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Gastroenterology

Volume 159, Issue 6, December 2020, Pages 2181-2192.e1
Gastroenterology

Original Research
Basic and Translational—Alimentary Tract
Clostridioides difficile Toxin A Remodels Membranes and Mediates DNA Entry Into Cells to Activate Toll-Like Receptor 9 Signaling

https://doi.org/10.1053/j.gastro.2020.08.038Get rights and content

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.

Section snippets

Machine-Learning Based Screening of Clostridioides difficile Toxin A for Membrane-Active Sequences

Using a previously published Support Vector Machine classifier from our laboratory,23 we screened the full amino acid sequence of TcdA from C difficile for the presence of membrane-active sequences (data not included). Candidate peptides were identified by scoring individual sequences with a variable size window. Of high-scoring sequence candidates, we identified a 24–amino acid fragment in the membrane targeting N-terminal 4-helix bundle, TcdA (57–80), which we refer to as TcdA57–80.

Small-Angle X-Ray Scattering Experiments for Peptide–Membrane Interaction

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Clostridioides difficile Toxin A Binds DNA and Facilitates Cellular Entry

Upon purification of TcdA from C difficile, we found that DNA is co-purified with the toxin at a 0.06% ± 0.02% DNA concentration (0.6 μg DNA per mg toxin protein). The presence of DNA was observed in both native and recombinant toxins from multiple academic and commercial sources. DNA was extracted, followed by polymerase chain reaction and sequencing, and identified to be C difficile genomic DNA and other contaminating DNAs. To examine the DNA binding ability of TcdA directly, DNA fragments

Discussion

In this report, we demonstrate that C difficile TcdA binds to DNA, facilitates DNA entry into human colonocytes, and organizes DNA for amplified TLR9-mediated immune activation. We find multiple cell penetrating peptide motifs in TcdA that can enhance endosomal access for the toxin. TLR9 signaling was required in TcdA-induced inflammatory responses in vitro and in vivo, and in human colonic muscosa. Finally, in the extreme case of TcdA digestion into peptide fragments in the protease-rich

Acknowledgments

The authors would like to thank Dr Alexander Dalpke from University of Heidelberg for his generous gifts of TLR9 mutant constructs. Author contributions: Xinhua Chen, Xiaotong Yang, Jaime de Anda, Gerard C. L. Wong, and Ciarán P. Kelly are involved study concept and design; acquisition of data; analysis and interpretation of data; drafting of the manuscript; Ciarán P. Kelly, Gerard C. L. Wong, and Xinhua Chen obtained funding and provided study supervision; Kelsey S. Shields, Joshua Hansen,

References (53)

  • B. Park et al.

    Granulin is a soluble cofactor for Toll-like receptor 9 signaling

    Immunity

    (2011)
  • C.P. Kelly et al.

    Clostridium difficile—more difficult than ever

    N Engl J Med

    (2008)
  • X. Chen et al.

    Overview of Clostridium difficile infection: implications for China

    Gastroenterol Rep (Oxf)

    (2013)
  • K.E. Burke et al.

    Clostridium difficile infection: a worldwide disease

    Gut Liver

    (2014)
  • I. Just et al.

    Glucosylation of Rho proteins by Clostridium difficile toxin B

    Nature

    (1995)
  • C.P. Kelly et al.

    The host immune response to Clostridium difficile

    J Med Microbiol

    (2011)
  • D.E. Voth et al.

    Clostridium difficile toxins: mechanism of action and role in disease

    Clin Microbiol Rev

    (2005)
  • G. Vedantam et al.

    Clostridium difficile infection: toxins and non-toxin virulence factors, and their contributions to disease establishment and host response

    Gut Microbes

    (2012)
  • Z. Zhang et al.

    Translocation domain mutations affecting cellular toxicity identify the Clostridium difficile toxin B pore

    Proc Natl Acad Sci U S A

    (2014)
  • J.L. Larabee et al.

    Intrinsic toxin-derived peptides destabilize and inactivate Clostridium difficile TcdB

    mBio

    (2017)
  • T. Hamza et al.

    Defective mutations within the translocation domain of Clostridium difficile toxin B impair disease pathogenesis

    Pathog Dis

    (2016)
  • K.E. Orrell et al.

    The C. difficile toxin B membrane translocation machinery is an evolutionarily conserved protein delivery apparatus

    Nat Commun

    (2020)
  • P. Papatheodorou et al.

    Cytotoxicity of Clostridium difficile toxins A and B requires an active and functional SREBP-2 pathway

    FASEB J

    (2019)
  • J. Ng et al.

    Clostridium difficile toxin-induced inflammation and intestinal injury are mediated by the inflammasome

    Gastroenterology

    (2010)
  • M. Warny et al.

    p38 MAP kinase activation by Clostridium difficile toxin A mediates monocyte necrosis, IL-8 production, and enteritis

    J Clin Invest

    (2000)
  • H. Xu et al.

    Innate immune sensing of bacterial modifications of Rho GTPases by the Pyrin inflammasome

    Nature

    (2014)

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    Conflicts of interest The authors disclose no conflicts.
    Funding This work was supported by Irving W. and Charlotte F. Rabb Award (to Xinhua Chen), Crohn’s and Colitis Foundation of America (to Xinhua Chen), Young Investigator Award for Probiotic Research (to Xinhua Chen), National Institutes of Health (NIH) National Institute of Allergy and Infectious Diseases RO1 AI095256, RO1 AI116596 (to Ciarán P. Kelly), NIH RO1 AI143730, NIH RO1 AI052453, National Science Foundation (NSF) DMR1808459 (to Gerard C. L. Wong), NSF Graduate Research Fellowship Program DGE-1650604 (to Jaime de Anda). Use of the Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, is supported by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences under contract no. DE-AC02-76SF00515. The Stanford Synchrotron Radiation Lightsource Structural Molecular Biology Program is supported by the U.S. Department of Energy, Office of Biological and Environmental Research, and by the NIH, National Institute of General Medical Sciences (including P41GM103393).
    Author names in bold designate shared co-first authorship.
    ∗
    Authors share co-first authorship.
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