Clostridioides difficile Toxin A Remodels Membranes and Mediates DNA Entry Into Cells to Activate Toll-Like Receptor 9 Signaling
Abstract
Background & Aims: Clostridioides difficile toxin A (TcdA) activates the innate immune response. TcdA co-purifies with DNA. Toll-like receptor 9 (TLR9) recognizes bacterial DNA to initiate inflammation. We investigated whether DNA bound to TcdA activates an inflammatory response in murine models of C difficile infection via activation of TLR9. Methods: We performed studies with human colonocytes and monocytes and macrophages from wild-type and TLR9 knockout mice incubated with TcdA or its antagonist (ODN TTAGGG) or transduced with vectors encoding TLR9 or small-interfering RNAs. Cytokine production was measured with enzyme-linked immunosorbent assay. We studied a transduction domain of TcdA (TcdA57-80), which was predicted by machine learning to have cell-penetrating activity and confirmed by synchrotron small-angle X-ray scattering. Intestines of CD1 mice, C57BL6J mice, and mice that express a form of TLR9 that is not activated by CpG DNA were injected with TcdA, TLR9 antagonist, or both. Enterotoxicity was estimated based on loop weight to length ratios. A TLR9 antagonist was tested in mice infected with C difficile. We incubated human colon explants with an antagonist of TLR9 and measured TcdA-induced production of cytokines. Results: The TcdA57-80 protein transduction domain had membrane remodeling activity that allowed TcdA to enter endosomes. TcdA-bound DNAmore »
- Authors:
-
- Harvard Medical School, Boston, MA (United States)
- Shanghai Normal University (China); Harvard Medical School, Boston, MA (United States)
- Univ. of California, Los Angeles, CA (United States)
- Harvard Medical School, Boston, MA (United States); Sun Yat-sen University, Guangzhou (China)
- Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); USDOE Joint Genome Institute (JGI), Berkeley, CA (United States)
- Stony Brook University Hospital, Stony Brook, NY (United States)
- University of Arkansas for Medical Sciences, Little Rock, AR (United States)
- University of Massachusetts Medical School, Worcester, MA (United States)
- Publication Date:
- Research Org.:
- Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); SLAC National Accelerator Lab., Menlo Park, CA (United States). Stanford Synchrotron Radiation Lightsource (SSRL)
- Sponsoring Org.:
- USDOE Office of Science (SC), Biological and Environmental Research (BER); Crohn's & Colitis Foundation of America; National Institutes of Health (NIH); National Institute of Allergy and Infectious Diseases (NIAID); National Science Foundation (NSF); USDOE Office of Science (SC), Basic Energy Sciences (BES); National Institute of General Medical Sciences (NIGMS)
- OSTI Identifier:
- 1844371
- Alternate Identifier(s):
- OSTI ID: 1734382
- Grant/Contract Number:
- AC02-05CH11231; AC02-76SF00515
- Resource Type:
- Accepted Manuscript
- Journal Name:
- Gastroenterology
- Additional Journal Information:
- Journal Volume: 159; Journal Issue: 6; Journal ID: ISSN 0016-5085
- Publisher:
- Elsevier
- Country of Publication:
- United States
- Language:
- English
- Subject:
- 59 BASIC BIOLOGICAL SCIENCES; SAXS; Antibiotic-Associated Diarrhea and Colitis; Intestinal Inflammation; Pore Formation
Citation Formats
Chen, Xinhua, Yang, Xiaotong, de Anda, Jaime, Huang, Jun, Li, Dan, Xu, Hua, Shields, Kelsey S., Džunková, Mária, Hansen, Joshua, Patel, Ishan J., Yee, Eric U., Golenbock, Douglas T., Grant, Marianne A., Wong, Gerard C. L., and Kelly, Ciarán P. Clostridioides difficile Toxin A Remodels Membranes and Mediates DNA Entry Into Cells to Activate Toll-Like Receptor 9 Signaling. United States: N. p., 2020.
Web. doi:10.1053/j.gastro.2020.08.038.
Chen, Xinhua, Yang, Xiaotong, de Anda, Jaime, Huang, Jun, Li, Dan, Xu, Hua, Shields, Kelsey S., Džunková, Mária, Hansen, Joshua, Patel, Ishan J., Yee, Eric U., Golenbock, Douglas T., Grant, Marianne A., Wong, Gerard C. L., & Kelly, Ciarán P. Clostridioides difficile Toxin A Remodels Membranes and Mediates DNA Entry Into Cells to Activate Toll-Like Receptor 9 Signaling. United States. https://doi.org/10.1053/j.gastro.2020.08.038
Chen, Xinhua, Yang, Xiaotong, de Anda, Jaime, Huang, Jun, Li, Dan, Xu, Hua, Shields, Kelsey S., Džunková, Mária, Hansen, Joshua, Patel, Ishan J., Yee, Eric U., Golenbock, Douglas T., Grant, Marianne A., Wong, Gerard C. L., and Kelly, Ciarán P. Sat .
"Clostridioides difficile Toxin A Remodels Membranes and Mediates DNA Entry Into Cells to Activate Toll-Like Receptor 9 Signaling". United States. https://doi.org/10.1053/j.gastro.2020.08.038. https://www.osti.gov/servlets/purl/1844371.
@article{osti_1844371,
title = {Clostridioides difficile Toxin A Remodels Membranes and Mediates DNA Entry Into Cells to Activate Toll-Like Receptor 9 Signaling},
author = {Chen, Xinhua and Yang, Xiaotong and de Anda, Jaime and Huang, Jun and Li, Dan and Xu, Hua and Shields, Kelsey S. and Džunková, Mária and Hansen, Joshua and Patel, Ishan J. and Yee, Eric U. and Golenbock, Douglas T. and Grant, Marianne A. and Wong, Gerard C. L. and Kelly, Ciarán P.},
abstractNote = {Background & Aims: Clostridioides difficile toxin A (TcdA) activates the innate immune response. TcdA co-purifies with DNA. Toll-like receptor 9 (TLR9) recognizes bacterial DNA to initiate inflammation. We investigated whether DNA bound to TcdA activates an inflammatory response in murine models of C difficile infection via activation of TLR9. Methods: We performed studies with human colonocytes and monocytes and macrophages from wild-type and TLR9 knockout mice incubated with TcdA or its antagonist (ODN TTAGGG) or transduced with vectors encoding TLR9 or small-interfering RNAs. Cytokine production was measured with enzyme-linked immunosorbent assay. We studied a transduction domain of TcdA (TcdA57-80), which was predicted by machine learning to have cell-penetrating activity and confirmed by synchrotron small-angle X-ray scattering. Intestines of CD1 mice, C57BL6J mice, and mice that express a form of TLR9 that is not activated by CpG DNA were injected with TcdA, TLR9 antagonist, or both. Enterotoxicity was estimated based on loop weight to length ratios. A TLR9 antagonist was tested in mice infected with C difficile. We incubated human colon explants with an antagonist of TLR9 and measured TcdA-induced production of cytokines. Results: The TcdA57-80 protein transduction domain had membrane remodeling activity that allowed TcdA to enter endosomes. TcdA-bound DNA entered human colonocytes. TLR9 was required for production of cytokines by cultured cells and in human colon explants incubated with TcdA. TLR9 was required in TcdA-induced mice intestinal secretions and in the survival of mice infected by C difficile. Even in a protease-rich environment, in which only fragments of TcdA exist, the TcdA57-80 domain organized DNA into a geometrically ordered structure that activated TLR9. Conclusions: TcdA from C difficile can bind and organize bacterial DNA to activate TLR9. TcdA and TcdA fragments remodel membranes, which allows them to access endosomes and present bacterial DNA to and activate TLR9. Rather than inactivating the ability of DNA to bind TLR9, TcdA appears to chaperone and organize DNA into an inflammatory, spatially periodic structure.},
doi = {10.1053/j.gastro.2020.08.038},
journal = {Gastroenterology},
number = 6,
volume = 159,
place = {United States},
year = {Sat Aug 22 00:00:00 EDT 2020},
month = {Sat Aug 22 00:00:00 EDT 2020}
}
Works referenced in this record:
Glucosylation of Rho proteins by Clostridium difficile toxin B
journal, June 1995
- Just, I.; Selzer, J.; Wilm, M.
- Nature, Vol. 375, Issue 6531
Identification of an Essential Region for Translocation of Clostridium difficile Toxin B
journal, August 2016
- Chen, Shuyi; Wang, Haiying; Gu, Huawei
- Toxins, Vol. 8, Issue 8
Innate immune sensing of bacterial modifications of Rho GTPases by the Pyrin inflammasome
journal, June 2014
- Xu, Hao; Yang, Jieling; Gao, Wenqing
- Nature, Vol. 513, Issue 7517
A Mouse Model of Clostridium difficile–Associated Disease
journal, December 2008
- Chen, Xinhua; Katchar, Kianoosh; Goldsmith, Jeffrey D.
- Gastroenterology, Vol. 135, Issue 6
Defective mutations within the translocation domain of Clostridium difficile toxin B impair disease pathogenesis
journal, October 2015
- Hamza, Therwa; Zhang, Zhifen; Melnyk, Roman A.
- Pathogens and Disease, Vol. 74, Issue 1
TLR-2 and TLR-9 are sensors of apoptosis in a mouse model of doxorubicin-induced acute inflammation
journal, February 2011
- Krysko, D. V.; Kaczmarek, A.; Krysko, O.
- Cell Death & Differentiation, Vol. 18, Issue 8
The C. difficile toxin B membrane translocation machinery is an evolutionarily conserved protein delivery apparatus
journal, January 2020
- Orrell, Kathleen E.; Mansfield, Michael J.; Doxey, Andrew C.
- Nature Communications, Vol. 11, Issue 1
Clostridium difficile Toxin–Induced Inflammation and Intestinal Injury Are Mediated by the Inflammasome
journal, August 2010
- Ng, Jeffrey; Hirota, Simon A.; Gross, Olaf
- Gastroenterology, Vol. 139, Issue 2
Clostridium difficile infection: Toxins and non-toxin virulence factors, and their contributions to disease establishment and host response
journal, March 2012
- Vedantam, Gayatri; Clark, Andrew; Chu, Michele
- Gut Microbes, Vol. 3, Issue 2
Molecular Motor Dnm1 Synergistically Induces Membrane Curvature To Facilitate Mitochondrial Fission
journal, November 2017
- Lee, Michelle W.; Lee, Ernest Y.; Lai, Ghee Hwee
- ACS Central Science, Vol. 3, Issue 11
Intrinsic Toxin-Derived Peptides Destabilize and Inactivate Clostridium difficile TcdB
journal, July 2017
- Larabee, Jason L.; Bland, Sarah J.; Hunt, Jonathan J.
- mBio, Vol. 8, Issue 3
Clostridium difficile — More Difficult Than Ever
journal, October 2008
- Kelly, Ciarán P.; LaMont, J. Thomas
- New England Journal of Medicine, Vol. 359, Issue 18
Mapping membrane activity in undiscovered peptide sequence space using machine learning
journal, November 2016
- Lee, Ernest Y.; Fulan, Benjamin M.; Wong, Gerard C. L.
- Proceedings of the National Academy of Sciences, Vol. 113, Issue 48
Binding and entry of Clostridium difficile toxin B is mediated by multiple domains
journal, November 2015
- Manse, Jared S.; Baldwin, Michael R.
- FEBS Letters, Vol. 589, Issue 24PartB
Purification and characterization of toxins A and B of Clostridium difficile
journal, March 1982
- Sullivan, N. M.; Pellett, S.; Wilkins, T. D.
- Infection and Immunity, Vol. 35, Issue 3
Control of infection by pyroptosis and autophagy: role of TLR and NLR
journal, March 2010
- Bortoluci, Karina R.; Medzhitov, Ruslan
- Cellular and Molecular Life Sciences, Vol. 67, Issue 10
Toxin Binding of Tolevamer, a Polyanionic Drug that Protects against Antibiotic-Associated Diarrhea
journal, July 2004
- Braunlin, William; Xu, Qiuwei; Hook, Patrick
- Biophysical Journal, Vol. 87, Issue 1
Cell-penetrating peptides derived from Clostridium difficile TcdB2 and a related large clostridial toxin
journal, February 2018
- Larabee, Jason L.; Hauck, Garrett D.; Ballard, Jimmy D.
- Journal of Biological Chemistry, Vol. 293, Issue 5
Granulin Is a Soluble Cofactor for Toll-like Receptor 9 Signaling
journal, April 2011
- Park, Boyoun; Buti, Ludovico; Lee, Sungwook
- Immunity, Vol. 34, Issue 4
Nika : software for two-dimensional data reduction
journal, March 2012
- Ilavsky, Jan
- Journal of Applied Crystallography, Vol. 45, Issue 2
Antimicrobial peptides and induced membrane curvature: Geometry, coordination chemistry, and molecular engineering
journal, August 2013
- Schmidt, Nathan W.; Wong, Gerard C. L.
- Current Opinion in Solid State and Materials Science, Vol. 17, Issue 4
Sulfated glycosaminoglycans and low-density lipoprotein receptor contribute to Clostridium difficile toxin A entry into cells
journal, June 2019
- Tao, Liang; Tian, Songhai; Zhang, Jie
- Nature Microbiology, Vol. 4, Issue 10
Cholesterol-dependent Pore Formation of Clostridium difficile Toxin A
journal, March 2006
- Giesemann, Torsten; Jank, Thomas; Gerhard, Ralf
- Journal of Biological Chemistry, Vol. 281, Issue 16
Overview of Clostridium difficile infection: implications for China
journal, November 2013
- Chen, X.; Lamont, J. T.
- Gastroenterology Report, Vol. 1, Issue 3
Malaria hemozoin is immunologically inert but radically enhances innate responses by presenting malaria DNA to Toll-like receptor 9
journal, January 2007
- Parroche, P.; Lauw, F. N.; Goutagny, N.
- Proceedings of the National Academy of Sciences, Vol. 104, Issue 6
Bacterial amyloid curli acts as a carrier for DNA to elicit an autoimmune response via TLR2 and TLR9
journal, April 2017
- Tursi, Sarah A.; Lee, Ernest Y.; Medeiros, Nicole J.
- PLOS Pathogens, Vol. 13, Issue 4
Identification of a conserved membrane localization domain within numerous large bacterial protein toxins
journal, March 2010
- Geissler, B.; Tungekar, R.; Satchell, K. J. F.
- Proceedings of the National Academy of Sciences, Vol. 107, Issue 12
Host S-nitrosylation inhibits clostridial small molecule–activated glucosylating toxins
journal, August 2011
- Savidge, Tor C.; Urvil, Petri; Oezguen, Numan
- Nature Medicine, Vol. 17, Issue 9
Clostridium difficile Infection: A Worldwide Disease
journal, January 2014
- Burke, Kristin E.; Lamont, J. Thomas
- Gut and Liver, Vol. 8, Issue 1
Externalized histone H4 orchestrates chronic inflammation by inducing lytic cell death
journal, May 2019
- Silvestre-Roig, Carlos; Braster, Quinte; Wichapong, Kanin
- Nature, Vol. 569, Issue 7755
Liquid-crystalline ordering of antimicrobial peptide–DNA complexes controls TLR9 activation
journal, June 2015
- Schmidt, Nathan W.; Jin, Fan; Lande, Roberto
- Nature Materials, Vol. 14, Issue 7
Gut inflammation provides a respiratory electron acceptor for Salmonella
journal, September 2010
- Winter, Sebastian E.; Thiennimitr, Parameth; Winter, Maria G.
- Nature, Vol. 467, Issue 7314
Proteases and the gut barrier
journal, March 2012
- Biancheri, Paolo; Di Sabatino, Antonio; Corazza, Gino R.
- Cell and Tissue Research, Vol. 351, Issue 2
CXCL4 assembles DNA into liquid crystalline complexes to amplify TLR9-mediated interferon-α production in systemic sclerosis
journal, May 2019
- Lande, Roberto; Lee, Ernest Y.; Palazzo, Raffaella
- Nature Communications, Vol. 10, Issue 1
Spore Formation and Toxin Production in Clostridium difficile Biofilms
journal, January 2014
- Semenyuk, Ekaterina G.; Laning, Michelle L.; Foley, Jennifer
- PLoS ONE, Vol. 9, Issue 1
Mechanisms of Disease: protease functions in intestinal mucosal pathobiology
journal, July 2007
- Antalis, Toni M.; Shea-Donohue, Terez; Vogel, Stefanie N.
- Nature Clinical Practice Gastroenterology & Hepatology, Vol. 4, Issue 7
Clostridium difficile Toxins: Mechanism of Action and Role in Disease
journal, April 2005
- Voth, D. E.; Ballard, J. D.
- Clinical Microbiology Reviews, Vol. 18, Issue 2
Identification of an N-Terminal Recognition Site in TLR9 That Contributes to CpG-DNA-Mediated Receptor Activation
journal, June 2009
- Peter, Mirjam E.; Kubarenko, Andriy V.; Weber, Alexander N. R.
- The Journal of Immunology, Vol. 182, Issue 12
Microbiota-liberated host sugars facilitate post-antibiotic expansion of enteric pathogens
journal, September 2013
- Ng, Katharine M.; Ferreyra, Jessica A.; Higginbottom, Steven K.
- Nature, Vol. 502, Issue 7469
Crystallinity of Double-Stranded RNA-Antimicrobial Peptide Complexes Modulates Toll-Like Receptor 3-Mediated Inflammation
journal, October 2016
- Lee, Ernest Y.; Takahashi, Toshiya; Curk, Tine
- ACS Nano, Vol. 11, Issue 12
The host immune response to Clostridium difficile
journal, August 2011
- Kelly, Ciarán P.; Kyne, Lorraine
- Journal of Medical Microbiology, Vol. 60, Issue 8
Therapeutical targeting of nucleic acid-sensing Toll-like receptors prevents experimental cerebral malaria
journal, February 2011
- Franklin, Bernardo S.; Ishizaka, Sally T.; Lamphier, Marc
- Proceedings of the National Academy of Sciences, Vol. 108, Issue 9
Cytotoxicity of Clostridium difficile toxins A and B requires an active and functional SREBP‐2 pathway
journal, December 2018
- Papatheodorou, Panagiotis; Song, Shuo; López‐Ureña, Diana
- The FASEB Journal, Vol. 33, Issue 4
Structure and mode of action of clostridial glucosylating toxins: the ABCD model
journal, May 2008
- Jank, Thomas; Aktories, Klaus
- Trends in Microbiology, Vol. 16, Issue 5
Translocation of HIV TAT peptide and analogues induced by multiplexed membrane and cytoskeletal interactions
journal, October 2011
- Mishra, A.; Lai, G. H.; Schmidt, N. W.
- Proceedings of the National Academy of Sciences, Vol. 108, Issue 41
Bacterial Toxin Effector-Membrane Targeting: Outside in, then Back Again
journal, January 2012
- Geissler, Brett
- Frontiers in Cellular and Infection Microbiology, Vol. 2
Helical antimicrobial peptides assemble into protofibril scaffolds that present ordered dsDNA to TLR9
journal, March 2019
- Lee, Ernest Y.; Zhang, Changsheng; Di Domizio, Jeremy
- Nature Communications, Vol. 10, Issue 1
TLR9 as a key receptor for the recognition of DNA☆
journal, April 2008
- Kumagai, Y.; Takeuchi, O.; Akira, S.
- Advanced Drug Delivery Reviews, Vol. 60, Issue 7
Regulation of Apoptosis and Innate Immune Stimuli in Inflammation-Induced Preterm Labor
journal, October 2013
- Jaiswal, Mukesh K.; Agrawal, Varkha; Mallers, Timothy
- The Journal of Immunology, Vol. 191, Issue 11
Low pH-induced Formation of Ion Channels by Clostridium difficile Toxin B in Target Cells
journal, January 2001
- Barth, Holger; Pfeifer, Gunther; Hofmann, Fred
- Journal of Biological Chemistry, Vol. 276, Issue 14
Translocation domain mutations affecting cellular toxicity identify the Clostridium difficile toxin B pore
journal, February 2014
- Zhang, Zhifen; Park, Minyoung; Tam, John
- Proceedings of the National Academy of Sciences, Vol. 111, Issue 10
p38 MAP kinase activation by Clostridium difficile toxin A mediates monocyte necrosis, IL-8 production, and enteritis
journal, April 2000
- Warny, Michel; Keates, Andrew C.; Keates, Sarah
- Journal of Clinical Investigation, Vol. 105, Issue 8