Title: 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 (TcdA_57-80), 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 TcdA_57-80 protein transduction domain had membrane remodeling activity that allowed TcdA to enter endosomes. TcdA-bound DNAmore » 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 TcdA_57-80 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.« less

Authors:

Chen, Xinhua  ^[1];

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Yang, Xiaotong ^[2]; de Anda, Jaime ^[3]; Huang, Jun ^[4]; Li, Dan ^[1]; Xu, Hua ^[1]; Shields, Kelsey S. ^[1]; Džunková, Mária ^[5]; Hansen, Joshua ^[1]; Patel, Ishan J. ^[6]; Yee, Eric U. ^[7]; Golenbock, Douglas T. ^[8]; Grant, Marianne A. ^[1]; Wong, Gerard C. L. ^[3]; Kelly, Ciarán P. ^[1]

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+ Show Author Affiliations

1. Harvard Medical School, Boston, MA (United States)
2. Shanghai Normal University (China); Harvard Medical School, Boston, MA (United States)
3. Univ. of California, Los Angeles, CA (United States)
4. Harvard Medical School, Boston, MA (United States); Sun Yat-sen University, Guangzhou (China)
5. Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); USDOE Joint Genome Institute (JGI), Berkeley, CA (United States)
6. Stony Brook University Hospital, Stony Brook, NY (United States)
7. University of Arkansas for Medical Sciences, Little Rock, AR (United States)
8. University of Massachusetts Medical School, Worcester, MA (United States)

Publication Date:

Sat Aug 22 00:00:00 EDT 2020

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