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Published in final edited form as: Curr Opin Microbiol. 2021 Nov 19;65:101–107. doi: 10.1016/j.mib.2021.11.001

Clostridioides difficile spore germination: initiation to DPA release

Marko Baloh 1, Joseph A Sorg 1,*
PMCID: PMC8792321  NIHMSID: NIHMS1756166  PMID: 34808546

Abstract

Germination by C. difficile spores is an essential step in pathogenesis. Spores are metabolically dormant forms of bacteria that resist severe conditions. Work over the last 10 years has elucidated that C. difficile spores germinate thorough a novel pathway. This review summarizes our understanding of C. difficile spore germination and the factors involved in germinant recognition, cortex degradation and DPA release.

Keywords: Clostridioides difficile, spore, Germination, pseudoprotease, DPA

Introduction

Germination by Clostridioides difficile spores is an essential step in the pathogenesis of this anaerobic, Gram-positive, spore-forming pathogen [1]. The small molecules that serve as signals to stimulate the germination process, germinants, are sensed by the subtilisin-like Csp proteins. In C. difficile, CspA and CspC are pseudoproteases whose activation upon germinant binding is hypothesized to relieve repression of the CspB protease. CspB then activates a cortex lytic enzyme, SleC, by cleaving the inhibitory pro-peptide. SleC then degrades the spore cortex layer (a protective layer of modified cell wall, degradation of which is critical for resumption of spore metabolic activity) which leads to core hydration and exit of spores from dormancy [2–4]. The disruption of the germination initiation pathway, either by triggering germination under sub-optimal conditions (i.e., in the presence of a protective gut microbiome), or by preventing germination altogether, could be a strategy to prevent the establishment of CDI. Indeed, this strategy is showing promise in animal models of CDI [5–8]. This review will summarize the current state of research into C. difficile spore germination, from the stage of spore dormancy up to the re-establishment of metabolic processes in a newly germinated cell, and how its disruption affects the treatment of CDI.

Spore structure

C. difficile spores have a complex structure that allows them to adhere to surfaces and resist adverse environmental conditions [9–11]. The outermost exosporium layer is composed of proteins and glycoproteins (i.e., BclA1, BclA2, BclA3, CdeA, CdeC, and CdeM). It is of variable thickness, and is believed to have a role in the spore interaction and attachment with the host intestinal epithelium [12–15]. The exosporium surrounds the coat layer, which is composed of layers of protein serving as a barrier for enzymes and damaging chemicals [16]. The coat is built upon the outer spore membrane, a membrane that is thought to be largely permeable during spore dormancy [17,18]. The outer spore membrane surrounds the cortex, a thick layer of modified peptidoglycan [19]. Under the cortex is the germ cell wall, a peptidoglycan layer destined to become the cell wall during outgrowth of a vegetative cell from a germinated spore, and the inner spore membrane, an immobile, low-permeability membrane that serves as a barrier to damaging chemicals [20,21]. The spore core contains DNA, RNA, proteins, and other components necessary for re-establishment of metabolic processes upon germination. The core has a low water content and increased levels of 1:1 chelate of Ca2+ with pyridine-2,6-dicarboxylic acid (CaDPA), which provide C. difficile (and spores derived from all other studied endospore forming bacteria) with remarkable heat resistance [22–27].

Germinants

Like all endospore-forming bacteria, C. difficile spores require the presence of small molecule compounds, germinants, to stimulate the cascade of events that trigger germination [28]. C. difficile spores activate germination in response to host-derived bile acids [29,30]. Bile acids are synthesized in the liver using cholesterol as a scaffold. The two primary bile acids, cholate and chenodeoxycholate, are further modified via conjugation with either taurine or glycine (e.g., taurocholate is generated from cholate and taurine) [31,32]. Cholate-derivatives are the most effective bile acid germinants, while chenodeoxycholate is a competitive inhibitor of cholic acid-mediated germination [29,30,33–37]. During GI transit, approximately 5% of the total amount of bile acids reach the large intestine where they are deconjugated and then metabolized by 7α-dehydroxylation by the native microbiota to generate secondary bile acids [31,32]. Upon antibiotic treatment, or during gut dysbiosis, the members of the colonic flora that mediate this metabolism are lost and this results in sufficient taurocholate concentrations (coupled with the increased pH) to germinate the spores [38–40]. It should be noted that certain conditions like obesity may significantly affect the severity of CDI by altering the ratio of primary to secondary bile acids present in the gut, with the increased levels of primary bile acids favoring the persistence of C. difficile and worsening the clinical outcomes [41]. Another way by which diet may influence CDI is by altering colonic pH levels. Recent work suggests that C. difficile germination is highly sensitive to even slight pH level variations, with the optimal pH for germination being greater than pH 6.2, and that particular diets, i.e., those high in fiber, may lower the colonic pH below the optimum, thereby reducing spore germination and outgrowth [38,42].

Though bile acids are necessary to stimulate C. difficile spore germination in vitro, they are not sufficient and a co-germinant signal is required [3,29,35,36,43,44]. Several amino acids can function as C. difficile spore co-germinants, but glycine is the most efficient amino acid co-germinant (calcium can also function as a co-germinant) [3,29,36,44]. Recent work by Leslie and colleagues [45] has shown that pre-colonization with a non-toxigenic C. difficile strain depletes glycine in the mouse gut and this prevents subsequent establishment of infection when spores derived from a toxigenic, C. difficile strain are used to infect. Their work suggests that despite the capability of other amino acids to stimulate spore germination in vitro, glycine is an important in vivo spore co-germinant.

Because spores, unlike vegetative cells, are insensitive to the toxic actions of antibiotics, one strategy to eradicate C. difficile in the gut is to initiate germination with the administration of germinants, while simultaneously administering antibiotics to kill the resulting vegetative cell [46]. This ‘germinate to eradicate’ strategy could be a potential avenue for the prevention of recurring CDI [47]. Alternatively, compounds that block the initiation of spore germination have potential in preventing C. difficile infection. By blocking the initiation of spore germination, all subsequent downstream events (e.g., outgrowth, vegetative growth, toxin production and sporulation) are prevented. This strategy has been shown to be effective in animal models of CDI [6–8].

Initiating C. difficile spore germination

CspC

In most endospore-forming bacteria, germinants are recognized at the inner spore membrane by Ger-type germinant receptors (e.g., GerAA – GerAB – GerAC) [48]. C. difficile does not encode the Ger-type germinant receptors and instead recognizes germinants using the CspA, CspB, and CspC proteins [1,37,43,49–53]. Prior to work done in C. difficile, the Csp proteins were best studied in Clostridium perfringens. In C. perfringens, CspA, CspB and CspC are subtilisin-like serine proteases and are hypothesized to remove the inhibitory propeptide from the cortex-degrading enzyme, pro-SleC [54–57]. Interestingly, in C. difficile, cspB and cspA have been translationally fused, and cspC is encoded downstream of the cspBA gene. Again, unlike what is found in C. perfringens, C. difficile CspA and CspC have lost their catalytic triad and are, thus, pseudoproteases [1,43,49–53].

In an ethylmethane sulfonate (EMS) screen to identify germination-null strains, single point mutations in C. difficile cspC were found to abrogate spore germination in response to bile acids and another (CspCG457R) altered germinant specificity by inducing germination in response to chenodeoxycholate, a primary bile acid that is normally an inhibitor of germination [1,30,33]. These results suggested that despite its loss of catalytic activity, the C. difficile CspC pseudoprotease still functioned to regulate germination in response to bile acids, potentially as a regulatory inhibitor of the CspB protease [1,37]. In recent work, Rohlfing and colleagues [50] crystallized the C. difficile CspC protein and used the data provided by this structure to probe regions of the protein and test how mutations in these regions altered C. difficile spore germination. Interestingly, they found that the CspCG457R mutation described previously was hypersensitive to both bile acid-mediated germination (i.e., taurocholate) and the co-germinant signal (i.e., glycine or arginine) [50]. Moreover, the authors found that the a strain harboring a mutation in a neighboring amino acid (CspCR456G) was also hypersensitive [50]. Despite this, not all cspC mutations altered sensitivities to both stimulatory molecules, and the authors found that CspCD429K led to increased bile acid sensitivity but not to sensitivity to the co-germinant signal [13]. Surprisingly, restoration of the catalytic site residues to CspC decreased protein stability and thus led to decreased germination [49]. These results suggest that CspC is a signaling point for C. difficile spore germination or that, potentially, CspC makes contact with the other germinant receptors and these mutations alter the binding to these proteins.

CspBA

Encoded upstream of C. difficile cspC is cspBA. CspBA is produced as a translational fusion between the cspB and cspA genes, and undergoes interdomain processing that involves the removal of a ~10 kDa long N-terminal domain separating CspBA into the CspB protease and the CspA pseudoprotease [52,53]. CspA undergoes further processing by the YabG protease [43,52]. Deletion or disruption of cspB or cspA prevents spore germination indicating that the CspA pseudoprotease, like the CspC pseudoprotease, is important for C. difficile spore germination [43,51]. The role of CspB in C. difficile spore germination is clear. It must cleave the inhibitory pro-peptide from the cortex degrading enzyme, pro-SleC, to trigger germination [51,53].

Mutations in C. difficile cspA also prevent spore germination. However, the mechanism by which C. difficile CspA functions during germination is complex. Disruption of the cspA portion of the cspBA gene prevents the incorporation of CspC into the developing spore suggesting that CspA and CspC may interact at some point during spore development, or that CspA is important for CspC stability in the dormant spore, or both [43,51,52]. Moreover, a C. difficile yabG mutant strain resulted in spores that no longer responded to a co-germinant and germinated in response to bile acids only [43]. In this strain, CspBA is no longer efficiently processed into the CspB and CspA forms (the interdomain processing of CspBA was weak in this strain) [43]. Moreover, small deletions in cspA, near the hypothesized YabG processing site within CspA [cspBAΔ537–571 or cspBAΔ581–584 (an SRQS deletion)], resulted in a strain whose spores could germinate in response to taurocholate only. However, unlike the yabG mutant, spores derived from a cspBAΔ537–571 strain could still respond to co-germinants [43]. This latter work led to the hypothesis that C. difficile CspA functions as the co-germinant receptor for C. difficile spore germination [43].

SleC

The C. difficile cortex lytic enzyme, SleC, is essential for spore germination [4]. SleC is deposited into the spore as an inactive zymogen (pro-SleC) [53,55]. Upon germinant and co-germinant recognition by CspA and CspC [1,37,43,49–52], CspB is activated (somehow) and CspB cleaves the inhibitory pro-peptide from SleC, thereby activating it [53]. SleC acts on the muramic-δ-lactam (MDL) residues that are uniquely found in the cortex layer and then degrades the cortex peptidoglycan layer. The degradation of the spore cortex permits the release of DPA from the spore core, in exchange for water [58,59]. Though SleC is the major spore cortex lytic enzyme, its ability to hydrolyze the cortex is dependent on the modification of the cortex peptidoglycan by GerS and CwlD, permitting SleC to recognize the substrate targeted for hydrolysis [60,61]. Interestingly, removal of the inhibitory pro-peptide is not required for in vitro degradation of muramic-δ-lactam-containing peptidoglycan – CspB activation of SleC may be required in C. difficile spores for proper lytic activity [62].

DPA release

In C. difficile and other spore-forming organisms, pyridine-2,6-dicarboxylic acid (dipicolinic acid [DPA]) is chelated in 1:1 ratio with Ca2+ (Ca-DPA)and packaged into the spore in large amounts at the expense of water. DPA is largely responsible for spore wet heat resistance, although it is not required for C. difficile spore formation and germination [20,21,25,27]. For the spore metabolic processes to resume, DPA must be released from the core in exchange for water. During dormancy, the cortex prevents the osmotic expansion of the spore core, thereby preventing the release of DPA [26,59,63]. In B. subtilis the proteins encoded by the spoVA operon (SpoVAA-AB-AC-AD-AEa-AEb-AF) play a role in DPA packaging and release. C. difficile encodes three orthologues of the spoVA operon, SpoVAC, SpoVAD, and SpoVAE. In B. subtilis, SpoVAD binds to DPA and is likely important in the packaging of DPA into the developing spore, a spoVAEa mutation causes only a slight germination defect, and the role of spoVAEb is unclear [64,65]. The mechanosensing protein, SpoVAC, is embedded in the inner spore membrane and, presumably, serves as the channel through which DPA is packaged into the spore core during sporulation and released during germination [26,66]. Recent work has shown that C. difficile SpoVAD and SpoVAE interact at inner spore membrane and, along with SpoVAC, hypothesized to form a complex required for packaging of DPA, which is perhaps applicable to other spore-forming organisms utilizing spoVA operon [67]. There is evidence that the DPA packaging into the spore core may be under regulation of other genes besides those in spoVA operon in C. difficile. Spores from strains lacking CD3298, an AAA+ ATPase, contain <1% of DPA found in spores of wild-type strains, similar to SpoVAC/SpoVAD/SpoVAE mutants, potentially indicating a role of CD3298 in DPA transport into the forespore during sporulation [44].Upon activation of SleC, degradation of the spore cortex results in the loss of ‘constraint’ on the inner spore membrane resulting in the activation of the mechanosensing SpoVAC protein and release of DPA from the core, in exchange for water (an ‘outside – in germination pathway) [26,59]. The rehydrated core then begins metabolic activity, and the spore eventually develops into a vegetative cell. This order of events is inverted from what is observed during B. subtilis spore germination. In B. subtilis, germinant recognition by the Ger-type germinant receptor leads to release of DPA and the released DPA can activate degradation of the cortex layer (an ‘inside – out’ germination pathway) because DPA directly activates the CwIJ cortex lytic enzyme [26,48,59,68].

Hypothesized model for C. difficile spore germination

In a working / potential model for C. difficile spore germination, the CspB protease is held in an inactive state by the CspA and CspC pseudoproteases (Figure 1A and 1B). In this complex, the interaction of the three proteins regulates spore germination in response to exogenous signals. Upon recognition of the bile acid by CspC and the co-germinant by CspA, these proteins disassociate from CspB (Figure 1A). The liberated CspB is free to process pro-SleC into its active form resulting in degradation of the spore cortex layer (Figure 1A and 1B). Based on the data provided by Rohlfing and colleagues [50], we hypothesize that CspCD429 is near the CspB and CspA binding interfaces and the D429K allele is hypersensitive to the TA germinant and calcium co-germinant due to instability of these surfaces (Figure 1A) [50]. The CspCR456 and CspCG457 amino acids may be located near the binding interface with CspA and with CspB because the CspCR456G and CspCG457R alleles are hypersensitive to both bile acid germinants and amino acid co-germinants [1,50]. We hypothesize that this leads to destabilization of the CspA/CspC and the CspC/CspB binding interface and thus weakening the overall complex. In a yabG mutant strain, CspA is not processed from CspB and thus is not positioned in the complex to regulate spore germination in response to co-germinants [43].

Figure 1. Working model for C. difficile spore germination.

Figure 1.

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(A) The CspC and CspA pseudoproteases bind to and inhibit the CspB protease from gaining access to the cortex-degrading SleC zymogen. Upon germinant sensing, CspC and CspA disassociate from CspB resulting in CspB cleaving the inhibitory pro-peptide from pro-SleC. Activated SleC degrades the spore cortex. (B) In a dormant spore, the mechanosensing membrane protein, SpoVAC, is in a closed state and prevents the release of the large amounts of DPA from the spore core. Upon degradation of the spore cortex layer, a change in osmolarity is perceived at the inner spore membrane and this results in SpoVAC opening to release DPA. Created with Biorender.com.

In a dormant spore, the SpoVAC mechanosensing protein is in a closed state and prevents the release of the large depot of CaDPA from the spore core (Figure 1B). Upon activation of germination, SleC degrades the spore cortex layer. This results in an osmotic shift that is perceived at the inner spore membrane and SpoVAC opens to release DPA from the core (Figure 1B).

Concluding remarks

The last 10 years have marked significant advances in our understanding of how C. difficile spores germinate. The identification of the proteins required to initiate the spore germination process and how these proteins / processes differ compared to what is observed in B. subtilis has led to a proposed novel “outside – in” germination pathway. However, despite these advances, much remains to be understood. It is unclear if CspB, CspA and CspC interact in dormant spores and how the identified alleles contribute to germinant recognition is biochemically unknown. Moreover, it is interesting that mutations in cspA can block the import or stability of CspC in the spore. Is CspA merely required for CspC stability or is CspA influencing the packaging of CspC into the spore? A biochemical understanding of spore germination is likely to shed light on many of these questions.

Highlights.

  • C. difficile spore germination is regulated by two pseudoproteases.

  • The CspC germinant receptor and pseudoprotease regulates bile acid and co-germinant recognition.

  • C. difficile spore germination proceeds through an “outside – in” pathway.

  • Inhibiting germination has shown promise in preventing disease in animal models.

Acknowledgements

We would like to thank other members of the Sorg laboratory for their helpful comments during the preparation of this manuscript. Due to space limitations, we could not address all aspects of C. difficile spore germination and thus apologize to those authors whose work was not included in this review. This work was supported by a grant from the National Institutes of Health (R01AI116895). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Footnotes

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Declaration of interests

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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