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 (EC 50 values; the concentrations that achieve a halfmaximum 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. IMPORTANCEClostridium 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. S pore formation and germination by Peptoclostridium difficile spores (1) (referred to here as Clostridium difficile for simplicity) are significant hurdles for overcoming the transmission of this pathogen within the hospital environment. Due to the strict anaerobic nature of C. difficile vegetative cells, spores are thought to be the main reservoir for transmission within the health care setting (2, 3). Prior antibiotic treatment ...

Germinants and Their Receptors in Clostridia

“…Some C. difficile strains are more primed to germinate in response to the taurocholic acid germinant than others (91). However, these differences do not appear to be linked to differences in the CspBA or CspC protein sequences, because any differences in protein sequence were ribotype specific, but the observed differences in germination were not (91).…”

The Design, Synthesis, and Characterizations of Spore Germination Inhibitors Effective against an Epidemic Strain of Clostridium difficile

“…31,32 Given this, we examined the sequences of CspC, from R20291, an epidemic strain that is not inhibited by CamSA and 630, a nonepidemic strain that is inhibited by CamSA with an IC 50 of 58 μM. To our surprise, we found that the sequences were 99.3% conserved between both strains.…”

Disease-Associated Changes in Bile Acid Profiles and Links to Altered Gut Microbiota

“…Bile acid signatures in the host GI tract have a significant influence upon the pathogenesis of C. difficile . Bile acids (in particular TCA) in the small intestine play a role in triggering the germination of spores into vegetative cells, while other bile acids (CDCA) inhibit this process [37,38] . Subsequently, the presence of secondary bile acids in the colon appears to be a major factor that influences host resistance to infection [9,10] .…”