Intestinal bile acids directly modulate the structure and function of C. difficile TcdB toxin John Tama, Simoun Ichoa,b, Evelyn Utamaa,b, Kathleen E. Orrellb, Rodolfo F. Gómez-Biagia,c, Casey M. Theriotd, Heather K. Krohe, Stacey A. Rutherforde, D. Borden Lacye, and Roman A. Melnyka,b,c,1 aMolecular Medicine, Research Institute, The Hospital for Sick Children, Toronto, ON M5G 0A4, Canada; bDepartment of Biochemistry, University of Toronto, Toronto, ON M5S 1A8, Canada; cSickKids Proteomics Analytics Robotics Chemical Biology Drug Discovery Facility, Research Institute, Hospital for Sick Children, Toronto, ON M5G 0A4, Canada; dDepartment of Population Health and Pathobiology, College of Veterinary Medicine, North Carolina State University, Raleigh, NC 27606; and eDepartment of Pathology, Microbiology, and Immunology, Vanderbilt University School of Medicine, Nashville, TN 37232 Edited by John Collier, Harvard Medical School, Boston, MA, and approved February 13, 2020 (received for review September 29, 2019) Intestinal bile acids are known to modulate the germination and C. difficile has been observed, particularly in hospitals and growth of Clostridioides difficile. Here we describe a role for in- healthcare settings (18–20). Though it is not known what factors testinal bile acids in directly binding and neutralizing TcdB toxin, are responsible for rendering an individual susceptible to infection the primary determinant of C. difficile disease. We show that in- and disease by C. difficile, several studies have shown that in- dividual primary and secondary bile acids reversibly bind and in- testinal bile acids play a role in modulating various aspects of the hibit TcdB to varying degrees through a mechanism that requires C. difficile lifecycle. For instance, it has been established that the the combined oligopeptide repeats region to which no function primary bile acid taurocholic acid and other cholic acid derivatives has previously been ascribed. We find that bile acids induce TcdB trigger germination of C. difficile spores into their toxin-producing “ ” into a compact balled up conformation that is no longer able to vegetative state via the germinant receptor CspC (21–23), whereas bind cell surface receptors. Lastly, through a high-throughput screen chenodeoxycholate derivatives and other secondary bile acids designed to identify bile acid mimetics we uncovered nonsteroidal inhibit cholate-induced germination (22, 24). Moreover, the small molecule scaffolds that bind and inhibit TcdB through a bile microbial-derived secondary bile acids, including deoxycholic acid acid-like mechanism. In addition to suggesting a role for bile acids in and lithocholic acid, are able to inhibit growth of C. difficile (25). C. difficile pathogenesis, these findings provide a framework for development of a mechanistic class of C. difficile antitoxins. In a recent study investigating the gut metabolome in mice before and after antibiotic exposure, it was shown that C. difficile bacte- C. difficile | toxin | bile acid | pathogenesis | structure rium can exploit specific metabolites that become more abundant in the mouse gut after antibiotics, including the primary bile acid taurocholate for spore germination (26). Further, in a landmark lostridioides difficile is the most frequent cause of infectious study, Buffie et al. were able to pinpoint the single bacterium, Cdiarrhea in hospitals and has emerged as a major public-health Clostridium scindens, which they showed enhances resistance concern in recent decades (1). Antibiotic-induced disruption of the protective gut microbiota triggers C. difficile infections (CDIs) by to C. difficile infection by producing key bile acids that directly creating an environment in the gut that enables C. difficile germi- inhibit C. difficile outgrowth (27). Finally, a recent study showed nation and growth. Virulent strains of C. difficile produce protein differences in bile acid composition between asymptomatic car- toxins that are responsible for the clinical symptoms of disease, riers of C. difficile and patients with CDI (28). Taken together, which can range from self-limiting diarrhea to pseudomembranous these studies highlight a complex interplay between C. difficile and colitis, and potentially death in severe cases (2). In particular, the homologous toxins TcdA and TcdB produced by pathogenic Significance strains of C. difficile are capable of causing disease in animal models (3), with TcdB appearing to be the primary determinant of Clostridioides difficile is a bacterial pathogen of global impor- disease in humans (4). TcdA and TcdB are large homologous tance that is a major cause of hospital-acquired diarrhea. toxins (sharing 48% sequence identity) with similar multidomain Antibiotic-mediated disruptions to the gut microbiota and as- architectures consisting of a glucosyltransferase domain (GTD), sociated metabolome promote C. difficile growth and infection an autoprocessing domain (APD), a translocation domain, and a through mechanisms that are poorly understood. Here, we C-terminal domain consisting of oligopeptide repeats, known as show that intestinal bile acids, which are known to play a role the CROP domain (5). After binding to their cell surface recep- in C. difficile germination and outgrowth, also directly bind and tors (6–9), TcdA and TcdB are internalized into acidified endo- inhibit TcdB toxin, the primary virulence determinant of somes, whereupon the central translocation domain forms C. difficile. Bile acid binding induces a major conformational transmembrane pores that are thought to mediate entry of the change in TcdB structure that prevents receptor binding and upstream GTD and APD into the cytosol (10). Processed and uptake into cells. In addition to suggesting a role for bile acids released GTD enzymatically glucosylates, and thereby inactivate in protecting against C. difficile pathogenesis, these findings intracellular Rho and Ras family GTPases (11, 12), leading first to highlight an approach to block C. difficile virulence. cytopathic effects (i.e., cell rounding) (13), and later cytotoxic effects (i.e., apoptosis and necrosis) (14, 15). Blocking the actions Author contributions: J.T., C.M.T., D.B.L., and R.A.M. designed research; J.T., S.I., E.U., of TcdB has emerged as a promising nonantibiotic-based strategy K.E.O., H.K.K., and S.A.R. performed research; J.T., R.F.G.-B., C.M.T., H.K.K., S.A.R., to treat CDI in recent years. Indeed, bezlotoxumab, a monoclonal D.B.L., and R.A.M. analyzed data; and J.T. and R.A.M. wrote the paper. antibody against TcdB, was recently approved for recurrent CDI The authors declare no competing interest. prevention in adults (4), and small molecules blocking TcdB ac- This article is a PNAS Direct Submission. tion have shown efficacy in preventing CDI in preclinical animal Published under the PNAS license. models (16, 17). 1To whom correspondence may be addressed. Email: [email protected]. Although toxins are responsible for symptomatic CDI, the mere This article contains supporting information online at https://www.pnas.org/lookup/suppl/ presence of toxigenic C. difficile in an individual, however, does doi:10.1073/pnas.1916965117/-/DCSupplemental. not portend disease. Indeed, asymptomatic carriage of toxigenic First published March 9, 2020. 6792–6800 | PNAS | March 24, 2020 | vol. 117 | no. 12 www.pnas.org/cgi/doi/10.1073/pnas.1916965117 Downloaded by guest on October 2, 2021 the host, which is dictated by the host-produced and microbiota- are more potent than the corresponding α7-hydroxylated primary modified bile acid composition. bile acids TCDCA, glycocholic acid (GCA), and taurocholic acid In this study, we describe an entirely unexpected role for bile (31), respectively. The binding site in TcdB can accommodate and acids in the C. difficile lifecycle as directly binding to and tolerate substitutions at R2 while the degree of hydroxylation at α7 inhibiting toxin uptake into cells. This work stems from a recent and α12, which lie on the “alpha face” of bile acids, is important high-throughput phenotypic screen that we conducted to identify for the binding interaction (SI Appendix,Fig.S1). The complete small molecules that prevent TcdB-induced toxicity, where methyl lack of binding of TcdB to dehydrocholic acid (dCA or dehydro- cholate—a synthetic methyl ester of cholic acid—was among the CA), oxidized at α3, α7, and α12, however, confirms that the ox- handful of hits in the primary screen that protected cells from idation state at these positions, and likely the stereochemistry of TcdB (29). Given that bile acids are abundant in the gut lumen the bile acid ring system, is important for binding. where C. difficile and its toxins act, we hypothesized that bile acids To evaluate the extent of protection by bile acids across a may, in addition to playing a role in spore germination and bac- range of different concentrations of TcdB that might be experi- terial viability, play a role in modulating virulence and therefore enced during an infection, cells were treated with a broad range disease. To explore the biological and therapeutic significance of TcdB concentrations at different fixed doses of TCDCA—a of this work we set out here to uncover the effects of natural highly soluble prototypic bile acid. In the absence of drug, TcdB bile acids on toxin pathogenesis and define the mechanism of dose-dependently rounds cells with an EC50 = 0.6 pM (Fig. 1D). inhibition. With increasing concentrations of TCDCA, the amount of TcdB required to reach