{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,15]],"date-time":"2026-08-15T20:58:38Z","timestamp":1786827518391,"version":"build-2736575974"},"reference-count":53,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2022,2,3]],"date-time":"2022-02-03T00:00:00Z","timestamp":1643846400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100001691","name":"Japan Society for the Promotion of Science","doi-asserted-by":"publisher","award":["20K17462"],"award-info":[{"award-number":["20K17462"]}],"id":[{"id":"10.13039\/501100001691","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001691","name":"Japan Society for the Promotion of Science","doi-asserted-by":"publisher","award":["18H02654"],"award-info":[{"award-number":["18H02654"]}],"id":[{"id":"10.13039\/501100001691","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100009619","name":"AMED","doi-asserted-by":"publisher","award":["20wm0325025h0001"],"award-info":[{"award-number":["20wm0325025h0001"]}],"id":[{"id":"10.13039\/100009619","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100007449","name":"Takeda Science Foundation","doi-asserted-by":"publisher","id":[{"id":"10.13039\/100007449","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Microbiol."],"abstract":"<jats:p><jats:italic>Clostridium botulinum<\/jats:italic> produces botulinum neurotoxin complexes that cause botulism. Previous studies elucidated the molecular pathogenesis of botulinum neurotoxin complexes; however, it currently remains unclear whether other components of the bacterium affect host cells. Recent studies provided insights into the role of bacterial membrane vesicles (MVs) produced by some bacterial species in host immunity and pathology. We herein examined and compared the cellular effects of MVs isolated from four strains of <jats:italic>C. botulinum<\/jats:italic> with those of closely related <jats:italic>Clostridium sporogenes<\/jats:italic> and two strains of the symbiont <jats:italic>Clostridium scindens<\/jats:italic>. MVs derived from all strains induced inflammatory cytokine expression in intestinal epithelial and macrophage cell lines. Cytokine expression was dependent on myeloid differentiation primary response (MyD) 88 and TIR-domain-containing adapter-inducing interferon-\u03b2 (TRIF), essential adaptors for toll-like receptors (TLRs), and TLR1\/2\/4. The inhibition of actin polymerization impeded the uptake of MVs in RAW264.7 cells, however, did not reduce the induction of cytokine expression. On the other hand, the inhibition of dynamin or phosphatidylinositol-3 kinase (PI3K) suppressed the induction of cytokine expression by MVs, suggesting the importance of these factors downstream of TLR signaling. MVs also induced expression of Reg3 family antimicrobial peptides <jats:italic>via<\/jats:italic> MyD88\/TRIF signaling in primary cultured mouse small intestinal epithelial cells (IECs). The present results indicate that MVs from <jats:italic>C. botulinum<\/jats:italic> and related clostridial species induce host innate immune responses.<\/jats:p>","DOI":"10.3389\/fmicb.2022.720308","type":"journal-article","created":{"date-parts":[[2022,2,3]],"date-time":"2022-02-03T08:52:11Z","timestamp":1643878331000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":12,"title":["Membrane Vesicles Derived From Clostridium botulinum and Related Clostridial Species Induce Innate Immune Responses via MyD88\/TRIF Signaling in vitro"],"prefix":"10.3389","volume":"13","author":[{"given":"Nobuhide","family":"Kobayashi","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kimihiro","family":"Abe","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sachiyo","family":"Akagi","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mayu","family":"Kitamura","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yoshitake","family":"Shiraishi","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Aki","family":"Yamaguchi","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Masahiro","family":"Yutani","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sho","family":"Amatsu","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Takuhiro","family":"Matsumura","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nobuhiko","family":"Nomura","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Noriyuki","family":"Ozaki","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nozomu","family":"Obana","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yukako","family":"Fujinaga","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1965","published-online":{"date-parts":[[2022,2,3]]},"reference":[{"key":"ref1","doi-asserted-by":"publisher","first-page":"e12080","DOI":"10.1002\/jev2.12080","article-title":"Staphylococcus aureus membrane vesicles contain immunostimulatory DNA, RNA and peptidoglycan that activate innate immune receptors and induce autophagy","volume":"10","author":"Bitto","year":"2021","journal-title":"J. Extracell. Vesicles"},{"key":"ref2","doi-asserted-by":"publisher","first-page":"965","DOI":"10.3389\/fimmu.2020.00965","article-title":"The network of colonic host defense peptides as an innate immune defense against enteropathogenic bacteria","volume":"11","author":"Blyth","year":"2020","journal-title":"Front. Immunol."},{"key":"ref3","doi-asserted-by":"publisher","first-page":"4154.e12","DOI":"10.1016\/j.cell.2021.07.001","article-title":"The microbiota coordinates diurnal rhythms in innate immunity with the circadian clock","volume":"184","author":"Brooks","year":"2021","journal-title":"Cell"},{"key":"ref4","doi-asserted-by":"publisher","first-page":"205","DOI":"10.1038\/nature13828","article-title":"Precision microbiome reconstitution restores bile acid mediated resistance to Clostridium difficile","volume":"517","author":"Buffie","year":"2015","journal-title":"Nature"},{"key":"ref5","doi-asserted-by":"publisher","first-page":"966","DOI":"10.4049\/jimmunol.164.2.966","article-title":"Lipopolysaccharide activates distinct signaling pathways in intestinal epithelial cell lines expressing toll-Like receptors","volume":"164","author":"Cario","year":"2000","journal-title":"J. Immunol."},{"key":"ref6","doi-asserted-by":"publisher","first-page":"303","DOI":"10.1016\/j.resmic.2014.10.010","article-title":"Genomes, neurotoxins and biology of clostridium botulinum group I and group II","volume":"166","author":"Carter","year":"2015","journal-title":"Res. Microbiol."},{"key":"ref7","doi-asserted-by":"publisher","first-page":"12018","DOI":"10.1128\/microbiolspec.psib-0001-2018","article-title":"Outer membrane vesicle-host cell interactions","volume":"7","author":"Cecil","year":"2019","journal-title":"Microbiol. Spectr."},{"key":"ref8","doi-asserted-by":"publisher","first-page":"e450","DOI":"10.1038\/emm.2017.282","article-title":"Akkermansia muciniphila-derived extracellular vesicles influence gut permeability through the regulation of tight junctions","volume":"50","author":"Chelakkot","year":"2018","journal-title":"Exp. Mol. Med."},{"key":"ref9","doi-asserted-by":"publisher","first-page":"12246","DOI":"10.1002\/anie.201204910","article-title":"Discovery of small-molecule inhibitors of the TLR1\/TLR2 complex","volume":"51","author":"Cheng","year":"2012","journal-title":"Angew. Chem. Int. Ed."},{"key":"ref10","doi-asserted-by":"publisher","first-page":"5","DOI":"10.1046\/j.1365-2672.1997.00313.x","article-title":"Phylogeny and taxonomy of the food-borne pathogen Clostridium botulinum and its neurotoxins","volume":"84","author":"Collins","year":"1998","journal-title":"J. Appl. Microbiol."},{"key":"ref11","doi-asserted-by":"publisher","first-page":"13","DOI":"10.1016\/j.toxicon.2017.10.020","article-title":"Translocation and dissemination of botulinum neurotoxin from the intestinal tract","volume":"147","author":"Fujinaga","year":"2018","journal-title":"Toxicon"},{"key":"ref12","doi-asserted-by":"publisher","first-page":"358","DOI":"10.1016\/S1471-4906(03)00139-X","article-title":"PI3K and negative regulation of TLR signaling","volume":"24","author":"Fukao","year":"2003","journal-title":"Trends Immunol."},{"key":"ref13","doi-asserted-by":"publisher","first-page":"21","DOI":"10.1007\/978-3-642-33570-9_2","article-title":"Assembly and function of the botulinum neurotoxin progenitor complex","volume":"364","author":"Gu","year":"2013","journal-title":"Curr. Top. Microbiol. Immunol."},{"key":"ref14","doi-asserted-by":"publisher","first-page":"81","DOI":"10.3390\/toxins12020081","article-title":"Adult intestinal toxemia botulism","volume":"12","author":"Harris","year":"2020","journal-title":"Toxins"},{"key":"ref15","doi-asserted-by":"publisher","first-page":"4872","DOI":"10.4049\/jimmunol.1003761","article-title":"Nucleotide-binding oligomerization domain 1 mediates recognition of Clostridium difficile and induces neutrophil recruitment and protection against the pathogen","volume":"186","author":"Hasegawa","year":"2011","journal-title":"J. Immunol."},{"key":"ref16","doi-asserted-by":"publisher","first-page":"1589","DOI":"10.1128\/iai.64.5.1589-1594.1996","article-title":"Molecular composition of Clostridium botulinum type A progenitor toxins","volume":"64","author":"Inoue","year":"1996","journal-title":"Infect. 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Microbiol."},{"key":"ref20","doi-asserted-by":"publisher","first-page":"4538","DOI":"10.1038\/sj.emboj.7600471","article-title":"Enterotoxigenic Escherichia coli vesicles target toxin delivery into mammalian cells","volume":"23","author":"Kesty","year":"2004","journal-title":"EMBO J."},{"key":"ref21","doi-asserted-by":"publisher","first-page":"2345","DOI":"10.3389\/fimmu.2019.02345","article-title":"The roles of Peyer\u2019s patches and microfold cells in the gut immune system: relevance to autoimmune diseases","volume":"10","author":"Kobayashi","year":"2019","journal-title":"Front. Immunol."},{"key":"ref22","doi-asserted-by":"publisher","first-page":"750","DOI":"10.1128\/iai.10.4.750-756.1974","article-title":"Purification and some properties of progenitor toxins of Clostridium botulinum type B","volume":"10","author":"Kozaki","year":"1974","journal-title":"Infect. 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Fluoresc."},{"key":"ref26","doi-asserted-by":"publisher","first-page":"1405","DOI":"10.1126\/science.1253823","article-title":"Molecular basis for disruption of E-cadherin adhesion by botulinum neurotoxin A complex","volume":"344","author":"Lee","year":"2014","journal-title":"Science"},{"key":"ref27","doi-asserted-by":"publisher","first-page":"481","DOI":"10.1080\/19490976.2018.1549420","article-title":"In vitro and in vivo characterization of Clostridium scindens bile acid transformations","volume":"10","author":"Marion","year":"2019","journal-title":"Gut Microbes"},{"key":"ref28","doi-asserted-by":"publisher","first-page":"6255","DOI":"10.1038\/ncomms7255","article-title":"Botulinum toxin A complex exploits intestinal M cells to enter the host and exert neurotoxicity","volume":"6","author":"Matsumura","year":"2015","journal-title":"Nat. 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Protoc."},{"key":"ref31","doi-asserted-by":"publisher","first-page":"6","DOI":"10.1016\/j.micpath.2017.03.006","article-title":"Clostridium difficile-derived membrane vesicles induce the expression of pro-inflammatory cytokine genes and cytotoxicity in colonic epithelial cells in vitro","volume":"107","author":"Nicholas","year":"2017","journal-title":"Microb. Pathog."},{"key":"ref32","doi-asserted-by":"publisher","first-page":"3209","DOI":"10.1074\/jbc.M114.593426","article-title":"A role for the adaptor proteins TRAM and TRIF in toll-like receptor 2 signaling","volume":"290","author":"Nilsen","year":"2015","journal-title":"J. Biol. Chem."},{"key":"ref33","doi-asserted-by":"publisher","first-page":"1508","DOI":"10.1111\/cmi.12655","article-title":"Mechanisms of outer membrane vesicle entry into host cells","volume":"18","author":"O\u2019Donoghue","year":"2016","journal-title":"Cell. Microbiol."},{"key":"ref34","doi-asserted-by":"publisher","first-page":"1","DOI":"10.1128\/IAI.00096-17","article-title":"Immunoactive clostridial membrane vesicle production is regulated by a sporulation factor","volume":"85","author":"Obana","year":"2017","journal-title":"Infect. Immun."},{"key":"ref35","doi-asserted-by":"publisher","first-page":"200","DOI":"10.1124\/pr.116.012658","article-title":"Botulinum neurotoxins: biology, pharmacology, and toxicology","volume":"69","author":"Pirazzini","year":"2017","journal-title":"Pharmacol. Rev."},{"key":"ref36","doi-asserted-by":"publisher","first-page":"1","DOI":"10.1186\/s12964-015-0102-1","article-title":"Dynasore \u2013 not just a dynamin inhibitor","volume":"13","author":"Preta","year":"2015","journal-title":"Cell Commun. Signal."},{"key":"ref37","doi-asserted-by":"publisher","first-page":"1","DOI":"10.3390\/toxins12110716","article-title":"Toxemia in human naturally acquired botulism","volume":"12","author":"Rasetti-Escargueil","year":"2020","journal-title":"Toxins"},{"key":"ref38","doi-asserted-by":"publisher","first-page":"19002","DOI":"10.1073\/pnas.1008843107","article-title":"Bacillus anthracis produces membrane-derived vesicles containing biologically active toxins","volume":"107","author":"Rivera","year":"2010","journal-title":"Proc. Natl. Acad. Sci."},{"key":"ref39","doi-asserted-by":"publisher","first-page":"91","DOI":"10.3389\/fcimb.2020.00091","article-title":"Secretion and delivery of intestinal pathogenic Escherichia coli virulence factors via outer membrane vesicles","volume":"10","author":"Rueter","year":"2020","journal-title":"Front. Cell. Infect. Microbiol."},{"key":"ref40","doi-asserted-by":"publisher","first-page":"33","DOI":"10.1385\/IR:34:1:33","article-title":"New players in TLR-mediated innate immunity: PI3K and small rho GTPases","volume":"34","author":"Ruse","year":"2006","journal-title":"Immunol. Res."},{"key":"ref41","doi-asserted-by":"publisher","first-page":"509","DOI":"10.1016\/j.chom.2012.08.004","article-title":"Outer membrane vesicles of a human commensal mediate immune regulation and disease protection","volume":"12","author":"Shen","year":"2012","journal-title":"Cell Host Microbe"},{"key":"ref42","doi-asserted-by":"publisher","first-page":"1506","DOI":"10.1210\/en.2019-00073","article-title":"REG3 proteins as gut hormones?","volume":"160","author":"Shin","year":"2019","journal-title":"Endocrinology"},{"key":"ref43","doi-asserted-by":"publisher","first-page":"54","DOI":"10.1186\/s40168-015-0119-0","article-title":"Characterizing the fecal microbiota of infants with botulism","volume":"3","author":"Shirey","year":"2015","journal-title":"Microbiome"},{"key":"ref44","doi-asserted-by":"publisher","first-page":"691","DOI":"10.1083\/jcb.200910119","article-title":"Botulinum hemagglutinin disrupts the intercellular epithelial barrier by directly binding E-cadherin","volume":"189","author":"Sugawara","year":"2010","journal-title":"J. Cell Biol."},{"key":"ref45","doi-asserted-by":"publisher","first-page":"e111170","DOI":"10.1371\/journal.pone.0111170","article-title":"Functional dissection of the Clostridium botulinum type B hemagglutinin complex: identification of the carbohydrate and E-cadherin binding sites","volume":"9","author":"Sugawara","year":"2014","journal-title":"PLoS One"},{"key":"ref46","doi-asserted-by":"publisher","first-page":"625","DOI":"10.1016\/S1567-5769(01)00010-8","article-title":"Toll-like receptors; their physiological role and signal transduction system","volume":"1","author":"Takeuchi","year":"2001","journal-title":"Int. Immunopharmacol."},{"key":"ref47","doi-asserted-by":"publisher","first-page":"13","DOI":"10.1038\/s41579-018-0112-2","article-title":"Types and origins of bacterial membrane vesicles","volume":"17","author":"Toyofuku","year":"2019","journal-title":"Nat. Rev. Microbiol."},{"key":"ref48","doi-asserted-by":"publisher","first-page":"1106","DOI":"10.1016\/j.cell.2016.04.015","article-title":"Bacterial outer membrane vesicles mediate cytosolic localization of LPS and caspase-11 activation","volume":"165","author":"Vanaja","year":"2016","journal-title":"Cell"},{"key":"ref49","doi-asserted-by":"publisher","first-page":"243","DOI":"10.1038\/nri.2017.136","article-title":"Peptidoglycan recognition by the innate immune system","volume":"18","author":"Wolf","year":"2018","journal-title":"Nat. Rev. Immunol."},{"key":"ref50","doi-asserted-by":"publisher","first-page":"640","DOI":"10.1126\/science.1087262","article-title":"Role of adaptor TRIF in the MyD88-independent toll-Like receptor signaling pathway","volume":"301","author":"Yamamoto","year":"2003","journal-title":"Science"},{"key":"ref51","doi-asserted-by":"publisher","first-page":"692.e7","DOI":"10.1016\/j.immuni.2019.02.001","article-title":"Dysregulated lung commensal bacteria drive interleukin-17B production to promote pulmonary fibrosis through their outer membrane vesicles","volume":"50","author":"Yang","year":"2019","journal-title":"Immunity"},{"key":"ref52","doi-asserted-by":"publisher","first-page":"1981","DOI":"10.1099\/00221287-132-7-1981","article-title":"Activation of Clostridium botulinum type E toxin purified by two different procedures","volume":"132","author":"Yokosawa","year":"2009","journal-title":"Microbiology"},{"key":"ref53","doi-asserted-by":"publisher","first-page":"225.e8","DOI":"10.1016\/j.chom.2019.12.002","article-title":"Outer membrane vesiculation facilitates surface exchange and in vivo adaptation of Vibrio cholerae","volume":"27","author":"Zingl","year":"2020","journal-title":"Cell Host Microbe"}],"container-title":["Frontiers in Microbiology"],"original-title":[],"link":[{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fmicb.2022.720308\/full","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2022,2,3]],"date-time":"2022-02-03T08:52:15Z","timestamp":1643878335000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fmicb.2022.720308\/full"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2022,2,3]]},"references-count":53,"alternative-id":["10.3389\/fmicb.2022.720308"],"URL":"https:\/\/doi.org\/10.3389\/fmicb.2022.720308","relation":{},"ISSN":["1664-302X"],"issn-type":[{"value":"1664-302X","type":"electronic"}],"subject":[],"published":{"date-parts":[[2022,2,3]]},"article-number":"720308"}}