Abt, M. C., Mckenney, P. T. & Pamer, E. G. Clostridium difficile colitis: pathogenesis and host defence. Nat. Rev. Microbiol. 14, 609–620 (2016).
Di Bella, S. et al. Clostridioides difficile infection: history, epidemiology, risk factors, prevention, clinical manifestations, treatment, and future options. Clin. Microbiol. Rev. 37, e0013523 (2024).
Wilcox, M. H. et al. Bezlotoxumab for prevention of recurrent Clostridium difficile infection. N. Engl. J. Med. 376, 305–317 (2017).
Stieglitz, F. et al. TcdB of Clostridioides difficile mediates RAS-dependent necrosis in epithelial cells. Int. J. Mol. Sci. 23, 4258 (2022).
Kuehne, S. A. et al. The role of toxin A and toxin B in Clostridium difficile infection. Nature 467, 711–713 (2010).
Kelly, C. P. & Kyne, L. The host immune response to Clostridium difficile. J. Med. Microbiol. 60, 1070–1079 (2011).
Zhou, R. et al. Molecular basis of TMPRSS2 recognition by Paeniclostridium sordellii hemorrhagic toxin. Nat. Commun. 15, 1976 (2024).
Chen, P. et al. Structural basis for recognition of frizzled proteins by Clostridium difficile toxin B. Science 360, 664–669 (2018).
Chen, P. et al. Structure of the full-length Clostridium difficile toxin B. Nat. Struct. Mol. Biol. 26, 712–719 (2019).
Shen, E. et al. Subtyping analysis reveals new variants and accelerated evolution of Clostridioides difficile toxin B. Commun. Biol. 3, 347 (2020).
Luo, J. et al. TFPI is a colonic crypt receptor for TcdB from hypervirulent clade 2 C. difficile. Cell 185, 980–994.e915 (2022).
Lawley, T. D. et al. Targeted restoration of the intestinal microbiota with a simple, defined bacteriotherapy resolves relapsing Clostridium difficile disease in mice. PLoS Pathog. 8, e1002995 (2012).
Cornely, O. A. Current and emerging management options for Clostridium difficile infection: what is the role of fidaxomicin? Clin. Microbiol. Infect. 18, 28–35 (2012).
Jiang, Y., Sarpong, E. M., Sears, P. & Obi, E. N. Correction to: Budget impact analysis of fidaxomicin versus vancomycin for the treatment of Clostridioides difficile infection in the United States. Infect. Dis. Ther. 11, 127 (2022).
Gurung, B., Stricklin, M. & Wang, S. Gut microbiota-gut metabolites and Clostridioides difficile infection: approaching sustainable solutions for therapy. Metabolites 14, 74 (2024).
Pourliotopoulou, E., Karampatakis, T. & Kachrimanidou, M. Exploring the toxin-mediated mechanisms in Clostridioides difficile infection. Microorganisms 12, 1004 (2024).
Brown, A. T. & Seifert, C. F. Effect of treatment variation on outcomes in patients with Clostridium difficile. Am. J. Med. 127, 865–870 (2014).
Wan, S. et al. Gut microbiome changes in mouse, Mongolian gerbil, and hamster models following Clostridioides difficile challenge. Front. Microbiol. 15, 1368194 (2024).
Muhammed, M. T. & Aki-Yalcin, E. Molecular docking: principles, advances, and its applications in drug discovery. Lett. Drug Des. Discov. 21, 480–495 (2024).
Federico, L. B. et al. Potential colchicine binding site inhibitors unraveled by virtual screening, molecular dynamics and MM/PBSA. Comput. Biol. Med. 137, 104817 (2021).
Ouyang, Z. et al. Differences in virulence and drug resistance between Clostridioides difficile ST37 and ST1 isolates. Virulence 16, 2502554 (2025).
Valiente, E., Cairns, M. D. & Wren, B. W. The Clostridium difficile PCR ribotype 027 lineage: a pathogen on the move. Clin. Microbiol Infect. 20, 396–404 (2014).
Pepin, J. et al. Increasing risk of relapse after treatment of Clostridium difficile colitis in Quebec, Canada. Clin. Infect. Dis. 40, 1591–1597 (2005).
Bartlett John, G. Bezlotoxumab—a new agent for Clostridium difficile infection. N. Engl. J. Med. 376, 381–382 (2017).
Hussack, G. & Tanha, J. Toxin-specific antibodies for the treatment of Clostridium difficile: current status and future perspectives. Toxins 2, 998–1018 (2010).
Hu, M. et al. Structural insights into the mechanism of human NPC1L1-mediated cholesterol uptake. Sci. Adv. 7, eabg3188 (2021).
Xu, X. et al. Genomic evolution and virulence association of Clostridioides difficile sequence type 37 (ribotype 017) in China. Emerg. Microbes Infect. 10, 1331–1345 (2021).
Yang, Z. et al. Molecular epidemiology and risk factors of Clostridium difficile ST81 infection in a teaching hospital in Eastern China. Front. Cell. Infect. Microbiol. 10, 578098 (2020).
Gu, W. et al. New ribotype Clostridioides difficile from ST11 group revealed higher pathogenic ability than RT078. Emerg. Microbes Infect. 10, 687–699 (2021).
Tang, C. et al. Epidemiology and risk factors for Clostridium difficile-associated diarrhea in adult inpatients in a university hospital in China. Am. J. Infect. Control 46, 285–290 (2018).
O’rourke, S. A., Shanley, L. C. & Dunne, A. The Nrf2-HO-1 system and inflammaging. Front. Immunol. 15, 1457010 (2024).
Bai, W. et al. Biliverdin modulates the Nrf2/A20/eEF1A2 axis to alleviate cerebral ischemia-reperfusion injury by inhibiting pyroptosis. Biomed. Pharmacother. 165, 115057 (2023).
Campbell, N. K., Fitzgerald, H. K. & Dunne, A. Regulation of inflammation by the antioxidant haem oxygenase 1. Nat. Rev. Immunol. 21, 411–425 (2021).
Sugimoto, R. et al. Preservation solution supplemented with biliverdin prevents lung cold ischaemia/reperfusion injury. Eur. J. Cardiothorac. Surg. 42, 1035–1041 (2012).
Toro, A. et al. A journey into the clinical relevance of heme oxygenase 1 for human inflammatory disease and viral clearance: why does it matter on the COVID-19 scene? Antioxidants 11, 276 (2022).
Luu Hoang, K. N., Anstee, J. E. & Arnold, J. N. The diverse roles of heme oxygenase-1 in tumor progression. Front. Immunol. 12, 658315 (2021).
Mancuso, C. Biliverdin reductase as a target in drug research and development: facts and hypotheses. Free Radic. Biol. Med. 172, 521–529 (2021).
Bulmer, A. C. et al. Bile pigment pharmacokinetics and absorption in the rat: therapeutic potential for enteral administration. Br. J. Pharm. 164, 1857–1870 (2011).
Mancuso, C. Biliverdin as a disease-modifying agent: an integrated viewpoint. Free Radic. Biol. Med. 207, 133–143 (2023).
Shiels, R. G. et al. Pharmacokinetics of bilirubin-10-sulfonate and biliverdin in the rat. Eur. J. Pharm. Sci. 159, 105684 (2021).
Shiels, R. G. et al. Unprecedented microbial conversion of biliverdin into bilirubin-10-sulfonate. Sci. Rep. 9, 2988 (2019).
He, J. et al. Exosomal targeting and its potential clinical application. Drug Deliv. Transl. Res. 12, 2385–2402 (2022).
Du, S., Zhou, X. & Zheng, B. Beyond traditional medicine: EVs-loaded hydrogels as a game changer in disease therapeutics. Gels 10, 162 (2024).
Larsen, J. B. et al. Membrane curvature enables N-Ras lipid anchor sorting to liquid-ordered membrane phases. Nat. Chem. Biol. 11, 192–194 (2015).
Midekessa, G. et al. Zeta potential of extracellular vesicles: toward understanding the attributes that determine colloidal stability. ACS Omega 5, 16701–16710 (2020).
Jain, A. K. & Thareja, S. In vitro and in vivo characterization of pharmaceutical nanocarriers used for drug delivery. Artif. Cells Nanomed. Biotechnol. 47, 524–539 (2019).
Mcdonagh, A. F. & Palma, L. A. Preparation and properties of crystalline biliverdin IX alpha. Simple methods for preparing isomerically homogeneous biliverdin and [14C[biliverdin by using 2,3-dichloro-5,6-dicyanobenzoquinone. Biochem J. 189, 193–208 (1980).
Mei, J. et al. Production of bilirubin by biotransformation of biliverdin using recombinant Escherichia coli cells. Bioprocess Biosyst. Eng. 45, 563–571 (2022).
Wienkers, L. C. & Heath, T. G. Predicting in vivo drug interactions from in vitro drug discovery data. Nat. Rev. Drug Discov. 4, 825–833 (2005).
Normington, C., Chilton, C. H. & Buckley, A. M. Clostridioides difficile infections; new treatments and future perspectives. Curr. Opin. Gastroenterol. 40, 7–13 (2024).
Sun, Y. et al. Gut firmicutes: Relationship with dietary fiber and role in host homeostasis. Crit. Rev. Food Sci. Nutr. 63, 12073–12088 (2023).
Jordan, C. K. I. et al. Symbiotic Firmicutes establish mutualism with the host via innate tolerance and resistance to control systemic immunity. Cell Host Microbe 31, 1433–1449.e1439 (2023).
Brown, R. L., Sequeira, R. P. & Clarke, T. B. The microbiota protects against respiratory infection via GM-CSF signaling. Nat. Commun. 8, 1512 (2017).
Ichinohe, T. et al. Microbiota regulates immune defense against respiratory tract influenza A virus infection. Proc. Natl Acad. Sci. USA 108, 5354–5359 (2011).
Abdel-Nour, M., Tsalikis, J., Kleinman, D. & Girardin, S. E. The emerging role of mTOR signalling in antibacterial immunity. Immunol. Cell Biol. 92, 346–353 (2014).
Yu, Y. et al. Tumor necrosis factor-α induces interleukin-34 expression through nuclear factor‑κB activation in MC3T3-E1 osteoblastic cells. Mol. Med. Rep. 10, 1371–1376 (2014).
Naz, F. & Petri, W. A. Host immunity and immunization strategies for Clostridioides difficile infection. Clin. Microbiol Rev. 36, e0015722 (2023).
Koh, A., De Vadder, F., Kovatcheva-Datchary, P. & Bäckhed, F. From dietary fiber to host physiology: short-chain fatty acids as key bacterial metabolites. Cell 165, 1332–1345 (2016).
Louis, P. & Flint, H. J. Formation of propionate and butyrate by the human colonic microbiota. Environ. Microbiol 19, 29–41 (2017).
Van Der Hee, B. & Wells, J. M. Microbial regulation of host physiology by short-chain fatty acids. Trends Microbiol 29, 700–712 (2021).
Yuille, S., Mackay, W. G., Morrison, D. J. & Tedford, M. C. Drivers of Clostridioides difficile hypervirulent ribotype 027 spore germination, vegetative cell growth and toxin production in vitro. Clin. Microbiol. Infect. 26, 941.e941–941.e947 (2020).
Fachi, J. L. et al. Butyrate protects mice from Clostridium difficile-induced colitis through an HIF-1-dependent mechanism. Cell Rep. 27, 750–761.e757 (2019).
Wang, S. et al. Butyrate protects against Clostridium difficile infection by regulating bile acid metabolism. Microbiol. Spectr. 11, e0447922 (2023).
Pensinger, D. A. et al. Butyrate differentiates permissiveness to Clostridioides difficile infection and influences growth of diverse C. difficile isolates. Infect. Immun. 91, e0057022 (2023).
Kulecka, M. et al. Diarrheal-associated gut dysbiosis in cancer and inflammatory bowel disease patients is exacerbated by Clostridioides difficile infection. Front. Cell. Infect. Microbiol. 13, 1190910 (2023).
Fachi, J. L. et al. Acetate coordinates neutrophil and ILC3 responses against C. difficile through FFAR2. J. Exp. Med. 217, e20190489 (2020).
Vital, M., Howe, A. C. & Tiedje, J. M. Revealing the bacterial butyrate synthesis pathways by analyzing (meta)genomic data. mBio 5, e00889 (2014).
Farrow, M. A. et al. Clostridium difficile toxin B-induced necrosis is mediated by the host epithelial cell NADPH oxidase complex. Proc. Natl. Acad. Sci. USA 110, 18674–18679 (2013).
Montassier, E. et al. 16S rRNA gene pyrosequencing reveals shift in patient faecal microbiota during high-dose chemotherapy as conditioning regimen for bone marrow transplantation. Micro. Ecol. 67, 690–699 (2014).
Shen, Z. et al. Insights into Roseburia intestinalis which alleviates experimental colitis pathology by inducing anti-inflammatory responses. J. Gastroenterol. Hepatol. 33, 1751–1760 (2018).
Wang, S. et al. Treatment with butyrate alleviates dextran sulfate sodium and Clostridium difficile-induced colitis by preventing activity of Th17 cells via regulation of SIRT1/mTOR in mice. J. Nutr. Biochem. 111, 109155 (2023).
Ferreyra, J. A. et al. Gut microbiota-produced succinate promotes C. difficile infection after antibiotic treatment or motility disturbance. Cell Host Microbe 16, 770–777 (2014).
Abramson, J. et al. Accurate structure prediction of biomolecular interactions with AlphaFold 3. Nature 630, 493–500 (2024).
Kim, S. et al. PubChem 2023 update. Nucleic Acids Res. 51, D1373–D1380 (2022).
Eberhardt, J., Santos-Martins, D., Tillack, A. F. & Forli, S. AutoDock Vina 1.2.0: new docking methods, expanded force field, and python bindings. J. Chem. Inf. Model. 61, 3891–3898 (2021).
Seeliger, D. & De Groot, B. L. Ligand docking and binding site analysis with PyMOL and Autodock/Vina. J. Comput Aided Mol. Des. 24, 417–422 (2010).
Abraham, M. et al. GROMACS 2024.2 Source code (2024.2). (2024).
Hornak, V. et al. Comparison of multiple Amber force fields and development of improved protein backbone parameters. Proteins Struct. Funct. Bioinform. 65, 712–725 (2006).
Jorgensen, W. L. et al. Comparison of simple potential functions for simulating liquid water. J. Chem. Phys. 79, 926–935 (1983).
Valdés-Tresanco, M. S., Valdés-Tresanco, M. E., Valiente, P. A. & Moreno, E. gmx_MMPBSA: a new tool to perform end-state free energy calculations with GROMACS. J. Chem. Theory Comput 17, 6281–6291 (2021).
Zhang, K. et al. TcdB from hypervirulent Clostridioides difficile induces neuronal loss and neurotransmitter alterations in the intrinsic enteric nervous system. J. Infect. Dis. 231, 1465–1477 (2025).
Yi, C. et al. Comprehensive mapping of binding hot spots of SARS-CoV-2 RBD-specific neutralizing antibodies for tracking immune escape variants. Genome Med. 13, 164 (2021).
Li, S. et al. Critical roles of Clostridium difficile toxin B enzymatic activities in pathogenesis. Infect. Immun. 83, 502–513 (2015).
Li, Y. et al. Inhibition of CISD2 promotes ferroptosis through ferritinophagy-mediated ferritin turnover and regulation of p62-Keap1-NRF2 pathway. Cell. Mol. Biol. Lett. 27, 81 (2022).
Théry, C. et al. Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines. J. Extracell. Vesicles 7, 1535750 (2018).
Wan, S. et al. Intestine epithelial cell-derived extracellular vesicles alleviate inflammation induced by Clostridioides difficile TcdB through the activity of TGF-β1. Mol. Cell. Toxicol. 19, 509–519 (2023).
Zhou, Y. et al. Extracellular vesicles encapsulated with caspase-1 inhibitor ameliorate experimental autoimmune myasthenia gravis through targeting macrophages. J. Control. Release 364, 458–472 (2023).
Li, Y. H. et al. Berberine ameliorates chronic relapsing dextran sulfate sodium-induced colitis in C57BL/6 mice by suppressing Th17 responses. Pharm. Res. 110, 227–239 (2016).
Jin, D. et al. Molecular epidemiology of Clostridium difficile infection in hospitalized patients in Eastern China. J. Clin. Microbiol. 55, 801–810 (2017).
Bäumler, A. J. & Sperandio, V. Interactions between the microbiota and pathogenic bacteria in the gut. Nature 535, 85–93 (2016).
Fu, L. et al. ADMETlab 3.0: an updated comprehensive online ADMET prediction platform enhanced with broader coverage, improved performance, API functionality and decision support. Nucleic Acids Res. 52, W422-w431 (2024).
Daina, A., Michielin, O. & Zoete, V. SwissADME: a free web tool to evaluate pharmacokinetics, drug-likeness and medicinal chemistry friendliness of small molecules. Sci. Rep. 7, 42717 (2017).
Mitteer, D. R. & Greer, B. D. Using GraphPad prism’s heat maps for efficient, fine-grained analyses of single-case data. Behav. Anal. Pract. 15, 505–514 (2022).
