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.
Our results support a mechanism whereby the antigermination effect of CamSA is responsible for preventing CDI signs. This approach represents a new paradigm in CDI treatment. Instead of further compromising the microbiota of CDI patients with strong antibiotics, antigermination therapy could serve as a microbiota surrogate to curtail C. difficile colonization of antibiotic-treated patients.
CoQ10 is able to suppress the UVR- or IL-1-induced inflammatory response in dermal fibroblasts. Furthermore, this compound can block the UVR induction of the matrix-eroding enzyme, MMP-1. Finally, the combination of carotenoids plus CoQ10 results in enhanced suppression of inflammation. The results suggest that the combination of carotenoids and CoQ10 in topical skin care products may provide enhanced protection from inflammation and premature aging caused by sun exposure.
Transcriptional silencing and anti-silencing mechanisms modulate bacterial physiology and virulence in many human pathogens. In Shigella species, many virulence plasmid genes are silenced by the histone-like nucleoid structuring protein H-NS and anti-silenced by the virulence gene regulator VirB. Despite the key role that these regulatory proteins play in Shigella virulence, their mechanisms of transcriptional control remain poorly understood. Here, we characterize the regulatory elements and their relative spacing requirements needed for the transcriptional silencing and anti-silencing of icsP, a locus that requires remotely located regulatory elements for both types of transcriptional control. Our findings highlight the flexibility of the regulatory elements' positions with respect to each other, and yet, a molecular roadblock docked between the VirB binding site and the upstream H-NS binding region abolishes transcriptional anti-silencing by VirB, providing insight into transcriptional anti-silencing. Our study also raises the need to re-evaluate the currently proposed VirB binding site. Models of transcriptional silencing and anti-silencing at this genetic locus are presented, and the implications for understanding these regulatory mechanisms in bacteria are discussed.
Clostridium difficile infection (CDI) is a leading cause of antibiotic-associated diarrhea, a major nosocomial complication. The infective form of C. difficile is the spore, a dormant and resistant structure that forms under stress. Although spore germination is the first committed step in CDI onset, the temporal and spatial distribution of ingested C. difficile spores is not clearly understood. We recently reported that CamSA, a synthetic bile salt analog, inhibits C. difficile spore germination in vitro and in vivo. In this study, we took advantage of the anti-germination activity of bile salts to determine the fate of ingested C. difficile spores. We tested four different bile salts for efficacy in preventing CDI. Since CamSA was the only anti-germinant tested able to prevent signs of CDI, we characterized CamSa’s in vitro stability, distribution, and cytotoxicity. We report that CamSA is stable to simulated gastrointestinal (GI) environments, but will be degraded by members of the natural microbiota found in a healthy gut. Our data suggest that CamSA will not be systemically available, but instead will be localized to the GI tract. Since in vitro pharmacological parameters were acceptable, CamSA was used to probe the mouse model of CDI. By varying the timing of CamSA dosage, we estimated that C. difficile spores germinated and established infection less than 10 hours after ingestion. We also showed that ingested C. difficile spores rapidly transited through the GI tract and accumulated in the colon and cecum of CamSA-treated mice. From there, C. difficile spores were slowly shed over a 96-hour period. To our knowledge, this is the first report of using molecular probes to obtain disease progression information for C. difficile infection.
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