"10.1172\/JCI117080","article-title":"Neutrophil recruitment in Clostridium difficile toxin A enteritis in the rabbit","volume":"93","author":"CP Kelly","year":"1994","journal-title":"J. Clin. Invest"},{"key":"ppat.1010323.ref034","doi-asserted-by":"crossref","first-page":"G178","DOI":"10.1152\/ajpgi.2001.280.2.G178","article-title":"Microbes and microbial toxins: paradigms for microbial- mucosal interactions II. The integrated response of the intestine to Clostridium difficile toxins","volume":"280","author":"C Pothoulakis","year":"2001","journal-title":"Am. J. Physiol. Gastrointest. Liver Physiol"},{"key":"ppat.1010323.ref035","doi-asserted-by":"crossref","first-page":"1186","DOI":"10.1016\/0016-5085(89)91689-2","article-title":"Comparative study of Clostridium difficile toxin A and cholera toxin in rabbit ileum","volume":"97","author":"G Triadafilopoulous","year":"1989","journal-title":"Gastroenterology"},{"key":"ppat.1010323.ref036","doi-asserted-by":"crossref","first-page":"618","DOI":"10.1093\/jaoac\/94.2.618","article-title":"Clospore: a liquid medium for producing high titers of semi-purified spores of Clostridium difficile","volume":"94","author":"J Perez","year":"2011","journal-title":"J. AOAC Int"},{"key":"ppat.1010323.ref037","doi-asserted-by":"crossref","first-page":"439","DOI":"10.1046\/j.1365-2958.2002.03134.x","article-title":"Conjugative transfer of clostridial shuttle vectors from Escherichia coli to Clostridium difficile through circumvention of the restriction barrier","volume":"46","author":"D Purdy","year":"2002","journal-title":"Mol. Microbiol"},{"key":"ppat.1010323.ref038","doi-asserted-by":"crossref","first-page":"455","DOI":"10.1089\/cmb.2012.0021","article-title":"SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing","volume":"19","author":"A Bankevich","year":"2012","journal-title":"J. Comput. Biol"},{"key":"ppat.1010323.ref039","doi-asserted-by":"crossref","first-page":"8365","DOI":"10.1038\/srep08365","article-title":"RASTtk: a modular and extensible implementation of the RAST algorithm for building custom annotation pipelines and annotating batches of genomes","volume":"5","author":"T Brettin","year":"2015","journal-title":"Sci. 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