Xinhua Chen1, Xiaotong Yang2, Jaime de Anda3, Jun Huang4, Dan Li5, Hua Xu5, Kelsey S Shields5, Mária Džunková6, Joshua Hansen5, Ishan J Patel7, Eric U Yee8, Douglas T Golenbock9, Marianne A Grant10, Gerard C L Wong11, Ciarán P Kelly5. 1. Division of Gastroenterology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts. Electronic address: xchen1@bidmc.harvard.edu. 2. Division of Gastroenterology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts; Institute of Microbiology and Immunology, College of Life Sciences, Shanghai Normal University, Shanghai, China. 3. Department of Bioengineering, Department of Chemistry and Biochemistry, California Nano Systems Institute, University of California, Los Angeles, Los Angeles, California. 4. Division of Gastroenterology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts; Department of Colorectal Surgery, The 6th Affiliated Hospital, Sun Yat-sen University, Guangzhou, China. 5. Division of Gastroenterology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts. 6. US Department of Energy Joint Genome Institute, Lawrence Berkeley National Laboratory, Berkeley, California. 7. Stony Brook University Hospital, Stony Brook, New York. 8. Department of Pathology, University of Arkansas for Medical Sciences, Little Rock, Arkansas. 9. Division of Infectious Diseases and Immunology, University of Massachusetts Medical School, Worcester, Massachusetts. 10. Division of Molecular and Vascular Medicine, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts. 11. Department of Bioengineering, Department of Chemistry and Biochemistry, California Nano Systems Institute, University of California, Los Angeles, Los Angeles, California. Electronic address: gclwong@seas.ucla.edu.
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 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.
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.
Authors: Abhijit Mishra; Ghee Hwee Lai; Nathan W Schmidt; Victor Z Sun; April R Rodriguez; Rong Tong; Li Tang; Jianjun Cheng; Timothy J Deming; Daniel T Kamei; Gerard C L Wong Journal: Proc Natl Acad Sci U S A Date: 2011-10-03 Impact factor: 11.205
Authors: Ernest Y Lee; Toshiya Takahashi; Tine Curk; Jure Dobnikar; Richard L Gallo; Gerard C L Wong Journal: ACS Nano Date: 2017-10-19 Impact factor: 15.881
Authors: Lauren K Nicastro; Jaime de Anda; Neha Jain; Kaitlyn C M Grando; Amanda L Miller; Shingo Bessho; Stefania Gallucci; Gerard C L Wong; Çagla Tükel Journal: PLoS Pathog Date: 2022-08-16 Impact factor: 7.464