The glucosyltransferase activity of C. difficile Toxin B is required for disease pathogenesis
Enzymatic inactivation of Rho-family GTPases by the glucosyltransferase domain of Clostridioides difficile Toxin B (TcdB) gives rise to various pathogenic effects in cells that are classically thought to be responsible for the disease symptoms associated with C. difficile infection (CDI). Recent in...
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| description | Enzymatic inactivation of Rho-family GTPases by the glucosyltransferase domain of Clostridioides difficile Toxin B (TcdB) gives rise to various pathogenic effects in cells that are classically thought to be responsible for the disease symptoms associated with C. difficile infection (CDI). Recent in vitro studies have shown that TcdB can, under certain circumstances, induce cellular toxicities that are independent of glucosyltransferase (GT) activity, calling into question the precise role of GT activity. Here, to establish the importance of GT activity in CDI disease pathogenesis, we generated the first described mutant strain of C. difficile producing glucosyltransferase-defective (GT-defective) toxin. Using allelic exchange (AE) technology, we first deleted tcdA in C. difficile 630Δerm and subsequently introduced a deactivating D270N substitution in the GT domain of TcdB. To examine the role of GT activity in vivo, we tested each strain in two different animal models of CDI pathogenesis. In the non-lethal murine model of infection, the GT-defective mutant induced minimal pathology in host tissues as compared to the profound caecal inflammation seen in the wild-type and 630ΔermΔtcdA (ΔtcdA) strains. In the more sensitive hamster model of CDI, whereas hamsters in the wild-type or ΔtcdA groups succumbed to fulminant infection within 4 days, all hamsters infected with the GT-defective mutant survived the 10-day infection period without primary symptoms of CDI or evidence of caecal inflammation. These data demonstrate that GT activity is indispensable for disease pathogenesis and reaffirm its central role in disease and its importance as a therapeutic target for small-molecule inhibition. |
| doi_str_mv | 10.1371/journal.ppat.1008852 |
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Recent in vitro studies have shown that TcdB can, under certain circumstances, induce cellular toxicities that are independent of glucosyltransferase (GT) activity, calling into question the precise role of GT activity. Here, to establish the importance of GT activity in CDI disease pathogenesis, we generated the first described mutant strain of C. difficile producing glucosyltransferase-defective (GT-defective) toxin. Using allelic exchange (AE) technology, we first deleted tcdA in C. difficile 630Δerm and subsequently introduced a deactivating D270N substitution in the GT domain of TcdB. To examine the role of GT activity in vivo, we tested each strain in two different animal models of CDI pathogenesis. In the non-lethal murine model of infection, the GT-defective mutant induced minimal pathology in host tissues as compared to the profound caecal inflammation seen in the wild-type and 630ΔermΔtcdA (ΔtcdA) strains. In the more sensitive hamster model of CDI, whereas hamsters in the wild-type or ΔtcdA groups succumbed to fulminant infection within 4 days, all hamsters infected with the GT-defective mutant survived the 10-day infection period without primary symptoms of CDI or evidence of caecal inflammation. These data demonstrate that GT activity is indispensable for disease pathogenesis and reaffirm its central role in disease and its importance as a therapeutic target for small-molecule inhibition.</description><identifier>ISSN: 1553-7374</identifier><identifier>ISSN: 1553-7366</identifier><identifier>EISSN: 1553-7374</identifier><identifier>DOI: 10.1371/journal.ppat.1008852</identifier><identifier>PMID: 32960931</identifier><language>eng</language><publisher>United States: Public Library of Science</publisher><subject>Animal models ; Animals ; Apoptosis ; Bacterial Proteins - genetics ; Bacterial Proteins - metabolism ; Bacterial Toxins - genetics ; Bacterial Toxins - metabolism ; Biology and Life Sciences ; Clostridioides difficile - enzymology ; Clostridioides difficile - genetics ; Clostridioides difficile - pathogenicity ; Clostridium difficile ; Cricetinae ; Deactivation ; Defective mutant ; Disease Models, Animal ; Domains ; Enterocolitis, Pseudomembranous - enzymology ; Enterocolitis, Pseudomembranous - genetics ; Enterocolitis, Pseudomembranous - pathology ; Female ; Funding ; Gene Deletion ; Genetic aspects ; Glucosyltransferase ; Glucosyltransferases - genetics ; Glucosyltransferases - metabolism ; Hamsters ; Health aspects ; Inactivation ; Infections ; Life sciences ; Male ; Medicine ; Medicine and Health Sciences ; Mice ; Pathogenesis ; Pathology ; Research and Analysis Methods ; Signs and symptoms ; Supervision ; Synthetic biology ; Toxicity ; Toxin B ; Toxins ; Transferases ; Virulence ; Virulence (Microbiology)</subject><ispartof>PLoS pathogens, 2020-09, Vol.16 (9), p.e1008852</ispartof><rights>COPYRIGHT 2020 Public Library of Science</rights><rights>2020 Bilverstone et al. This is an open access article distributed under the terms of the Creative Commons Attribution License: http://creativecommons.org/licenses/by/4.0/ (the “License”), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License.</rights><rights>2020 Bilverstone et al 2020 Bilverstone et al</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c5762-851fe5e9e962aa832bd643eac73fc57c8afbd06740467f64e51fb6a5188249973</citedby><cites>FETCH-LOGICAL-c5762-851fe5e9e962aa832bd643eac73fc57c8afbd06740467f64e51fb6a5188249973</cites><orcidid>0000-0002-0972-5978 ; 0000-0003-3753-4412 ; 0000-0001-6790-8433 ; 0000-0002-3564-2340 ; 0000-0002-9277-1261 ; 0000-0003-1284-0229 ; 0000-0002-7187-2362</orcidid></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC7531778/pdf/$$EPDF$$P50$$Gpubmedcentral$$Hfree_for_read</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC7531778/$$EHTML$$P50$$Gpubmedcentral$$Hfree_for_read</linktohtml><link.rule.ids>230,314,723,776,780,860,881,2095,2914,23846,27903,27904,53769,53771,79346,79347</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/32960931$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><contributor>McClane, Bruce A.</contributor><creatorcontrib>Bilverstone, Terry W</creatorcontrib><creatorcontrib>Garland, Megan</creatorcontrib><creatorcontrib>Cave, Rory J</creatorcontrib><creatorcontrib>Kelly, Michelle L</creatorcontrib><creatorcontrib>Tholen, Martina</creatorcontrib><creatorcontrib>Bouley, Donna M</creatorcontrib><creatorcontrib>Kaye, Philip</creatorcontrib><creatorcontrib>Minton, Nigel P</creatorcontrib><creatorcontrib>Bogyo, Matthew</creatorcontrib><creatorcontrib>Kuehne, Sarah A</creatorcontrib><creatorcontrib>Melnyk, Roman A</creatorcontrib><title>The glucosyltransferase activity of C. difficile Toxin B is required for disease pathogenesis</title><title>PLoS pathogens</title><addtitle>PLoS Pathog</addtitle><description>Enzymatic inactivation of Rho-family GTPases by the glucosyltransferase domain of Clostridioides difficile Toxin B (TcdB) gives rise to various pathogenic effects in cells that are classically thought to be responsible for the disease symptoms associated with C. difficile infection (CDI). Recent in vitro studies have shown that TcdB can, under certain circumstances, induce cellular toxicities that are independent of glucosyltransferase (GT) activity, calling into question the precise role of GT activity. Here, to establish the importance of GT activity in CDI disease pathogenesis, we generated the first described mutant strain of C. difficile producing glucosyltransferase-defective (GT-defective) toxin. Using allelic exchange (AE) technology, we first deleted tcdA in C. difficile 630Δerm and subsequently introduced a deactivating D270N substitution in the GT domain of TcdB. To examine the role of GT activity in vivo, we tested each strain in two different animal models of CDI pathogenesis. In the non-lethal murine model of infection, the GT-defective mutant induced minimal pathology in host tissues as compared to the profound caecal inflammation seen in the wild-type and 630ΔermΔtcdA (ΔtcdA) strains. In the more sensitive hamster model of CDI, whereas hamsters in the wild-type or ΔtcdA groups succumbed to fulminant infection within 4 days, all hamsters infected with the GT-defective mutant survived the 10-day infection period without primary symptoms of CDI or evidence of caecal inflammation. These data demonstrate that GT activity is indispensable for disease pathogenesis and reaffirm its central role in disease and its importance as a therapeutic target for small-molecule inhibition.</description><subject>Animal models</subject><subject>Animals</subject><subject>Apoptosis</subject><subject>Bacterial Proteins - genetics</subject><subject>Bacterial Proteins - metabolism</subject><subject>Bacterial Toxins - genetics</subject><subject>Bacterial Toxins - metabolism</subject><subject>Biology and Life Sciences</subject><subject>Clostridioides difficile - enzymology</subject><subject>Clostridioides difficile - genetics</subject><subject>Clostridioides difficile - pathogenicity</subject><subject>Clostridium difficile</subject><subject>Cricetinae</subject><subject>Deactivation</subject><subject>Defective mutant</subject><subject>Disease Models, Animal</subject><subject>Domains</subject><subject>Enterocolitis, Pseudomembranous - enzymology</subject><subject>Enterocolitis, Pseudomembranous - genetics</subject><subject>Enterocolitis, Pseudomembranous - pathology</subject><subject>Female</subject><subject>Funding</subject><subject>Gene Deletion</subject><subject>Genetic aspects</subject><subject>Glucosyltransferase</subject><subject>Glucosyltransferases - genetics</subject><subject>Glucosyltransferases - metabolism</subject><subject>Hamsters</subject><subject>Health aspects</subject><subject>Inactivation</subject><subject>Infections</subject><subject>Life sciences</subject><subject>Male</subject><subject>Medicine</subject><subject>Medicine and Health Sciences</subject><subject>Mice</subject><subject>Pathogenesis</subject><subject>Pathology</subject><subject>Research and Analysis Methods</subject><subject>Signs and symptoms</subject><subject>Supervision</subject><subject>Synthetic biology</subject><subject>Toxicity</subject><subject>Toxin B</subject><subject>Toxins</subject><subject>Transferases</subject><subject>Virulence</subject><subject>Virulence 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difficile Toxin B is required for disease pathogenesis</atitle><jtitle>PLoS pathogens</jtitle><addtitle>PLoS Pathog</addtitle><date>2020-09-22</date><risdate>2020</risdate><volume>16</volume><issue>9</issue><spage>e1008852</spage><pages>e1008852-</pages><issn>1553-7374</issn><issn>1553-7366</issn><eissn>1553-7374</eissn><abstract>Enzymatic inactivation of Rho-family GTPases by the glucosyltransferase domain of Clostridioides difficile Toxin B (TcdB) gives rise to various pathogenic effects in cells that are classically thought to be responsible for the disease symptoms associated with C. difficile infection (CDI). Recent in vitro studies have shown that TcdB can, under certain circumstances, induce cellular toxicities that are independent of glucosyltransferase (GT) activity, calling into question the precise role of GT activity. Here, to establish the importance of GT activity in CDI disease pathogenesis, we generated the first described mutant strain of C. difficile producing glucosyltransferase-defective (GT-defective) toxin. Using allelic exchange (AE) technology, we first deleted tcdA in C. difficile 630Δerm and subsequently introduced a deactivating D270N substitution in the GT domain of TcdB. To examine the role of GT activity in vivo, we tested each strain in two different animal models of CDI pathogenesis. In the non-lethal murine model of infection, the GT-defective mutant induced minimal pathology in host tissues as compared to the profound caecal inflammation seen in the wild-type and 630ΔermΔtcdA (ΔtcdA) strains. In the more sensitive hamster model of CDI, whereas hamsters in the wild-type or ΔtcdA groups succumbed to fulminant infection within 4 days, all hamsters infected with the GT-defective mutant survived the 10-day infection period without primary symptoms of CDI or evidence of caecal inflammation. 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| ispartof | PLoS pathogens, 2020-09, Vol.16 (9), p.e1008852 |
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| source | MEDLINE; DOAJ Directory of Open Access Journals; Elektronische Zeitschriftenbibliothek - Frei zugängliche E-Journals; PubMed Central Open Access; Public Library of Science (PLoS); PubMed Central |
| subjects | Animal models Animals Apoptosis Bacterial Proteins - genetics Bacterial Proteins - metabolism Bacterial Toxins - genetics Bacterial Toxins - metabolism Biology and Life Sciences Clostridioides difficile - enzymology Clostridioides difficile - genetics Clostridioides difficile - pathogenicity Clostridium difficile Cricetinae Deactivation Defective mutant Disease Models, Animal Domains Enterocolitis, Pseudomembranous - enzymology Enterocolitis, Pseudomembranous - genetics Enterocolitis, Pseudomembranous - pathology Female Funding Gene Deletion Genetic aspects Glucosyltransferase Glucosyltransferases - genetics Glucosyltransferases - metabolism Hamsters Health aspects Inactivation Infections Life sciences Male Medicine Medicine and Health Sciences Mice Pathogenesis Pathology Research and Analysis Methods Signs and symptoms Supervision Synthetic biology Toxicity Toxin B Toxins Transferases Virulence Virulence (Microbiology) |
| title | The glucosyltransferase activity of C. difficile Toxin B is required for disease pathogenesis |
| url | https://sfx.bib-bvb.de/sfx_tum?ctx_ver=Z39.88-2004&ctx_enc=info:ofi/enc:UTF-8&ctx_tim=2025-01-23T22%3A00%3A12IST&url_ver=Z39.88-2004&url_ctx_fmt=infofi/fmt:kev:mtx:ctx&rfr_id=info:sid/primo.exlibrisgroup.com:primo3-Article-gale_plos_&rft_val_fmt=info:ofi/fmt:kev:mtx:journal&rft.genre=article&rft.atitle=The%20glucosyltransferase%20activity%20of%20C.%20difficile%20Toxin%20B%20is%20required%20for%20disease%20pathogenesis&rft.jtitle=PLoS%20pathogens&rft.au=Bilverstone,%20Terry%20W&rft.date=2020-09-22&rft.volume=16&rft.issue=9&rft.spage=e1008852&rft.pages=e1008852-&rft.issn=1553-7374&rft.eissn=1553-7374&rft_id=info:doi/10.1371/journal.ppat.1008852&rft_dat=%3Cgale_plos_%3EA638004611%3C/gale_plos_%3E%3Curl%3E%3C/url%3E&disable_directlink=true&sfx.directlink=off&sfx.report_link=0&rft_id=info:oai/&rft_pqid=2451547363&rft_id=info:pmid/32960931&rft_galeid=A638004611&rft_doaj_id=oai_doaj_org_article_a157c6602b164a1b842cd03189db0e84&rfr_iscdi=true |