Fight or flee, a vital choice for Clostridioides difficile

Abstract Clostridioides difficile is a leading cause of healthcare‐associated infections, causing billions of economic losses every year. Its symptoms range from mild diarrhea to life‐threatening damage to the colon. Transmission and recurrence of C. difficile infection (CDI) are mediated by the metabolically dormant spores, while the virulence of C. difficile is mainly due to the two large clostridial toxins, TcdA and TcdB. Producing toxins or forming spores are two different strategies for C. difficile to cope with harsh environmental conditions. It is of great significance to understand the molecular mechanisms for C. difficile to skew to either of the cellular processes. Here, we summarize the current understanding of the regulation and connections between toxin production and sporulation in C. difficile and further discuss the potential solutions for yet‐to‐be‐answered questions.


INTRODUCTION
Clostridioides difficile is an obligate, anaerobic, Grampositive bacterium that can produce toxins and form spores 1 .C. difficile infection (CDI) has been listed as an urgent threat of antibiotic resistance by the Centers for Disease Control and Prevention (CDC) 2 .About 0.25-0.5 million infection cases are diagnosed in America every year, including 13,000-20,000 in-hospital deaths [1][2][3] .The first line of treatment for CDI is antibiotics; however, about 15%-35% of patients relapse, and ~40%-65% of relapsed patients would have multiple recurrences 3,4 .The last resort for treating recurrent CDI is fecal microbiota transplantation (FMT).However, the safety of FMT is under debate 5 .Consequently, a better and safer therapy for recurrent CDI is always in demand.
The virulence is mainly contributed by two large clostridial toxins, toxin A (TcdA) and toxin B (TcdB), and, in some strains, the third binary toxin CDT 2,6,7 .TcdA and TcdB contain an N-terminal glucosyltransferase domain (GTD), a cysteine protease domain (CPD), an intermingled membrane translocation delivery and receptor-binding domain (DRBD), and a C-terminal combined repetitive oligopeptides (CROPs) domain (Figure 1) 2 .The two toxins enter the cell via receptormediated endocytosis 8,9 .Translocation of the GTD and CPD domains is then induced by the reduction of pH within endosomes, followed by autoproteolytic cleavage of the GTD domain that glycosylates and inactivates small GTPases 2 .The severity of the illness of CDI is directly associated with the production of toxins.The tcdA and tcdB genes are located in a 19.6 kb pathogenic locus (PaLoc), which also contains tcdR, tcdE, and tcdC (Figure 1) 10,11 .TcdR is a sigma factor that binds to RNA polymerase to promote the expression of tcdA and tcdB 10,11 .TcdC is an anti-sigma factor of TcdR, while TcdE may be involved in the secretion of toxins 12 .Toxin expression is largely determined by TcdR, which is regulated, directly or indirectly, by CcpA, CodY, and so forth 10,11 .The C. difficile transferase (CDT) is a poreforming toxin that catalyzes the ADP-ribosylation of actin 7 .CdtR, a LytTR family regulator within the CDT locus (CDTLoc), positively regulates CDT production 7 .However, little is known about other regulatory factors to CDTLoc 7 .
It is commonly believed that the high recurrence and transmission of C. difficile are generally due to the metabolically inactive spores, which may persist in gut epithelial cells and thus serve as a source of recurrence 13 .Spores are highly resistant to multiple factors, such as heat, UV light, and chemical insults 14 .The spore is composed of an inner partially dehydrated core, inner membrane, germ cell wall, cortex peptidoglycan layer, outer membrane, spore coat, 1 School of Biomedical and Pharmaceutical Sciences, Guangdong University of Technology, Guangzhou, China. 2 Department of Brest Surgery, Panyu Central Hospital, Guangzhou, China. 3Department of Microbiology, Harvard Medical School, Boston, Massachusetts, USA. 4 Department of Urology, Boston Children's Hospital, Harvard Medical School, Boston, Massachusetts, USA. 5 Department of Microbiology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, China. 6Advanced Medical Technology Center, The First Affiliated Hospital, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, China. 7Key Laboratory of Tropical Diseases Control (Sun Yat-sen University), Ministry of Education, Guangzhou, China. 8School of Medicine, Xizang Minzu University, Xianyang, China. 9Center for Infectious Disease Research, Westlake Laboratory of Life Sciences and Biomedicine, Westlake University, Hangzhou, China. 10Research Center for Industries of the Future, School of Life Sciences, Westlake University, Hangzhou, China.and outer exosporium 15 .Sporulation in C. difficile has been arbitrarily divided into seven stages (Figure 1) 3,15 .At stage 0, C. difficile integrates complex environmental signals and phosphorylates the sporulation master regulator Spo0A.Upon phosphorylation, Spo0A upregulates early-sporulation genes and starts the irreversible genetic program.At stages I and II, the C. difficile genome duplicates, and a polar septum divides the C. difficile cell into a larger mother cell and a smaller forespore.From stage III to stage V, the mother cell engulfs the forespore, a thick layer of cortex peptidoglycan is synthesized between the two membranes surrounding the forespore, the cortex layer is modified, and many proteins localize and spread along the forespore outer membrane to build the proteinaceous spore coat.At stages VI and VII, the outermost exosporium layer forms, the mother cell is lysed, and the forespore is finally released into the environment (Figure 1).
Toxin production and sporulation are two actions taken by C. difficile when nutrients are limited 2,3 .As both processes consume a large amount of energy and resources, it is critical for C. difficile to make the right decision at the right time.A wrong decision affects not only the fate of a single cell but also the integrity and survival rate of the bacterial population 2,16 .Quite a few reports suggest an existing linkage between these two bacterial processes.Yet, it is not clear whether these two processes are mutually exclusive and what the molecular mechanisms are for bacteria.Characterization of the molecular mechanism would help to design the treatment protocol during infection.In the following part, we summarize important regulatory factors that tune toxin production and sporulation similarly or differentially, which may provide new insights into the pathogenesis of C. difficile, and treatments of CDI and recurrent infections.

REGULATORS CONTROL TOXIN PRODUCTION AND SPORULATION CODIRECTIONALLY CcpA
CcpA is a LacI/GalR family protein, composed of a signalreceiving and dimerization domain and a DNA binding domain 17 .It is the master regulator of carbon catabolite repression (CCR), a mechanism to control the sequential consumption of carbohydrates in low G+C Gram-positive bacteria 17 .The active transport of carbohydrates is achieved by a phosphoenolpyruvate-dependent phosphotransferase system (PTS), composed of enzyme I (EI), histidine-containing phospho-carrier protein (HPr), and enzyme II (EII).In the presence of the preferred carbon source, glucose, for example, the PTS enhances CcpA binding to the promoter region of genes involved in sugar uptake, alternative metabolism, sporulation, and toxin production 18 .
A global mapping of CcpA targets in C. difficile, cultured in tryptone-yeast extract (TY) with or without 0.5% glucose, demonstrates that CcpA directly controls ~140 genes by recognizing an "RRGAAAANGTTTTCWW" motif 18 .This analysis reveals that repression of toxin production by CcpA is through direct interactions with the promoter regions of tcdR, tcdA, and tcdB.Glucose repression of toxin production is mediated by CcpA, such that deletion of ccpA in C. difficile causes constitutive toxin production in the presence of glucose.However, more regulatory factors seem to be involved in the CcpA regulation of toxin production, as the toxin titer in the ccpA mutant is lower than in the wild-type with glucose.Besides toxin production, CcpA also reduces the expression of spo0A, which encodes the master regulator of sporulation, via direct binding to its promoter region 18 .Unlike toxin production, glucose suppresses sporulation independent of CcpA, and the regulation of sporulation by CcpA and glucose has no synergistic effects.

CodY
CodY is a highly conserved global transcriptional regulator in low G+C Gram-positive bacteria, repressing genes involved in various alternative metabolic pathways during the exponential phase, and the repression is relieved in the stationary phase 19 .CodY is a dimeric protein that consists of an N-terminal effector binding domain and a winged C-terminal DNA binding helix-turn-helix domain 19 .Upon binding to its effectors, branched-chain amino acids (BCAAs) and GTP, the affinity of CodY to its targets increases, leading to the repression of genes involved in alternative metabolisms 19 .When BCAA and GTP levels decrease, which serves as an indication of the poor nutritional state, CodY no longer binds to these effectors and de-represses its regulon.
More than 100 genes are regulated directly or indirectly by CodY 20 .Specifically, CodY binds to the promoter region of tcdR and represses toxin production in the presence of GTP or BCAAs 21 .Yet, only weak interactions have been observed between CodY and the promoter regions of tcdA and tcdB, suggesting the CodY-mediated toxin regulation is primarily through the toxin-specific sigma factor TcdR 21 .CodY is also a negative regulator of sporulation, as deletion of codY increases the sporulation frequency in C. difficile, although the fold change is strain-specific 22 .Besides, the expression of sporulation-specific genes in codY mutants is increased.The mechanism of CodYmediated sporulation regulation remains unclear since the spo0A gene is not directly regulated by CodY 20 .CodY may regulate sporulation by modulating the expression of the opp operon, which encodes oligopeptide transporters, and/or sinR, which encodes a regulator of sporulation described below 22 .

SinR and SinR′
The sinRI locus in Bacillus subtilis is comprised of SinR, a master regulator of biofilm formation and inhibitor to sporulation, and SinI, the SinR antagonist 23 .SinR in B. subtilis is a tetrameric repressor composed of an N-terminal helix-turnhelix DNA binding domain and a C-terminal multimerization domain 23 .However, the sinRR′ locus in C. difficile contains two BsSinR homologs, SinR and SinR′, without SinI, suggesting an analogous physiological role 24,25 .RNA sequencing shows SinRR′ in C. difficile controls a wide range of genes involved in sporulation, toxin synthesis, motility, and various metabolic pathways.Specifically, overexpression of sinR promotes sporulation and toxin production, while SinR′ interacts with SinR and inhibits its function 24,25 .The regulatory effect of SinR on sporulation or toxin production may be through CodY, as SinR binds to the upstream region of codY and CodY expression increases 3-30-fold in sinRR′ mutants 24,25 .Interestingly, CodY also binds to the sinRR′ promoter region and inhibits its transcription, forming a toggle switch in C. difficile 20,24,25 .When entering the stationary phase, CodY-mediated repression of sinRR′ is alleviated as BCAAs and GTP become less abundant.This leads to increased production of SinR, which can further repress the expression of codY and thus alter sporulation initiation and toxin production.In addition, sin locus expression is repressed by Spo0A, which directly binds to the promoter region of the locus 26 .

REGULATORS CONTROL TOXIN PRODUCTION AND SPORULATION IN OPPOSITE DIRECTIONS SigH
Numerous bacteria have more than one sigma factor to adapt to the environmental and cellular nutritional state changes.SigH is a conserved factor in many Firmicutes that is produced during the transition from exponential to stationary phase, which governs gene expression related to motility, sporulation, virulence, and so forth 27 .SigH positively regulates spo0A and genes involved in sporulation by recognizing a (A/G)NAGGA(A/T)3-N11-12-(A/G)NNGAAT motif.Accordingly, the inactivation of SigH completely abolishes sporulation in C. difficile 27 .Additionally, SigH and Spo0A reciprocally activate each other, creating positive feedback to reinforce the sporulation 27,28 .Conversely, the C. difficile PaLoc is negatively regulated by SigH, such that the expression of tcdR, tcdA, tcdB, and tcdE are increased in a sigH mutant.The SigH recognition sequence is not identified in the promoters of these genes, indicating that the regulation is likely indirect 27 .

Spo0A
Spo0A is a highly conserved master regulator of sporulation in both the Bacilli and Clostridia 29,30 .In B. subtilis, Spo0A is phosphorylated by sensor histidine kinases (KinA, KinB, and KinC) via phosphorelay 30 .Upon phosphorylation, Spo0A is activated and initiates the sporulation process 30 .
In C. difficile, the phosphorelay is missing, but Spo0A is phosphorylated by a yet-to-be-identified organ kinase, which then starts the sporulation process similar to B. subtilis 31 .Unlike sporulation, the role of Spo0A in toxin production is controversial.One report demonstrates inactivation of spo0A results in a significant decrease in toxin production in C. difficile 630Δerm (RT012/ST54), an erythromycinsensitive derivative of the reference strain 630 32,33 .On the contrary, another study shows that the effects of Spo0A on toxin production are strain-specific, such that Spo0A is a negative regulator of toxins in certain C. difficile strains such as R20291 (RT027/ST1), M7404 (RT027), and JGS6133 (RT078), but not in 630Δerm 34 .This could be due to the genome variation among different strains, as the C. difficile with a core gene set only ~20% 34 .

TcdR
TcdR is a σ 70 family RNA polymerase sigma factor that recruits RNA polymerase to activate toxin genes and itself 35 .Therefore, the deletion of tcdR severely downregulates toxin production by decreasing the expression of tcdA, tcdB, tcdR, and tcdE 36 .However, the effect of TcdR on sporulation is also strain-specific.The sporulation efficiency in the tcdR mutant of C. difficile 630 is almost a twofold increase over the parental strain 36 .On the contrary, in C. difficile R20291, a nearly threefold reduction of sporulation efficiency is observed in the tcdR mutant compared to its parental strain 36 .Consistently, the expression of sporulation-related genes is downregulated in the absence of TcdR in R20291.Interestingly, it has been demonstrated that a subset of C. difficile cells is able to sporulate and produce TcdA 37,38 .The possibility cannot be ruled out that toxin and sporulation gene expression are sequential events, such that toxin production is interrupted by sporulation, and, therefore, a subset of cells appears to both induce toxin gene expression and sporulate 37 .In addition, C. difficile growth on a solid surface of TY plates or SMC plates seems to reduce the population of both toxin production and sporulation 38 .This may suggest that quorum sensing plays a role in creating a division of labor between toxin production and sporulation since autoinducers are almost evenly distributed in the growth medium with constant shaking, whereas these molecules may have gradients inside colonies growing on the plate.

RstA
RstA is a multifunctional transcriptional regulator that contains a conserved N-terminal helix-turn-helix DNA-binding domain, five TPR repeat regions, and a C-terminal putative quorum sensing domain 39 .In C. difficile 630Δerm, the expression of rstA is auto-regulated by recognizing a 29-bp imperfect repeat within its −10-consensus sequence 39,40 .RtsA positively regulates spore formation, such that the rstA mutant expresses sporulation-related genes at reduced levels compared to the WT strain 38,39 .The specific regulatory mechanism is unknown, but the TPR and C-terminal quorum sensing domain likely plays a role since complementation of RstAΔHTH in rstA mutant can partially restore C. difficile sporulation 39 .On the contrary, RstA negatively regulates toxin production.Studies in C. difficile 630Δerm show that RstA directly binds to the promoter regions of tcdA, tcdB, tcdR, and sigD and represses the expression of these genes [38][39][40] .Additionally, the regulation by RstA is strain-specific, as the regulatory effects of RstA on sporulation and toxin production are similar but more robust in C. difficile R20291 than 630Δerm strain 41 .This may be caused by stronger autoregulation of RstA in R20291 41 .

Agr system
The prototypical Gram-positive bacterial accessory gene regulator (Agr) system in Staphylococcus aureus contains AgrB, AgrD, AgrC, and AgrA 42 .The transmembrane protease AgrB processes the AgrD peptide to AIP, which is secreted to the extracellular space and bound to AgrC, a membraneassociated sensor histidine kinase 42 .Upon binding to AIP, AgrC is auto-phosphorylated and activated, which subsequently phosphorylates the response transcriptional regulator, AgrA.Interestingly, three different Agr systems have been identified in varied C. difficile strains 43 : Agr1 only contains AgrB1 and AgrD1, which are found in all sequenced C. difficile strains; Agr2, found in R20291, contains the entire prototypical ABCD genes; and Agr3, found in C. difficile NAP07 (RT078/ST11) and NAP08 (RT078/ST11) strains, contains AgrC3, AgrB3, and AgrD3, but lacks the AgrA3 response regulator.Deletion of AgrB1, AgrD1, or both in C. difficile 630 strain causes a significant reduction in the sporulation rate, but supernatant from wild-type cultures restores sporulation.The regulatory effects of Agr1 on toxin production remain elusive.One report demonstrates that the argB1D1 mutant is not able to produce TcdA and TcdB and is consistently avirulent in mice 44 .However, another report shows that the deletion of argB1 leads to a significant increase in tcdR and tcdA transcript levels 43 .

SigD
SigD regulates ~100 genes, including genes involved in motility, metabolism, and regulation 45 .Specifically, SigD positively regulates tcdA, tcdB, and tcdR via direct binding to the promoter region of tcdR 45 .The presence of high levels of C-di-GMP reduces toxin production through SigD 46 .The regulatory effects of SigD on sporulation remain unclear.However, the expression of sinR is increased by ~4-fold in a sigD mutant, suggesting a negative regulation on sporulation 45 .

INTERNAL CONNECTIONS BETWEEN TOXIN PRODUCTION AND SPORULATION IN C. DIFFICILE
C. difficile infection represents a great health burden in recent years, especially recurrent infections 4 .While toxins are the major pathogenic factors, spores are important causes of persistence and possibly recurrence, and plenty of research suggests a link exists between these two processes 10,11,19 .
Two important questions are yet to be answered.First, are these two cellular processes mutually exclusive?Both toxin production and sporulation require a large amount of energy investment.Therefore, it is intuitively contradictory for a sporulating C. difficile cell to produce toxins.However, there are two types of relationships between toxin production and sporulation.In the type I relationship, toxin production is inversely related to sporulation.For example, the sporulation and toxin production in Bacillus anthracis are inversely linked via AtxA 47 .Another example is B. thuringiensis, which follows the division of labor pattern 48 .In the type II relationship, toxin production is associated with sporulation 49 .For instance, the production of Clostridium perfringens enterotoxin (CPE) in the mother cell is coupled with spore formation, via SigE and SigK-dependent promoters 49 .Consistently, CPE release relies on the lysis of the mother cell at the final stage of sporulation.In C. difficile, most of the cells choose to either sporulate or produce toxin, following a division of labor mode 37,38 .However, a small population is able to express TcdA while sporulating 37,38 .Toxins of this small population may be released with spores when the mother cell is lysed.Alternatively, a possible explanation is that toxin and sporulation gene expression are sequential events; therefore, it is difficult to exclude the residue gene expression of the previous cellular process.Further experiments at the singlecell level may answer this question and reveal the precise mechanisms.
The second question is, what are the environmental cues for toxin production and sporulation?The regulatory effects of key factors on toxin production and sporulation are summarized in Table 1 and Figure 1.It is not surprising that transcriptional regulators involved in carbohydrate and amino acids metabolism tune the two cellular processes in the same direction at the onset of the stationary phase 12,17,18,[20][21][22] .However, the environmental cues to differentially regulate these two processes are yet to be revealed.Quorum sensing may be one of the targets.First, the quorum sensing-related Agr1 system and RstA regulate these two processes differentially [38][39][40][41][42][43] .Second, the division of labor is more obvious in C. difficile on a solid surface than in a liquid culture 38 .The autoinducer in the liquid medium is evenly distributed and may not be able to form a gradient as the colony or biofilm does, which may be required to decisively make the decision.Last but not least, most of the research has been done in a laboratory setting.However, the environment in a host is very different from a lab medium.Therefore, toxin production and sporulation mode of C. difficile cells need to be tested in a murine infection model or using ex vivo samples.Better study tools and methods may be required to characterize the toxin production and sporulation in an infection-relevant context.

Figure 1 .
Figure 1.The regulatory network between sporulation and toxin production.The central panel depicts important regulatory factors.CcpA, CodY, and SinR tune sporulation and toxin production codirectionally, while SigD, SigH, RstA, and Agr1 tune these two processes differentially.TcdR and Spo0A possibly suppress each other.Arrows indicate upregulation; the blunt ended line indicates downregulation; the dashed line indicates uncertainty or conflict reports.The left panel depicts the Clostridioides difficile PaLoc and the domains of TcdA and TcdB.The right panel depicts the stage II to stage VII of spore formation and the regulators in the forespore and mother cell.

Table 1 .
Regulatory factors that affect sporulation and toxin production.