Mapping interactions between germinants and Clostridium difficile spores

Abstract

Germination of Clostridium difficile spores is the first required step in establishing C. difficile-associated disease (CDAD). Taurocholate (a bile salt) and glycine (an amino acid) have been shown to be important germinants of C. difficile spores. In the present study, we tested a series of glycine and taurocholate analogs for the ability to induce or inhibit C. difficile spore germination. Testing of glycine analogs revealed that both the carboxy and amino groups are important epitopes for recognition and that the glycine binding site can accommodate compounds with more widely separated termini. The C. difficile germination machinery also recognizes other hydrophobic amino acids. In general, linear alkyl side chains are better activators of spore germination than their branched analogs. However, L-phenylalanine and L-arginine are also good germinants and are probably recognized by distinct binding sites. Testing of taurocholate analogs revealed that the 12-hydroxyl group of taurocholate is necessary, but not sufficient, to activate spore germination. In contrast, the 6- and 7-hydroxyl groups are required for inhibition of C. difficile spore germination. Similarly, C. difficile spores are able to detect taurocholate analogs with shorter, but not longer, alkyl amino sulfonic acid side chains. Furthermore, the sulfonic acid group can be partially substituted with other acidic groups. Finally, a taurocholate analog with an m-aminobenzenesulfonic acid side chain is a strong inhibitor of C. difficile spore germination. In conclusion, C. difficile spores recognize both amino acids and taurocholate through multiple interactions that are required to bind the germinants and/or activate the germination machinery.

FIG. 1.

Germination kinetic graphs showing agonistic and antagonistic behavior of molecules with C. difficile spores. (A) Activation of germination. C. difficile spores were treated with a fixed concentration of taurocholate (6 mM), and glycine was added at 0 mM (○), 8 mM (•), 10 mM (□), 12 mM (▪), and 14 mM (▵) final concentrations. For clarity, the data are shown at 5-min intervals and for only five glycine concentrations. (B) Inhibition of germination. C. difficile spores were incubated with 0 mM (○), 0.0005 mM (•), 0.001 mM (□), 0.075 mM (▪), and 7.5 mM (▵) concentrations of CAmSA (T15) and supplemented with taurocholate (6 mM) and glycine (12 mM). For clarity, the data are shown at 5-min intervals and for only five CAmSA (T15) concentrations. Although data were collected for 90 min, only 75 min are shown in both graphs for clarity. The error bars indicate standard deviations.

FIG. 2.

Amino acids assessed for activation or inhibition of glycine-mediated germination in C. difficile spores. A01, glycine; A02, β-alanine; A03, γ-aminobutyric acid; A04, aminomethylphosphonic acid; A05, glycine methyl ester; A06, glycine ethyl ester; A07, glycinamide; A08, glycine hydroxamate; A09, diglycine; A10, glycine anhydride; A11, sarcosine; A12, N,N-dimethylglycine; A13, betaine; A14, N-acetylglycine; A15, nitrilotriacetic acid; A16, l-alanine; A17, d-alanine; A18, l-2-aminobutyric acid; A19, l-norvaline; A20, l-valine; A21, l-isoleucine; A22, l-leucine; A23, l-cysteine; A24, l-serine A25, l-methionine; A26, l-phenylalanine; A27, l-arginine; A28, l-lysine; A29, l-histidine; A30, l-aspartic acid.

FIG. 3.

Comparison of amino acids as agonists of C. difficile spore germination. Spores were treated with taurocholate (6 mM) and amino acid analogs at 12 mM. Germination was determined by the decrease in the OD₅₈₀ for 90 min at 30°C. The percent germination for each analog was calculated based on glycine/taurocholate germination set as 100%. The error bars indicate standard deviations.

FIG. 4.

Germination kinetic graph showing behavior of C. difficile spores and germinants in buffer and complex media. C. difficile spores were resuspended in germination buffer and treated with l-phenylalanine, l-arginine, and glycine (each at 12 mM) (○) or l-phenylalanine, l-arginine, glycine (each at 12 mM), and chenodeoxycholate (6 mM) (•). Purified spores were also suspended in BHIS medium (□), BHIS supplemented with 12 mM taurocholate (▴), or BHIS supplemented with 12 mM taurocholate and 12 mM chenodeoxycholate (▵). For clarity, data are shown at 5-min intervals and only for 75 min. The error bars indicate standard deviations.

FIG. 5.

Taurocholate analogs assessed for activation or inhibition of taurocholate-mediated germination in C. difficile spores. T01, taurocholate; T02, taurodeoxycholate; T03, taurochenodeoxycholate; T04, tauroursodeoxycholate; T05, taurolithocholate; T06, taurocholanate; T07, taurohycholate; T08, taurohyodeoxycholate; T09, 3-methoxy-7,12-dihydroxytaurocholate; T10, 3,7-dimethoxy-12-hydroxytaurocholate; T11, CAAMSA; T12, CAAPSA; T13, CApSA; T14, CAoSA; T15, CAmSA; T16, hypotaurocholate; T17, glycocholate; T18, CAAPA; T19, CA2APA; T20, CAABA; T21, CA2ABA; T22, CAHESA.