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.

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 ...

Clostridium difficile spore germination is initiated in response to certain bile acids and amino acids (e.g., glycine). Though the amino acid-recognizing germinant receptor is unknown, the bile acid germinant receptor is the germination-specific, subtilisin-like pseudoprotease, CspC. In C. difficile the CspB, CspA, and CspC proteins are involved in spore germination. Of these, only CspB is predicted to have catalytic activity because the residues important for catalysis are mutated in the cspA and cspC sequence. The CspB, CspA, and CspC proteins are likely localized to the outer layers of the spore (e.g., the cortex or the coat layers) and not the inner membrane where the Ger-type germinant receptors are located. In C. difficile, germination proceeds in an “outside-in” direction, instead of the “‘inside-out” direction observed during the germination of Bacillus subtilis spores. During C. difficile spore germination, cortex fragments are released prior to the release of 2,4-dipicolinic acid (DPA) from the spore core. This is opposite to what occurs during B. subtilis spore germination. To understand if the mechanism C. difficile spore germination is unique or if spores from other organisms germinate in a similar fashion, we analyzed the germination of Paraclostridium bifermentans spores. We find that P. bifermentans spores release cortex fragments prior to DPA during germination and the DPA release from the P. bifermentans spore core can be blocked by high concentrations of osmolytes. Moreover, we find that P. bifermentans spores do not respond to steroid-like compounds (unlike the related C. difficile and P. sordellii organisms), indicating that the mere presence of the Csp proteins does permit germination in response to steroid compounds. Our findings indicate that the “outside in” mechanism of spore germination observed in C. difficile can be found in other bacteria suggesting that this mechanism is a novel pathway for endospore germination.

An important risk factor for acquiring Clostridioides difficile infection is antibiotic use. Therefore, a detailed knowledge of the physiology and the virulence factors can help drive the development of new diagnostic tools and nonantibiotic therapeutic agents to combat these organisms. Several genetic systems are available to study C. difficile in the laboratory environment, and all rely on stably replicating or segregationally unstable plasmids. Currently, the transfer of plasmids into C. difficile can only be performed by conjugation using Escherichia coli or Bacillus subtilis as conjugal donors. Here we report a method to introduce plasmid DNA into C. difficile using electroporation and test factors that might contribute to higher transformation efficiencies: osmolyte used to stabilize weakened cells, DNA concentration, and recovery time postelectroporation. Depending on the C. difficile strain and plasmid used, this transformation protocol achieves between 20 and 200 colonies per microgram of DNA and is mostly influenced by the recovery time postelectroporation. Based on our findings, we recommend that each strain be tested for the optimum recovery time in each lab. IMPORTANCE Understanding the underlying biology of pathogens is essential to develop novel treatment options. To drive this understanding, genetic tools are essential. In recent years, the genetic toolbox available to Clostridioides difficile researchers has expanded significantly but still requires the conjugal transfer of DNA from a donor strain into C. difficile. Here we describe an electroporation-based transformation protocol that was effective at introducing existing genetic tools into different C. difficile strains.

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Clostridia are a group of medically important anaerobes as both commensals and pathogens. Recently, a new class of Erysipelotrichia containing a number of reassigned clostridial species has emerged, including Clostridium innocuum .

Clostridia are a polyphyletic group of Gram-positive, spore-forming anaerobes in the Firmicutes phylum that significantly impact metabolism and functioning of human gastrointestinal tract. Recently, Clostridia were divided into two separate classes, Clostridia and Erysipelotrichia, based on phenotypic and 16S rRNA gene-based differences. While Clostridia include many well-known pathogenic bacteria, Erysipelotrichia remain relatively uncharacterized, particularly regarding their role as a pathogen vs. commensal. Despite wide recognition as a commensal, the Erysipelotrichial species, Clostridium innocuum, has recently been associated with various disease states. To further understand the ecological and potential virulent role of C. innocuum, we conducted a genomic comparison across 38 C. innocuum isolates and 75 publicly available genomes. Based on colony morphology, we isolated multiple C. innocuum cultivars from the feces of healthy human volunteers (n=5). Comparison of the 16S rRNA gene of our isolates against publicly available microbiota datasets in healthy individuals suggests a high prevalence of C. innocuum across the human population (> 80%). Analysis of single nucleotide polymorphisms (SNPs) across core genes and average nucleotide identify (ANI) revealed the presence of 4 clades among all available unique genomes (n=108 total). Investigation of carbohydrate and protein utilization pathways, including comparison against the carbohydrate-activating-enzyme (CAZyme) database, demonstrated inter-and intra-clade differences that were further substantiated in vitro. Collectively, these data indicate genetic variance within the C. innocuum species that may help clarify its role in human disease and health.IMPORTANCEClostridia are a group of medically important anaerobes as both commensals and pathogens. Recently, a new class of Erysipelotrichia containing a number of re-assigned Clostridial species has emerged, including Clostridium innocuum. Recent studies have implicated C. innocuum as a potential causative agent of diarrhea in patients from whom Clostridioides difficile could not be isolated. Using genomic and in vitro comparison, this study sought to characterize C. innocuum in the healthy human gut. Our analyses suggest that C. innocuum is a highly prevalent and diverse species, demonstrating clade-specific differences in metabolism and potential virulence. Collectively, this study is the first investigation into a broader description of C. innocuum as a human gut inhabitant.

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