Clostridioides difficile infection (CDI) is the major identifiable cause of antibiotic-associated diarrhea and has been declared an urgent threat by the CDC. C. difficile forms dormant and resistant spores that serve as infectious vehicles for CDI. To cause disease, C. difficile spores recognize taurocholate and glycine to trigger the germination process. In contrast to other sporulating bacteria, C. difficile spores are postulated to use a protease complex, CspABC, to recognize its germinants. Since spore germination is required for infection, we have developed anti-germination approaches for CDI prophylaxis. Previously, the bile salt analog CaPA (an aniline-substituted cholic acid) was shown to block spore germination and protect rodents from CDI caused by multiple C. difficile strains and isolates. In this study, we found that CaPA is an alternative substrate inhibitor of C. difficile spore germination. By competing with taurocholate for binding, CaPA delays C. difficile spore germination and reduces spore viability, thus diminishing the number of outgrowing vegetative bacteria. We hypothesize that the reduction of toxin-producing bacterial burden explains CaPA’s protective activity against murine CDI. Previous data combined with our results suggests that CaPA binds tightly to C. difficile spores in a CspC-dependent manner and irreversibly traps spores in an alternative, time-delayed, and low yield germination pathway. Our results are also consistent with kinetic data suggesting the existence of at least two distinct bile salt binding sites in C. difficile spores.
This is a preview of subscription content, access via your institution
Subscribe to this journal
Receive 12 print issues and online access
$259.00 per year
only $21.58 per issue
Rent or buy this article
Prices vary by article type
Prices may be subject to local taxes which are calculated during checkout
Dicks LMT, Mikkelsen LS, Brandsborg E, Marcotte H. Clostridium difficile, the Difficult “Kloster” Fuelled by Antibiotics. Curr Microbiol. 2019;76:774–82.
CDC. Antibiotic Resistance Threats in the United States 2019. In: Services USDoHaH, editor. Atlanta, GA.
Mylonakis E, Ryan ET, Calderwood SB. Clostridium difficile-associated diarrhea: a review. Arch Intern Med. 2001;161:525–33.
Ghose C. Clostridium difficile infection in the twenty-first century. Emerg Microbes Infect 2013;2:e62-e.
Olsen MA, Young-Xu Y, Stwalley D, Kelly CP, Gerding DN, Saeed MJ, et al. The Burden of Clostridium difficile Infection: Estimates of the Incidence of CDI from U.S. Administrative Databases. BMC Infect Dis. 2016;16:177.
Burns DA, Heap JT, Minton NP. Clostridium difficile spore germination: an update. Res Microbiol. 2010;161:730–4.
Giel JL, Sorg JA, Sonenshein AL, Zhu J. Metabolism of bile salts in mice influences spore germination in Clostridium difficile. PLOS ONE. 2010;5:e8740.
Francis MB, Allen CA, Shrestha R, Sorg JA. Bile acid recognition by the Clostridium difficile germinant receptor, CspC, is important for establishing infection. PLOS Pathog. 2013;9:e1003356.
Sorg JA, Sonenshein AL. Chenodeoxycholate is an inhibitor of Clostridium difficile spore germination. J Bacteriol. 2009;191:1115–7.
Sorg JA, Sonenshein AL. Bile salts and glycine as Cogerminants for Clostridium difficile spores. J Bacteriol. 2008;190:2505–12.
Howerton A, Ramirez N, Abel-Santos E. Mapping interactions between Germinants and Clostridium difficile spores. J Bacteriol. 2011;193:274–82.
Ramirez N, Liggins M, Abel-Santos E. Kinetic evidence for the presence of putative germination receptors in C. difficile spores. J Bacteriol. 2010;192:4215–22.
Ross C, Abel-Santos E. The Ger receptor family in sporulating bacteria. Curr Issues Mol Biol. 2010;12:147–58.
Setlow P. Spore germination. Curr Opin Microbiol. 2003;6:550–6.
Moir A, Corfe BM, Behravan J. Spore germination. Cell Mol Life Sci. 2002;59:403–9.
Ross CA, Abel-Santos E. Guidelines for nomenclature assignment of ger receptors. Res Microbiol. 2010;161:830–7.
Knight DR, Elliott B, Chang BJ, Perkins TT, Riley TV. Diversity and evolution in the genome of Clostridium difficile. Clin Microbiol Rev. 2015;28:721–41.
Bhattacharjee D, Francis MB, Ding X, McAllister KN, Shrestha R, Sorg JA, et al. Reexamining the germination phenotypes of several Clostridium difficile strains suggests another role for the CspC germinant receptor. J Bacteriol. 2016;198:777–86.
Shrestha R, Cochran AM, Sorg JA. The requirement for Co-germinants During Clostridium difficile spore germination is influenced by mutations in yabG and cspA. PLOS Pathog. 2019;15:e1007681.
Kevorkian Y, Shen A. Revisiting the role of Csp family proteins in regulating Clostridium difficile spore germination. J Bacteriol. 2017;199:e00266–17.
Rohlfing AE, Eckenroth BE, Forster ER, Kevorkian Y, Donnelly ML, Benito de la Puebla H, et al. The CspC pseudoprotease regulates germination of Clostridioides difficile spores in response to multiple environmental signals. PLoS Genet. 2019;15:e1008224.
Heeg D, Burns DA, Cartman ST, Minton NP. Spores of Clostridium difficile clinical isolates display a diverse germination response to bile salts. PLoS ONE. 2012;7:e32381.
Alvarez Z, Abel-Santos E. Potential use of inhibitors of bacteria spore germination in the prophylactic treatment of anthrax and Clostridium difficile-associated disease. Expert Rev Anti-infective Ther. 2007;5:783–92.
Stoltz KL, Erickson R, Staley C, Weingarden AR, Romens E, Steer CJ, et al. Synthesis and biological evaluation of bile acid analogues inhibitory to Clostridium difficile spore germination. J Med Chem. 2017;60:3451–71.
Sorg JA, Sonenshein AL. Inhibiting the Initiation of Clostridium difficile spore germination using analogs of chenodeoxycholic acid, a bile acid. J Bacteriol. 2010;192:4983–90.
Sharma SK, Yip C, Simon MP, Phan J, Abel-Santos E, Firestine SM. Studies on the importance of the 7α-, and 12α- hydroxyl groups of N-Aryl-3α,7α,12α-trihydroxy-5β-cholan-24-amides on their Antigermination Activity Against a Hypervirulent Strain of Clostridioides (Clostridium) difficile. Bioorg Med Chem. 2021;52:116503.
Sharma SK, Yip C, Esposito EX, Sharma PV, Simon MP, Abel-Santos E, et al. The design, synthesis, and characterizations of spore germination inhibitors effective against an epidemic strain of Clostridium difficile. J Med Chem. 2018;61:6759–78.
Liggins M, Ramirez N, Magnuson N, Abel-Santos E. Progesterone analogs influence germination of Clostridium sordellii and Clostridium difficile Spores In Vitro. J Bacteriol. 2011;193:2776–83.
Weingarden AR, Chen C, Zhang N, Graiziger CT, Dosa PI, Steer CJ, et al. Ursodeoxycholic acid inhibits Clostridium difficile spore germination and vegetative growth, and prevents the recurrence of ileal pouchitis associated with the infection. J Clin Gastroenterol. 2016;50:624–30.
Winston JA, Rivera AJ, Cai J, Thanissery R, Montgomery SA, Patterson AD, et al. Ursodeoxycholic Acid (UDCA) mitigates the host inflammatory response during Clostridioides difficile infection by altering gut bile acids. Infect Immun. 2020;88:e00045-20.
Thanissery R, Winston JA, Theriot CM. Inhibition of spore germination, growth, and toxin activity of clinically relevant C. difficile strains by gut microbiota derived secondary bile acids. Anaerobe. 2017;45:86–100.
Francis MB, Allen CA, Sorg JA. Muricholic acids Inhibit Clostridium difficile spore germination and growth. PLoS One. 2013;8:e73653.
Phan JR, Do DM, Truong MC, Ngo C, Phan JH, Sharma SK, et al. An aniline-substituted bile salt analog protects both mice and hamsters from multiple Clostridioides difficile strains. Antimicrobial Agents Chemother. 2022;66:e01435–21.
Howerton A, Seymour CO, Murugapiran SK, Liao Z, Phan JR, Estrada A, et al. Effect of the synthetic bile salt analog CamSA on the hamster model of Clostridium difficile infection. Antimicrobial Agents Chemother. 2018;62:e02251-17.
Howerton A, Patra M, Abel-Santos E. Fate of ingested Clostridium difficile spores in mice. PloS one 2013;8:e72620-e.
Howerton A, Patra M, Abel-Santos E. A new strategy for the prevention of Clostridium difficile infection. J Infect Dis. 2013;207:1498–504.
Yip C, Okada NC, Howerton A, Amei A, Abel-Santos E. Pharmacokinetics of Camsa, a potential prophylactic compound against Clostridioides difficile infections. Biochem Pharmacol. 2021;183:114314.
Sattar A, Thommes P, Payne L, Warn P, Vickers RJ. SMT19969 for Clostridium difficile infection (CDI): In Vivo Efficacy Compared with Fidaxomicin and Vancomycin in the Hamster Model of CDI. J Antimicrobial Chemother. 2015;70:1757–62.
Mormak DA, Casida LE. Study of Bacillus subtilis endospores in soil by use of a modified endospore stain. Appl Environ Microbiol. 1985;49:1356–60.
Desrosier NW, Heiligman F. Heat activation of bacterial spores. Food Res. 1956;21:54–62.
Ehsaan M, Kuehne SA, Minton NP Clostridium difficile Genome Editing Using pyrE Alleles. In: A. R, P. M, editors. Clostridium difficile Methods in Molecular Biology. 1476. New York, NY: Humana Press; 2016. 35–52.
Cartman ST, Kelly ML, Heeg D, Heap JT, Minton NP. Precise manipulation of the Clostridium difficile chromosome reveals a lack of association between the tcdC Genotype and toxin production. Appl Environ Microbiol. 2012;78:4683–90.
Hindle A, Hall E. Dipicolinic acid (DPA) assay revisited and appraised for spore detection. Analyst 1999;124:1599–604.
Wilson KH. Efficiency of various bile salt preparations for stimulation Of Clostridium Difficile spore germination. J Clin Microbiol. 1983;18:1017–9.
Segel IH Enzyme kinetics behavior and analysis of rapid equilibrium and steady-state enzyme systems. Wiley Classics Library Edition ed. New York, NY: Wiley Interscience Publication; 1993. p. 793–813.
Sebaihia M, Wren BW, Mullany P, Fairweather NF, Minton N, Stabler R, et al. The multidrug-resistant human pathogen Clostridium difficile has a highly mobile, mosaic genome. Nat Genet. 2006;38:779–86.
Fromm HJ Product, Substrate, and Alternative Substrate Inhibition. In: Fromm HJ, editor. Initial Rate Enzyme Kinetics. Berlin, Heidelberg: Springer Berlin Heidelberg; 1975. p. 121–60.
Kokkonen P, Beier A, Mazurenko S, Damborsky J, Bednar D, Prokop Z. Substrate inhibition by the blockage of product release and its control by tunnel engineering. RSC Chem Biol. 2021;2:645–55.
Luu H, Akoachere M, Patra M. Abel-Santos E. cooperativity and interference of germination pathways in Bacillus anthracis Spores. J Bacteriol. 2011;193:4192–8.
Ramirez N, Abel-Santos E. Requirements for germination of Clostridium sordellii spores in vitro. J Bacteriol. 2010;192:418–25.
Akoachere M, Squires RC, Nour AM, Angelov L, Brojatsch J, Abel-Santos EV. Identification of an in vivo inhibitor of Bacillus anthracis sterne spore germination. J Biol Chem. 2007;282:12112–8.
This work was supported by the National Institute of Health [grant numbers R01-AI109139 and GM103440]. The authors thank Prof. Steve Firestine from Wayne State University for the synthesis of CaPA, Prof. Nigel Minton from University of Nottingham for providing clinical C. difficile isolates, and Prof. Aimee Shen from Tufts University for the training and materials for C. difficile knock-out system.
Conflict of interest
The authors declare no competing interests.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
About this article
Cite this article
Yip, C., Phan, J.R. & Abel-Santos, E. Mechanism of germination inhibition of Clostridioides difficile spores by an aniline substituted cholate derivative (CaPA). J Antibiot 76, 335–345 (2023). https://doi.org/10.1038/s41429-023-00612-3