scholarly journals Inhibiting the Initiation of Clostridium difficile Spore Germination using Analogs of Chenodeoxycholic Acid, a Bile Acid

2010 ◽  
Vol 192 (19) ◽  
pp. 4983-4990 ◽  
Author(s):  
Joseph A. Sorg ◽  
Abraham L. Sonenshein
Keyword(s):  
Bile Acid ◽  
Gastrointestinal Tract ◽  
Clostridium Difficile ◽  
Spore Germination ◽  
Competitive Inhibitor ◽  
Apparent Affinity ◽  
Core Member ◽  
Normal Human ◽  
Menten Kinetic ◽  
Colonic Environment

ABSTRACT To cause disease, Clostridium difficile spores must germinate in the host gastrointestinal tract. Germination is initiated upon exposure to glycine and certain bile acids, e.g., taurocholate. Chenodeoxycholate, another bile acid, inhibits taurocholate-mediated germination. By applying Michaelis-Menten kinetic analysis to C. difficile spore germination, we found that chenodeoxycholate is a competitive inhibitor of taurocholate-mediated germination and appears to interact with the spores with greater apparent affinity than does taurocholate. We also report that several analogs of chenodeoxycholate are even more effective inhibitors. Some of these compounds resist 7α-dehydroxylation by Clostridium scindens, a core member of the normal human colonic microbiota, suggesting that they are more stable than chenodeoxycholate in the colonic environment.

scholarly journals Spore Cortex Hydrolysis Precedes Dipicolinic Acid Release during Clostridium difficile Spore Germination

2015 ◽  
Vol 197 (14) ◽  
pp. 2276-2283 ◽  
Author(s):  
Michael B. Francis ◽  
Charlotte A. Allen ◽  
Joseph A. Sorg
Keyword(s):  
Bacillus Subtilis ◽  
Bile Acid ◽  
Clostridium Difficile ◽  
Spore Germination ◽  
Dipicolinic Acid ◽  
Model Organism ◽  
Stage I ◽  
Content Type ◽  
The Core ◽  
Dormant Spore

ABSTRACTBacterial spore germination is a process whereby a dormant spore returns to active, vegetative growth, and this process has largely been studied in the model organismBacillus subtilis. InB. subtilis, the initiation of germinant receptor-mediated spore germination is divided into two genetically separable stages. Stage I is characterized by the release of dipicolinic acid (DPA) from the spore core. Stage II is characterized by cortex degradation, and stage II is activated by the DPA released during stage I. Thus, DPA release precedes cortex hydrolysis duringB. subtilisspore germination. Here, we investigated the timing of DPA release and cortex hydrolysis duringClostridium difficilespore germination and found that cortex hydrolysis precedes DPA release. Inactivation of either the bile acid germinant receptor,cspC, or the cortex hydrolase,sleC, prevented both cortex hydrolysis and DPA release. Because both cortex hydrolysis and DPA release duringC. difficilespore germination are dependent on the presence of the germinant receptor and the cortex hydrolase, the release of DPA from the core may rely on the osmotic swelling of the core upon cortex hydrolysis. These results have implications for the hypothesized glycine receptor and suggest that the initiation of germinant receptor-mediatedC. difficilespore germination proceeds through a novel germination pathway.IMPORTANCEClostridium difficileinfects antibiotic-treated hosts and spreads between hosts as a dormant spore. In a host, spores germinate to the vegetative form that produces the toxins necessary for disease.C. difficilespore germination is stimulated by certain bile acids and glycine. We recently identified the bile acid germinant receptor as the germination-specific, protease-like CspC. CspC is likely cortex localized, where it can transmit the bile acid signal to the cortex hydrolase, SleC. Due to the differences in location of CspC compared to theBacillus subtilisgerminant receptors, we hypothesized that there are fundamental differences in the germination processes between the model organism andC. difficile. We found thatC. difficilespore germination proceeds through a novel pathway.

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

2015 ◽  
Vol 198 (5) ◽  
pp. 777-786 ◽  
Author(s):  
Disha Bhattacharjee ◽  
Michael B. Francis ◽  
Xicheng Ding ◽  
Kathleen N. McAllister ◽  
Ritu Shrestha ◽  
...  
Keyword(s):  
Bile Acid ◽  
Clostridium Difficile ◽  
Bile Acids ◽  
Spore Germination ◽  
Germination Rate ◽  
Taurocholic Acid ◽  
Receptor Complex ◽  
Health Care Environment ◽  
Content Type ◽  
Downstream Processes

ABSTRACTClostridium difficilespore germination is essential for colonization and disease. The signals that initiateC. difficilespore 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 preventC. difficileinfection or reduce recurring disease. However, a recent report called into question the utility of such a strategy to prevent infection by describingC. difficilestrains that germinated in the apparent absence of bile acids or germinated in the presence of the CDCA inhibitor. Because the mechanisms ofC. difficilespore germination are beginning to be elucidated, the mechanism of germination in these particular strains could yield important information on howC. difficilespores 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 (EC50values; 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.IMPORTANCEClostridium difficileforms metabolically dormant spores that persist in the health care environment. In susceptible hosts,C. difficilespores germinate in response to certain bile acids and glycine. Blocking germination byC. difficilespores is an attractive strategy to prevent the initiation of disease or to block recurring infection. However, certainC. difficilestrains have been identified whose spores germinate in the absence of bile acids or are not blocked by known inhibitors ofC. difficilespore 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 theC. difficilebile acid germinant receptor.

scholarly journals Antibiotic-Induced Alterations of the Gut Microbiota Alter Secondary Bile Acid Production and Allow for Clostridium difficile Spore Germination and Outgrowth in the Large Intestine

mSphere ◽  
2016 ◽  
Vol 1 (1) ◽  
Author(s):  
Casey M. Theriot ◽  
Alison A. Bowman ◽  
Vincent B. Young
Keyword(s):  
Bile Acid ◽  
Clostridium Difficile ◽  
Gut Microbiota ◽  
Bile Acids ◽  
Large Intestine ◽  
Spore Germination ◽  
Secondary Bile Acid ◽  
Content Type ◽  
Secondary Bile Acids

ABSTRACT Antibiotics alter the gastrointestinal microbiota, allowing for Clostridium difficile infection, which is a significant public health problem. Changes in the structure of the gut microbiota alter the metabolome, specifically the production of secondary bile acids. Specific bile acids are able to initiate C. difficile spore germination and also inhibit C. difficile growth in vitro, although no study to date has defined physiologically relevant bile acids in the gastrointestinal tract. In this study, we define the bile acids C. difficile spores encounter in the small and large intestines before and after various antibiotic treatments. Antibiotics that alter the gut microbiota and deplete secondary bile acid production allow C. difficile colonization, representing a mechanism of colonization resistance. Multiple secondary bile acids in the large intestine were able to inhibit C. difficile spore germination and growth at physiological concentrations and represent new targets to combat C. difficile in the large intestine. It is hypothesized that the depletion of microbial members responsible for converting primary bile acids into secondary bile acids reduces resistance to Clostridium difficile colonization. To date, inhibition of C. difficile growth by secondary bile acids has only been shown in vitro. Using targeted bile acid metabolomics, we sought to define the physiologically relevant concentrations of primary and secondary bile acids present in the murine small and large intestinal tracts and how these impact C. difficile dynamics. We treated mice with a variety of antibiotics to create distinct microbial and metabolic (bile acid) environments and directly tested their ability to support or inhibit C. difficile spore germination and outgrowth ex vivo. Susceptibility to C. difficile in the large intestine was observed only after specific broad-spectrum antibiotic treatment (cefoperazone, clindamycin, and vancomycin) and was accompanied by a significant loss of secondary bile acids (deoxycholate, lithocholate, ursodeoxycholate, hyodeoxycholate, and ω-muricholate). These changes were correlated to the loss of specific microbiota community members, the Lachnospiraceae and Ruminococcaceae families. Additionally, physiological concentrations of secondary bile acids present during C. difficile resistance were able to inhibit spore germination and outgrowth in vitro. Interestingly, we observed that C. difficile spore germination and outgrowth were supported constantly in murine small intestinal content regardless of antibiotic perturbation, suggesting that targeting growth of C. difficile will prove most important for future therapeutics and that antibiotic-related changes are organ specific. Understanding how the gut microbiota regulates bile acids throughout the intestine will aid the development of future therapies for C. difficile infection and other metabolically relevant disorders such as obesity and diabetes. IMPORTANCE Antibiotics alter the gastrointestinal microbiota, allowing for Clostridium difficile infection, which is a significant public health problem. Changes in the structure of the gut microbiota alter the metabolome, specifically the production of secondary bile acids. Specific bile acids are able to initiate C. difficile spore germination and also inhibit C. difficile growth in vitro, although no study to date has defined physiologically relevant bile acids in the gastrointestinal tract. In this study, we define the bile acids C. difficile spores encounter in the small and large intestines before and after various antibiotic treatments. Antibiotics that alter the gut microbiota and deplete secondary bile acid production allow C. difficile colonization, representing a mechanism of colonization resistance. Multiple secondary bile acids in the large intestine were able to inhibit C. difficile spore germination and growth at physiological concentrations and represent new targets to combat C. difficile in the large intestine.

scholarly journals The requirement for the amino acid co-germinant duringC. difficilespore germination is influenced by mutations inyabGandcspA

2018 ◽  
Author(s):  
Ritu Shrestha ◽  
Joseph A. Sorg
Keyword(s):  
Amino Acids ◽  
Bile Acid ◽  
Amino Acid ◽  
Clostridium Difficile ◽  
Cholic Acid ◽  
Spore Germination ◽  
Ems Mutagenesis ◽  
Bile Acid Receptor ◽  
Transmission Of Disease ◽  
Cholic Acid Derivatives

AbstractClostridium difficilespore germination is critical for the transmission of disease.C. difficilespores germinate in response to cholic acid derivatives, such as taurocholate (TA), and amino acids, such as glycine or alanine. Although the bile acid germinant receptor is known, the amino acid germinant receptor has remained elusive. Here, we used EMS mutagenesis to generate mutants with altered requirements for the amino acid co-germinant, similar to the strategy used previously to identify the bile acid receptor, CspC. Surprisingly, we identified strains that do not require amino acids as co-germinants, and the mutant spores germinated in response to TA alone. Upon sequencing these mutants, we identified different mutations inyabG.InC. difficile, yabGexpression is required for the processing of CspBA to CspB and CspA and preproSleC to proSleC during spore formation. A definedyabGmutant exacerbated the EMS mutant phenotype. Moreover, we found that various mutations incspAcaused spores to germinate in the presence of TA alone without the requirement of an amino acid. Thus, our study provides evidence that apart from regulating the CspC levels in the spore, CspA is important for recognition of amino acids as co-germinants duringC. difficilespore germination and that two pseudoproteases (CspC and CspA) function as theC. difficilegerminant receptors.

scholarly journals Updates to Clostridium difficile Spore Germination

2018 ◽  
Vol 200 (16) ◽  
Author(s):  
Travis J. Kochan ◽  
Matthew H. Foley ◽  
Michelle S. Shoshiev ◽  
Madeline J. Somers ◽  
Paul E. Carlson ◽  
...  
Keyword(s):  
Gastrointestinal Tract ◽  
Clostridium Difficile ◽  
Bacillus Anthracis ◽  
Clostridium Perfringens ◽  
Spore Germination ◽  
Bile Salts ◽  
Content Type ◽  
Receptor Complexes ◽  
Recent Advances

ABSTRACT Germination of Clostridium difficile spores is a crucial early requirement for colonization of the gastrointestinal tract. Likewise, C. difficile cannot cause disease pathologies unless its spores germinate into metabolically active, toxin-producing cells. Recent advances in our understanding of C. difficile spore germination mechanisms indicate that this process is both complex and unique. This review defines unique aspects of the germination pathways of C. difficile and compares them to those of two other well-studied organisms, Bacillus anthracis and Clostridium perfringens. C. difficile germination is unique, as C. difficile does not contain any orthologs of the traditional GerA-type germinant receptor complexes and is the only known sporeformer to require bile salts in order to germinate. While recent advances describing C. difficile germination mechanisms have been made on several fronts, major gaps in our understanding of C. difficile germination signaling remain. This review provides an updated, in-depth summary of advances in understanding of C. difficile germination and potential avenues for the development of therapeutics, and discusses the major discrepancies between current models of germination and areas of ongoing investigation.

scholarly journals Characterization of the Dynamic Germination of Individual Clostridium difficile Spores Using Raman Spectroscopy and Differential Interference Contrast Microscopy

2015 ◽  
Vol 197 (14) ◽  
pp. 2361-2373 ◽  
Author(s):  
Shiwei Wang ◽  
Aimee Shen ◽  
Peter Setlow ◽  
Yong-qing Li
Keyword(s):  
Raman Spectroscopy ◽  
Gastrointestinal Tract ◽  
Clostridium Difficile ◽  
Spore Germination ◽  
Bile Salts ◽  
Differential Interference Contrast ◽  
Gram Positive ◽  
Content Type ◽  
Kinetics Of ◽  
Dic Microscopy

ABSTRACTThe Gram-positive spore-forming anaerobeClostridium difficileis a leading cause of nosocomial diarrhea. Spores ofC. difficileinitiate infection when triggered to germinate by bile salts in the gastrointestinal tract. We analyzed germination kinetics of individualC. difficilespores using Raman spectroscopy and differential interference contrast (DIC) microscopy. Similar toBacillusspores, individualC. difficilespores germinating with taurocholate plus glycine began slow leakage of a ∼15% concentration of a chelate of Ca2+and dipicolinic acid (CaDPA) at a heterogeneous timeT1, rapidly released CaDPA atTlag, completed CaDPA release atTrelease, and finished peptidoglycan cortex hydrolysis atTlysis.T1andTlagvalues for individual spores were heterogeneous, but ΔTreleaseperiods (Trelease−Tlag) were relatively constant. In contrast toBacillusspores, heat treatment did not stimulate spore germination in the twoC. difficilestrains tested.C. difficilespores did not germinate with taurocholate or glycine alone, and different bile salts differentially promoted spore germination, with taurocholate and taurodeoxycholate being best. Transient exposure of spores to taurocholate plus glycine was sufficient to commit individual spores to germinate.C. difficilespores did not germinate with CaDPA, in contrast toB. subtilisandC. perfringensspores. However, the detergent dodecylamine inducedC. difficilespore germination, and rates were increased by spore coat removal although cortex hydrolysis did not followTrelease, in contrast withB. subtilis.C. difficilespores lacking the cortex-lytic enzyme, SleC, germinated extremely poorly, and cortex hydrolysis was not observed in the fewsleCspores that partially germinated. Overall, these findings indicate thatC. difficileandB. subtilisspore germination exhibit key differences.IMPORTANCESpores of the Gram-positive anaerobeClostridium difficileare responsible for initiating infection by this important nosocomial pathogen. When exposed to germinants such as bile salts,C. difficilespores return to life through germination in the gastrointestinal tract and cause disease, but their germination has been studied only with population-wide measurements. In this work we used Raman spectroscopy and DIC microscopy to monitor the kinetics of germination of individualC. difficilespores, the commitment of spores to germination, and the effect of germinant type and concentration, sublethal heat shock, and spore decoating on germination. Our data suggest that the order of germination events inC. difficilespores differs from that inBacillusspores and provide new insights intoC. difficilespore germination.

Purification and characterization of the yeast-expressed erythropoietin mutant Epo (R103A), a specific inhibitor of human primary hematopoietic cell erythropoiesis

Blood ◽  
2002 ◽  
Vol 99 (12) ◽  
pp. 4400-4405 ◽  
Author(s):  
Suzanne Burns ◽  
Murat O. Arcasoy ◽  
Li Li ◽  
Elizabeth Kurian ◽  
Katri Selander ◽  
...  
Keyword(s):  
Animal Models ◽  
Specific Inhibitor ◽  
Competitive Inhibitor ◽  
Erythropoietin Receptor ◽  
Single Amino Acid ◽  
Human Cd34 ◽  
Unit Formation ◽  
Dependent Cell ◽  
Normal Human

A drug that specifically inhibits erythropoiesis would be clinically useful. The erythropoietin (Epo) mutant Epo (R103A) could potentially be used for this purpose. Epo (R103A) has a single amino acid substitution of alanine for arginine at position 103. Because of this mutation, Epo (R103A) is only able to bind to one of the 2 subunits of the erythropoietin receptor (EpoR) homodimer and is thus a competitive inhibitor of Epo activity. To produce large quantities of Epo (R103A) to test in animal models of thalassemia and sickle cell disease, we expressed and purified recombinant Epo (R103A) from the yeast Pichia pastoris. Using this method milligram quantities of highly purified Epo (R103A) are obtained. The yeast-expressed Epo (R103A) is properly processed and glycosylated and specifically inhibits Epo-dependent cell growth and125I-Epo binding. Epo (R103A) does not, however, directly induce apoptosis in 32D cells expressing EpoR. Epo (R103A) inhibits erythropoiesis of human CD34+ hematopoietic cells and completely blocks erythroid burst-forming unit formation in normal human bone marrow colony assays. Yeast-expressed Epo (R103A) is a specific inhibitor of primary erythropoiesis suitable for testing in animal models.

scholarly journals Regulation of Clostridium difficile spore germination by the CspA pseudoprotease domain

Biochimie ◽  
2016 ◽  
Vol 122 ◽  
pp. 243-254 ◽  
Author(s):  
Yuzo Kevorkian ◽  
David J. Shirley ◽  
Aimee Shen
Keyword(s):  
Clostridium Difficile ◽  
Spore Germination
Sign in / Sign up

Export Citation Format

Share Document