Germinants and Their Receptors in Clostridia

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 of Bacillus subtilis. However, recent studies in Clostridium difficile shed 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

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

ABSTRACT 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

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

Bacterial spore germination is a process whereby a dormant spore returns to active, vegetative growth, and this process has largely been studied in the model organism Bacillus subtilis. In B. 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 during B. subtilis spore germination. Here, we investigated the timing of DPA release and cortex hydrolysis during Clostridium difficile spore 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 during C. difficile spore 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-mediated C. difficile spore germination proceeds through a novel germination pathway. IMPORTANCE Clostridium difficile infects 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. difficile spore 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 the Bacillus subtilis germinant receptors, we hypothesized that there are fundamental differences in the germination processes between the model organism and C. difficile. We found that C. difficile spore germination proceeds through a novel pathway

The Use of Financial Literacy for Growing Personal Finance

Financial literacy played an important role for everyone in managing personal finances.This research aimed to determine how the level of financial literacy in students S1 Facultyof Economics and Business, Universitas Pasundan and investigate what factors are influencingit. The observed respondents were students from the Faculty of Economics andBusiness, Universitas Pasundan. The research data was collected through questionnaires,descriptive analysis, and test multinomial logit. Based on the results of the researchshowed that the level of financial literacy from undergraduate students UniversitasPasundan was in the low category. Financial literacy was determined by gender, GreaterAcademic Achievement (GPA), parental education level, and parental income level;,whereas for age, year of study and residence do not contribute to the research model. Theresults of this study were expected to support personal financial planning of students inimproving the skills of reading, analyzing, and managing their own finances, thus avoidingthe daily financial problems

Effect of tcdR Mutation on Sporulation in the Epidemic Clostridium difficile Strain R20291

Citation: Girinathan, B. P., Monot, M., Boyle, D., McAllister, K. N., Sorg, J. A., Dupuy, B., & Govind, R. (2017). Effect of tcdR Mutation on Sporulation in the Epidemic Clostridium difficile Strain R20291. Msphere, 2(1), 14. doi:10.1128/mSphere.00383-16Clostridium difficile is an important nosocomial pathogen and the leading cause of hospital-acquired diarrhea. Antibiotic use is the primary risk factor for the development of C. difficile-associated disease because it disrupts normally protective gut flora and enables C. difficile to colonize the colon. C. difficile damages host tissue by secreting toxins and disseminates by forming spores. The toxin-encoding genes, tcdA and tcdB, are part of a pathogenicity locus, which also includes the tcdR gene that codes for TcdR, an alternate sigma factor that initiates transcription of tcdA and tcdB genes. We created a tcdR mutant in epidemic-type C. difficile strain R20291 in an attempt to identify the global role of tcdR. A site-directed mutation in tcdR affected both toxin production and sporulation in C. difficile R20291. Spores of the tcdR mutant were more heat sensitive than the wild type (WT). Nearly 3-fold more taurocholate was needed to germinate spores from the tcdR mutant than to germinate the spores prepared from the WT strain. Transmission electron microscopic analysis of the spores also revealed a weakly assembled exosporium on the tcdR mutant spores. Accordingly, comparative transcriptome analysis showed many differentially expressed sporulation genes in the tcdR mutant compared to the WT strain. These data suggest that regulatory networks of toxin production and sporulation in C. difficile strain R20291 are linked with each other. IMPORTANCE C. difficile infects thousands of hospitalized patients every year, causing significant morbidity and mortality. C. difficile spores play a pivotal role in the transmission of the pathogen in the hospital environment. During infection, the spores germinate, and the vegetative bacterial cells produce toxins that damage host tissue. Thus, sporulation and toxin production are two important traits of C. difficile. In this study, we showed that a mutation in tcdR, the toxin gene regulator, affects both toxin production and sporulation in epidemic-type C. difficile strain R20291

Effects of Surotomycin on Clostridium difficile Viability and Toxin Production In Vitro

The increasing incidence and severity of infection by Clostridium difficile have stimulated attempts to develop new antimicrobial therapies. We report here the relative abilities of two antibiotics (metronidazole and vancomycin) in current use for treating C. difficile infection and of a third antimicrobial, surotomycin, to kill C. difficile cells at various stages of development and to inhibit the production of the toxin proteins that are the major virulence factors. The results indicate that none of the drugs affects the viability of spores at 8× MIC or 80× MIC and that all of the drugs kill exponential-phase cells when provided at 8× MIC. In contrast, none of the drugs killed stationary-phase cells or inhibited toxin production when provided at 8× MIC and neither vancomycin nor metronidazole killed stationary-phase cells when provided at 80× MIC. Surotomycin, on the other hand, did kill stationary-phase cells when provided at 80× MIC but did so without inducing lysis

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

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 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

Cleavage of an engulfment peptidoglycan hydrolase by a sporulation signature protease in Clostridioides difficile

Funding Information: This work was supported by the European Union Marie Sklodowska Curie Innovative Training Networks (contract number 642068) to AOH and AF was the recipient of a PhD fellowship under that contract. This project was supported by award PTDC/BIA\u2010MIC/29293/2017 to MS. This work was also financially supported by Project LISBOA\u201001\u20100145\u2010FEDER\u2010007660 (\u201CMicrobiologia Molecular, Estrutural e Celular\u201D) funded by FEDER funds through COMPETE2020\u2014\u201CPrograma Operacional Competitividade e Internacionaliza\u00E7\u00E3o\u201D (POCI), by national funds through the FCT (\u201CFunda\u00E7\u00E3o para a Ci\u00EAncia e a Tecnologia\u201D). DM is the recipient of a PhD fellowship (PD/BD/143148/2019) within the scope of the PhD program INTERFACE funded by FCT. This project was supported by the Medical Research Council (grant number MR/V032151/1) awarded to PSS and ABS and the BBSRC (BB/W005557/1, BB/W013630/1) to WV. CGR is supported by a Barbour Foundation PhD Studentship from Faculty of Medical Science, Newcastle University. This project was also supported by awards R01AI116895 and R01AI172043 from the National Institute of Allergy and Infectious Diseases to JAS. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIAID. The funders had no role in study design, data collection, and interpretation, or the decision to submit the work for publication. Publisher Copyright: © 2024 The Author(s). Molecular Microbiology published by John Wiley & Sons Ltd.In the model organism Bacillus subtilis, a signaling protease produced in the forespore, SpoIVB, is essential for the activation of the sigma factor σK, which is produced in the mother cell as an inactive pro-protein, pro-σK. SpoIVB has a second function essential to sporulation, most likely during cortex synthesis. The cortex is composed of peptidoglycan (PG) and is essential for the spore's heat resistance and dormancy. Surprisingly, the genome of the intestinal pathogen Clostridioides difficile, in which σK is produced without a pro-sequence, encodes two SpoIVB paralogs, SpoIVB1 and SpoIVB2. Here, we show that spoIVB1 is dispensable for sporulation, while a spoIVB2 in-frame deletion mutant fails to produce heat-resistant spores. The spoIVB2 mutant enters sporulation, undergoes asymmetric division, and completes engulfment of the forespore by the mother cell but fails to synthesize the spore cortex. We show that SpoIIP, a PG hydrolase and part of the engulfasome, the machinery essential for engulfment, is cleaved by SpoIVB2 into an inactive form. Within the engulfasome, the SpoIIP amidase activity generates the substrates for the SpoIID lytic transglycosylase. Thus, following engulfment completion, the cleavage and inactivation of SpoIIP by SpoIVB2 curtails the engulfasome hydrolytic activity, at a time when synthesis of the spore cortex peptidoglycan begins. SpoIVB2 is also required for normal late gene expression in the forespore by a currently unknown mechanism. Together, these observations suggest a role for SpoIVB2 in coordinating late morphological and gene expression events between the forespore and the mother cell.publishersversionpublishe

Stressor- and Corticotropin releasing Factor-induced Reinstatement and Active Stress-related Behavioral Responses are Augmented Following Long-access Cocaine Self-administration by Rats

Rationale Stressful events during periods of drug abstinence likely contribute to relapse in cocaine-dependent individuals. Excessive cocaine use may increase susceptibility to stressor-induced relapse through alterations in brain corticotropin-releasing factor (CRF) responsiveness. Objectives This study examined stressor- and CRF-induced cocaine seeking and other stress-related behaviors in rats with different histories of cocaine self-administration (SA). Materials and methods Rats self-administered cocaine under short-access (ShA; 2 h daily) or long-access (LgA; 6 h daily) conditions for 14 days or were provided access to saline and were tested for reinstatement by a stressor (electric footshock), cocaine or an icv injection of CRF and for behavioral responsiveness on the elevated plus maze, in a novel environment and in the light–dark box after a 14- to 17-day extinction/withdrawal period. Results LgA rats showed escalating patterns of cocaine SA and were more susceptible to reinstatement by cocaine, EFS, or icv CRF than ShA rats. Overall, cocaine SA increased activity in the center field of a novel environment, on the open arms of the elevated plus maze, and in the light compartment of a light–dark box. In most cases, the effects of cocaine SA were dependent on the pattern/amount of cocaine intake with statistically significant differences from saline self-administering controls only observed in LgA rats. Conclusions When examined after several weeks of extinction/ withdrawal, cocaine SA promotes a more active pattern of behavior during times of stress that is associated with a heightened susceptibility to stressor-induced cocaine-seeking behavior and may be the consequence of augmented CRF regulation of addiction-related neurocircuitry