scholarly journals Dipicolinic Acid Release by Germinating Clostridium difficile Spores Occurs through a Mechanosensing Mechanism

mSphere ◽  
2016 ◽  
Vol 1 (6) ◽  
Author(s):  
Michael B. Francis ◽  
Joseph A. Sorg
Keyword(s):  
Clostridium Difficile ◽  
Bile Acids ◽  
Spore Germination ◽  
Dipicolinic Acid ◽  
Model Organism ◽  
Lytic Enzyme ◽  
Prior Work ◽  
Spore Form ◽  
Content Type ◽  
Dormant Spore

ABSTRACT Clostridium difficile is transmitted between hosts in the form of a dormant spore, and germination by C. difficile spores is required to initiate infection, because the toxins that are necessary for disease are not deposited on the spore form. Importantly, the C. difficile spore germination pathway represents a novel pathway for bacterial spore germination. Prior work has shown that the order of events during C. difficile spore germination (cortex degradation and DPA release) is flipped compared to the events during B. subtilis spore germination, a model organism. Here, we further characterize the C. difficile spore germination pathway and summarize our findings indicating that DPA release by germinating C. difficile spores occurs through a mechanosensing mechanism in response to the degradation of the spore cortex. Classically, dormant endospores are defined by their resistance properties, particularly their resistance to heat. Much of the heat resistance is due to the large amount of dipicolinic acid (DPA) stored within the spore core. During spore germination, DPA is released and allows for rehydration of the otherwise-dehydrated core. In Bacillus subtilis, 7 proteins are encoded by the spoVA operon and are important for DPA release. These proteins receive a signal from the activated germinant receptor and release DPA. This DPA activates the cortex lytic enzyme CwlJ, and cortex degradation begins. In Clostridium difficile, spore germination is initiated in response to certain bile acids and amino acids. These bile acids interact with the CspC germinant receptor, which then transfers the signal to the CspB protease. Activated CspB cleaves the cortex lytic enzyme, pro-SleC, to its active form. Subsequently, DPA is released from the core. C. difficile encodes orthologues of spoVAC, spoVAD, and spoVAE. Of these, the B. subtilis SpoVAC protein was shown to be capable of mechanosensing. Because cortex degradation precedes DPA release during C. difficile spore germination (opposite of what occurs in B. subtilis), we hypothesized that cortex degradation would relieve the osmotic constraints placed on the inner spore membrane and permit DPA release. Here, we assayed germination in the presence of osmolytes, and we found that they can delay DPA release from germinating C. difficile spores while still permitting cortex degradation. Together, our results suggest that DPA release during C. difficile spore germination occurs though a mechanosensing mechanism. IMPORTANCE Clostridium difficile is transmitted between hosts in the form of a dormant spore, and germination by C. difficile spores is required to initiate infection, because the toxins that are necessary for disease are not deposited on the spore form. Importantly, the C. difficile spore germination pathway represents a novel pathway for bacterial spore germination. Prior work has shown that the order of events during C. difficile spore germination (cortex degradation and DPA release) is flipped compared to the events during B. subtilis spore germination, a model organism. Here, we further characterize the C. difficile spore germination pathway and summarize our findings indicating that DPA release by germinating C. difficile spores occurs through a mechanosensing mechanism in response to the degradation of the spore cortex.

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 Clostridium difficileLipoprotein GerS Is Required for Cortex Modification and Thus Spore Germination

mSphere ◽  
2018 ◽  
Vol 3 (3) ◽  
Author(s):  
Oscar R. Diaz ◽  
Cameron V. Sayer ◽  
David L. Popham ◽  
Aimee Shen
Keyword(s):  
Clostridium Difficile ◽  
Spore Germination ◽  
Protective Layer ◽  
Thick Layer ◽  
Lytic Enzyme ◽  
Gram Positive ◽  
Content Type ◽  
Clostridioides Difficile ◽  
Antibiotic Associated Diarrhea ◽  
Biochemical Analyses

ABSTRACTClostridium difficile, also known asClostridioides difficile, is a Gram-positive, spore-forming bacterium that is a leading cause of antibiotic-associated diarrhea.C. difficileinfections begin when its metabolically dormant spores germinate to form toxin-producing vegetative cells. Successful spore germination depends on the degradation of the cortex, a thick layer of modified peptidoglycan that maintains dormancy. Cortex degradation is mediated by the SleC cortex lytic enzyme, which is thought to recognize the cortex-specific modification muramic-δ-lactam.C. difficilecortex degradation also depends on thePeptostreptococcaceae-specific lipoprotein GerS for unknown reasons. In this study, we tested whether GerS regulates production of muramic-δ-lactam and thus controls the ability of SleC to recognize its cortex substrate. By comparing the muropeptide profiles of ΔgerSspores to those of spores lacking either CwlD or PdaA, both of which mediate cortex modification inBacillus subtilis, we determined thatC. difficileGerS, CwlD, and PdaA are all required to generate muramic-δ-lactam. Both GerS and CwlD were needed to cleave the peptide side chains from N-acetylmuramic acid, suggesting that these two factors act in concert. Consistent with this hypothesis, biochemical analyses revealed that GerS and CwlD directly interact and that CwlD modulates GerS incorporation into mature spores. Since ΔgerS, ΔcwlD, and ΔpdaAspores exhibited equivalent germination defects, our results indicate thatC. difficilespore germination depends on cortex-specific modifications, reveal GerS as a novel regulator of these processes, and highlight additional differences in the regulation of spore germination inC. difficilerelative toB. subtilisand other spore-forming organisms.IMPORTANCEThe Gram-positive, spore-forming bacteriumClostridium difficileis a leading cause of antibiotic-associated diarrhea. BecauseC. difficileis an obligate anaerobe, its aerotolerant spores are essential for transmitting disease, and their germination into toxin-producing cells is necessary for causing disease. Spore germination requires the removal of the cortex, a thick layer of modified peptidoglycan that maintains spore dormancy. Cortex degradation is mediated by the SleC hydrolase, which is thought to recognize cortex-specific modifications. Cortex degradation also requires the GerS lipoprotein for unknown reasons. In our study, we tested whether GerS is required to generate cortex-specific modifications by comparing the cortex composition of ΔgerSspores to the cortex composition of spores lacking two putative cortex-modifying enzymes, CwlD and PdaA. These analyses revealed that GerS, CwlD, and PdaA are all required to generate cortex-specific modifications. Since loss of these modifications in ΔgerS, ΔcwlD, and ΔpdaAmutants resulted in spore germination and heat resistance defects, the SleC cortex lytic enzyme depends on cortex-specific modifications to efficiently degrade this protective layer. Our results further indicate that GerS and CwlD are mutually required for removing peptide chains from spore peptidoglycan and revealed a novel interaction between these proteins. Thus, our findings provide new mechanistic insight intoC. difficilespore germination.

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 Revisiting the Role of Csp Family Proteins in Regulating Clostridium difficile Spore Germination

2017 ◽  
Vol 199 (22) ◽  
Author(s):  
Yuzo Kevorkian ◽  
Aimee Shen
Keyword(s):  
Health Care ◽  
Clostridium Difficile ◽  
Bile Salt ◽  
Spore Germination ◽  
Gastrointestinal Disease ◽  
Spore Form ◽  
Content Type ◽  
Allelic Exchange ◽  
Sequencing Errors ◽  
The Individual

ABSTRACT Clostridium difficile causes considerable health care-associated gastrointestinal disease that is transmitted by its metabolically dormant spore form. Upon entering the gut, C. difficile spores germinate and outgrow to produce vegetative cells that release disease-causing toxins. C. difficile spore germination depends on the Csp family of (pseudo)proteases and the cortex hydrolase SleC. The CspC pseudoprotease functions as a bile salt germinant receptor that activates the protease CspB, which in turn proteolytically activates the SleC zymogen. Active SleC degrades the protective cortex layer, allowing spores to outgrow and resume metabolism. We previously showed that the CspA pseudoprotease domain, which is initially produced as a fusion to CspB, controls the incorporation of the CspC germinant receptor in mature spores. However, study of the individual Csp proteins has been complicated by the polar effects of TargeTron-based gene disruption on the cspBA-cspC operon. To overcome these limitations, we have used pyrE-based allelic exchange to create individual deletions of the regions encoding CspB, CspA, CspBA, and CspC in strain 630Δerm. Our results indicate that stable CspA levels in sporulating cells depend on CspB and confirm that CspA maximizes CspC incorporation into spores. Interestingly, we observed that csp and sleC mutants spontaneously germinate more frequently in 630Δerm than equivalent mutants in the JIR8094 and UK1 strain backgrounds. Analyses of this phenomenon suggest that only a subpopulation of C. difficile 630Δerm spores can spontaneously germinate, in contrast with Bacillus subtilis spores. We also show that C. difficile clinical isolates that encode truncated CspBA variants have sequencing errors that actually produce full-length CspBA. IMPORTANCE Clostridium difficile is a leading cause of health care-associated infections. Initiation of C. difficile infection depends on spore germination, a process controlled by Csp family (pseudo)proteases. The CspC pseudoprotease is a germinant receptor that senses bile salts and activates the CspB protease, which activates a hydrolase required for germination. Previous work implicated the CspA pseudoprotease in controlling CspC incorporation into spores but relied on plasmid-based overexpression. Here we have used allelic exchange to study the functions of CspB and CspA. We determined that CspA production and/or stability depends on CspB and confirmed that CspA maximizes CspC incorporation into spores. Our data also suggest that a subpopulation of C. difficile spores spontaneously germinates in the absence of bile salt germinants and/or Csp proteins.

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 Slow Leakage of Ca-Dipicolinic Acid from Individual Bacillus Spores during Initiation of Spore Germination

2015 ◽  
Vol 197 (6) ◽  
pp. 1095-1103 ◽  
Author(s):  
Shiwei Wang ◽  
Peter Setlow ◽  
Yong-qing Li
Keyword(s):  
Spore Germination ◽  
Dipicolinic Acid ◽  
Lytic Enzyme ◽  
Early Event ◽  
Laser Tweezers ◽  
Wild Type ◽  
Content Type ◽  
Rapid Release ◽  
Contrast Microscopy ◽  
Bacillus Spores

When exposed to nutrient or nonnutrient germinants, individualBacillusspores can return to life through germination followed by outgrowth. Laser tweezers, Raman spectroscopy, and either differential interference contrast or phase-contrast microscopy were used to analyze the slow dipicolinic acid (DPA) leakage (normally ∼20% of spore DPA) from individual spores that takes place prior to the lag time,Tlag, when spores begin rapid release of remaining DPA. Major conclusions from this work withBacillus subtilisspores were as follows: (i) slow DPA leakage from wild-type spores germinating with nutrients did not begin immediately after nutrient exposure but only at a later heterogeneous timeT1; (ii) the period of slow DPA leakage (ΔTleakage=Tlag−T1) was heterogeneous among individual spores, although the amount of DPA released in this period was relatively constant; (iii) increases in germination temperature significantly decreasedT1times but increased values of ΔTleakage; (iv) upon germination withl-valine for 10 min followed by addition ofd-alanine to block further germination, all germinated spores hadT1times of less than 10 min, suggesting thatT1is the time when spores become committed to germinate; (v) elevated levels of SpoVA proteins involved in DPA movement in spore germination decreasedT1andTlagtimes but not the amount of DPA released in ΔTleakage; (vi) lack of the cortex-lytic enzyme CwlJ increased DPA leakage during germination due to longer ΔTleakagetimes in which more DPA was released; and (vii) there was slow DPA leakage early in germination ofB. subtilisspores by the nonnutrients CaDPA and dodecylamine and in nutrient germination ofBacillus cereusandBacillus megateriumspores. Overall, these findings have identified and characterized a new early event inBacillusspore germination.

scholarly journals Characterization of Clostridium difficile Spores Lacking Either SpoVAC or Dipicolinic Acid Synthetase

2016 ◽  
Vol 198 (11) ◽  
pp. 1694-1707 ◽  
Author(s):  
M. Lauren Donnelly ◽  
Kelly A. Fimlaid ◽  
Aimee Shen
Keyword(s):  
Clostridium Difficile ◽  
Heat Resistance ◽  
Spore Germination ◽  
Dipicolinic Acid ◽  
The United States ◽  
Spore Formation ◽  
Obligate Anaerobe ◽  
Wild Type ◽  
Content Type ◽  
Wet Heat

ABSTRACTThe spore-forming obligate anaerobeClostridium difficileis a leading cause of antibiotic-associated diarrhea around the world. In order forC. difficileto cause infection, its metabolically dormant spores must germinate in the gastrointestinal tract. During germination, spores degrade their protective cortex peptidoglycan layers, release dipicolinic acid (DPA), and hydrate their cores. InC. difficile, cortex hydrolysis is necessary for DPA release, whereas inBacillus subtilis, DPA release is necessary for cortex hydrolysis. Given this difference, we tested whether DPA synthesis and/or release was required forC. difficilespore germination by constructing mutations in eitherspoVACordpaAB, which encode an ion channel predicted to transport DPA into the forespore and the enzyme complex predicted to synthesize DPA, respectively.C. difficilespoVACanddpaABmutant spores lacked DPA but could be stably purified and were more hydrated than wild-type spores; in contrast,B. subtilisspoVACanddpaABmutant spores were unstable. AlthoughC. difficilespoVACanddpaABmutant spores exhibited wild-type germination responses, they were more readily killed by wet heat. Cortex hydrolysis was not affected by this treatment, indicating that wet heat inhibits a stage downstream of this event. Interestingly,C. difficilespoVACmutant spores were significantly more sensitive to heat treatment thandpaABmutant spores, indicating that SpoVAC plays additional roles in conferring heat resistance. Taken together, our results demonstrate that SpoVAC and DPA synthetase controlC. difficilespore resistance and reveal differential requirements for these proteins among theFirmicutes.IMPORTANCEClostridium difficileis a spore-forming obligate anaerobe that causes ∼500,000 infections per year in the United States. Although spore germination is essential forC. difficileto cause disease, the factors required for this process have been only partially characterized. This study describes the roles of two factors, DpaAB and SpoVAC, which control the synthesis and release of dipicolinic acid (DPA), respectively, from bacterial spores. Previous studies of these proteins in other spore-forming organisms indicated that they are differentially required for spore formation, germination, and resistance. We now show that the proteins are dispensable forC. difficilespore formation and germination but are necessary for heat resistance. Thus, our study further highlights the diverse functions of DpaAB and SpoVAC in spore-forming organisms.

scholarly journals Germinant Synergy FacilitatesClostridium difficileSpore Germination under Physiological Conditions

mSphere ◽  
2018 ◽  
Vol 3 (5) ◽  
Author(s):  
Travis J. Kochan ◽  
Michelle S. Shoshiev ◽  
Jessica L. Hastie ◽  
Madeline J. Somers ◽  
Yael M. Plotnick ◽  
...  
Keyword(s):  
Amino Acids ◽  
Clostridium Difficile ◽  
Spore Germination ◽  
Bile Salts ◽  
Gi Tract ◽  
Infectious Diarrhea ◽  
Content Type ◽  
Increased Risk ◽  
Calcium Reabsorption

ABSTRACTClostridium difficileis a Gram-positive obligate anaerobe that forms spores in order to survive for long periods in the unfavorable environment outside a host.C. difficileis the leading cause of nosocomial infectious diarrhea worldwide.C. difficileinfection (CDI) arises after a patient treated with broad-spectrum antibiotics ingests infectious spores. The first step inC. difficilepathogenesis is the metabolic reactivation of dormant spores within the gastrointestinal (GI) tract through a process known as germination. In this work, we aim to elucidate the specific conditions and the location within the GI tract that facilitate this process. Our data suggest thatC. difficilegermination occurs through a two-step biochemical process that is regulated by pH and bile salts, amino acids, and calcium present within the GI tract. Maximal germination occurs at a pH ranging from 6.5 to 8.5 in the terminal small intestine prior to bile salt and calcium reabsorption by the host. Germination can be initiated by lower concentrations of germinants when spores are incubated with a combination of bile salts, calcium, and amino acids, and this synergy is dependent on the availability of calcium. The synergy described here allows germination to proceed in the presence of inhibitory bile salts and at physiological concentrations of germinants, effectively decreasing the concentrations of nutrients required to initiate an essential step of pathogenesis.IMPORTANCEClostridium difficileis an anaerobic spore-forming human pathogen that is the leading cause of nosocomial infectious diarrhea worldwide. Germination of infectious spores is the first step in the development of aC. difficileinfection (CDI) after ingestion and passage through the stomach. This study investigates the specific conditions that facilitateC. difficilespore germination, including the following: location within the gastrointestinal (GI) tract, pH, temperature, and germinant concentration. The germinants that have been identified in culture include combinations of bile salts and amino acids or bile salts and calcium, butin vitro, these function at concentrations that far exceed normal physiological ranges normally found in the mammalian GI tract. In this work, we describe and quantify a previously unreported synergy observed when bile salts, calcium, and amino acids are added together. These germinant cocktails improve germination efficiency by decreasing the required concentrations of germinants to physiologically relevant levels. Combinations of multiple germinant types are also able to overcome the effects of inhibitory bile salts. In addition, we propose that the acidic conditions within the GI tract regulateC. difficilespore germination and could provide a biological explanation for why patients taking proton pump inhibitors are associated with increased risk of developing a CDI.

scholarly journals Germinants and Their Receptors in Clostridia

2016 ◽  
Vol 198 (20) ◽  
pp. 2767-2775 ◽  
Author(s):  
Disha Bhattacharjee ◽  
Kathleen N. McAllister ◽  
Joseph A. Sorg
Keyword(s):  
Infectious Disease ◽  
Spore Germination ◽  
Environmental Stressors ◽  
Spore Form ◽  
Content Type ◽  
Clinical Environments ◽  
Clostridial Species ◽  
Acetone Butanol Ethanol ◽  
Optimum Production ◽  
Shed Light

Many anaerobic spore-forming clostridial species are pathogenic, and some are industrially useful. Although many are strict anaerobes, the bacteria persist under aerobic and growth-limiting conditions as multilayered metabolically dormant spores. For many pathogens, the spore form is what most commonly transmits the organism between hosts. After the spores are introduced into the host, certain proteins (germinant receptors) recognize specific signals (germinants), inducing spores to germinate and subsequently grow into metabolically active cells. Upon germination of the spore into the metabolically active vegetative form, the resulting bacteria can colonize the host and cause disease due to the secretion of toxins from the cell. Spores are resistant to many environmental stressors, which make them challenging to remove from clinical environments. Identifying the conditions and the mechanisms of germination in toxin-producing species could help develop affordable remedies for some infections by inhibiting germination of the spore form. Unrelated to infectious disease, spore formation in species used in the industrial production of chemicals hinders the optimum production of the chemicals due to the depletion of the vegetative cells from the population. Understanding spore germination in acetone-butanol-ethanol-producing species can help boost the production of chemicals, leading to cheaper ethanol-based fuels. Until recently, clostridial spore germination is assumed to be similar to that ofBacillus subtilis. However, recent studies inClostridium difficileshed light on a mechanism of spore germination that has not been observed in any endospore-forming organisms to date. In this review, we focus on the germinants and the receptors recognizing these germinants in various clostridial species.

scholarly journals Both Fidaxomicin and Vancomycin Inhibit Outgrowth of Clostridium difficile Spores

2012 ◽  
Vol 57 (1) ◽  
pp. 664-667 ◽  
Author(s):  
Charlotte A. Allen ◽  
Farah Babakhani ◽  
Pam Sears ◽  
Ly Nguyen ◽  
Joseph A. Sorg
Keyword(s):  
Clostridium Difficile ◽  
Clinical Response ◽  
Recurrence Rate ◽  
Spore Germination ◽  
Vegetative Cells ◽  
Content Type

ABSTRACTFidaxomicin (FDX) is approved to treatClostridium difficile-associated diarrhea and is superior to vancomycin in providing a sustained clinical response (cure without recurrence in the subsequent 25 days). The mechanism(s) behind the low recurrence rate of FDX-treated patients could be multifactorial. Here, we tested effects of FDX, its metabolite OP-1118, and vancomycin on spore germination and determined that none affected the initiation of spore germination but all inhibited outgrowth of vegetative cells from germinated spores.

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