Reexamining the Germination Phenotypes of Several Clostridium difficile Strains Suggests Another Role for the CspC Germinant Receptor

Abstract

Clostridium difficile spore germination is essential for colonization and disease. The signals that initiate C. difficile spore germination are a combination of taurocholic acid (a bile acid) and glycine. Interestingly, the chenodeoxycholic acid class (CDCA) bile acids competitively inhibit taurocholic acid-mediated germination, suggesting that compounds that inhibit spore germination could be developed into drugs that prophylactically prevent C. difficile infection or reduce recurring disease. However, a recent report called into question the utility of such a strategy to prevent infection by describing C. difficile strains that germinated in the apparent absence of bile acids or germinated in the presence of the CDCA inhibitor. Because the mechanisms of C. difficile spore germination are beginning to be elucidated, the mechanism of germination in these particular strains could yield important information on how C. difficile spores initiate germination. Therefore, we quantified the interaction of these strains with taurocholic acid and CDCA, the rates of spore germination, the release of DPA from the spore core, and the abundance of the germinant receptor complex (CspC, CspB, and SleC). We found that strains previously observed to germinate in the absence of taurocholic acid correspond to more potent 50% effective concentrations (EC50 values; the concentrations that achieve a half-maximum germination rate) of the germinant and are still inhibited by CDCA, possibly explaining the previous observations. By comparing the germination kinetics and the abundance of proteins in the germinant receptor complex, we revised our original model for CspC-mediated activation of spore germination and propose that CspC may activate spore germination and then inhibit downstream processes.

Importance: Clostridium difficile forms metabolically dormant spores that persist in the health care environment. In susceptible hosts, C. difficile spores germinate in response to certain bile acids and glycine. Blocking germination by C. difficile spores is an attractive strategy to prevent the initiation of disease or to block recurring infection. However, certain C. difficile strains have been identified whose spores germinate in the absence of bile acids or are not blocked by known inhibitors of C. difficile spore germination (calling into question the utility of such strategies). Here, we further investigate these strains and reestablish that bile acid activators and inhibitors of germination affect these strains and use these data to suggest another role for the C. difficile bile acid germinant receptor.

FIG 1

Bile acid-mediated spore germination of several C. difficile strains. Purified C. difficile spores were suspended in BHIS medium alone (●) or medium supplemented with 2 mM (■), 5 mM (▲), 10 mM (▼), 20 mM (◆), or 50 mM (○) TA. Germination was monitored at OD₆₀₀ as described previously. (A) C. difficile UK1; (B) C. difficile 5108111; (C) C. difficile CD2315; (D) C. difficile DH1834; (E) C. difficile DH1858; (F) C. difficile M68; (G) C. difficile M120. A representative sample from three independent experiments is shown. For transparency, all plots are shown in Fig. S1 in the supplemental material.

FIG 2

Chenodeoxycholic acid inhibits C. difficile spore germination. Purified C. difficile spores were suspended in BHIS medium supplemented with 1 mM CDCA (●) or medium supplemented with 2 mM (■), 5 mM (▲), 10 mM (▼), 20 mM (◆), or 50 mM (○) TA and 1 mM CDCA. Germination was monitored at OD₆₀₀ as described previously. (A) C. difficile UK1; (B) C. difficile 5108111; (C) C. difficile CD2315; (D) C. difficile DH1834; (E) C. difficile DH1858; (F) C. difficile M68; (G) C. difficile M120. A representative sample from three independent experiments is shown. For transparency, all plots are shown in Fig. S3 in the supplemental material.

FIG 3

DPA release by several C. difficile strains. Purified C. difficile spores were suspended in HEPES buffer (●) or buffer supplemented with 10 mM TA (■), 100 mM glycine (▲), or 10 mM TA and 100 mM glycine (▼). DPA release during spore germination was monitored using Tb³⁺ fluorescence, as described previously. (A) C. difficile UK1; (B) C. difficile 5108111; (C) C. difficile CD2315; (D) C. difficile DH1834; (E) C. difficile DH1858; (F) C. difficile M68; (G) C. difficile M120. (H) Purified C. difficile spores were suspended in buffer and incubated at 100°C for 30 min. Total DPA content was normalized to the amount of DPA found in C. difficile UK1. All data represent the average from three independent experiments, and error bars represent the standard deviations from the mean.

FIG 4

Quantifying the abundance of CspB, CspC, and SleC in C. difficile spores. A total of 10⁹ C. difficile UK1 spores were extracted with NuPAGE buffer, as described previously. NuPAGE-soluble protein from three independent extracts was loaded onto a 10% SDS-polyacrylamide gel, along with recombinantly expressed and purified protein, as a protein standard. Samples were separated and detected as described in Materials and Methods. Signal intensities were quantified and used to generate a standard curve for CspB (A), CspC (B), and SleC (C). Signal intensities in the spore extract samples were quantified, and the generated standard curve was used to quantify the abundance of the specified proteins in the spore extracts. Independent standard curves were generated for each strain, and the results of the quantification can be found in Table 2. **, Full-length pre-pro-SleC; *, pro-SleC.

FIG 5

Correlating the abundance of C. difficile proteins with the kinetics of spore germination. (A) The calculated average per spore abundance of CspC was plotted versus the calculated rates of spore germination. (B) The ratio of SleC to CspC was plotted versus the calculated rates of spore germination. For all plots, GraphPad Prism was used to generate the linear fit to the curves, and the listed R² values represent the fit of the curve to the data.