A conserved switch controls virulence, sporulation, and motility in C. difficile.

Spore formation is required for environmental survival and transmission of the human enteropathogenic Clostridioides difficile. In all bacterial spore formers, sporulation is regulated through activation of the master response regulator, Spo0A. However, the factors and mechanisms that directly regulate C. difficile Spo0A activity are not defined. In the well-studied Bacillus species, Spo0A is directly inactivated by Spo0E, a small phosphatase. To understand Spo0E function in C. difficile, we created a null mutation of the spo0E ortholog and assessed sporulation and physiology. The spo0E mutant produced significantly more spores, demonstrating Spo0E represses C. difficile sporulation. Unexpectedly, the spo0E mutant also exhibited increased motility and toxin production, and enhanced virulence in animal infections. We uncovered that Spo0E interacts with both Spo0A and the toxin and motility regulator, RstA. Direct interactions between Spo0A, Spo0E, and RstA constitute a previously unknown molecular switch that coordinates sporulation with motility and toxin production. Reinvestigation of Spo0E function in B. subtilis revealed that Spo0E induced motility, demonstrating Spo0E regulation of motility and sporulation among divergent species. Further, 3D structural analyses of Spo0E revealed specific and exclusive interactions between Spo0E and binding partners in C. difficile and B. subtilis that provide insight into the conservation of this regulatory mechanism among different species.

The impact of orphan histidine kinases and phosphotransfer proteins on the regulation of clostridial sporulation initiation.

Sporulation is an important feature of the clostridial life cycle, facilitating survival of these bacteria in harsh environments, contributing to disease transmission for pathogenic species, and sharing common early steps that are also involved in regulating industrially important solvent production by some non-pathogenic species. Initial genomics studies suggested that Clostridia lack the classical phosphorelay that phosphorylates Spo0A and initiates sporulation in Bacillus, leading to the hypothesis that sporulation in Clostridia universally begins when Spo0A is phosphorylated by orphan histidine kinases (OHKs). However, components of the classical Bacillus phosphorelay were recently identified in some Clostridia. Similar Bacillus phosphorelay components have not yet been found in the pathogenic Clostridia or the solventogenic Clostridia of industrial importance. For some of those Clostridia lacking a classical phosphorelay, the involvement of OHKs in sporulation initiation has received support from genetic studies demonstrating the involvement of several apparent OHKs in their sporulation. In addition, several clostridial OHKs directly phosphorylate Spo0A in vitro. Interestingly, there is considerable protein domain diversity among the sporulation-associated OHKs in Clostridia. Further adding to the emergent complexity of sporulation initiation in Clostridia, several candidate OHK phosphotransfer proteins that were OHK candidates were shown to function as phosphatases that reduce sporulation in some Clostridia. The mounting evidence indicates that no single pathway explains sporulation initiation in all Clostridia and supports the need for further study to fully understand the unexpected and biologically fascinating mechanistic diversity of this important process among these medically and industrially important bacteria.

The pH-responsive SmrR-SmrT system modulates C. difficile antimicrobial resistance, spore formation, and toxin production.

Clostridioides difficile is an anaerobic gastrointestinal pathogen that spreads through the environment as dormant spores. To survive, replicate, and sporulate in the host intestine, C. difficile must adapt to a variety of conditions in its environment, including changes in pH, the availability of metabolites, host immune factors, and a diverse array of other species. Prior studies showed that changes in intestinal conditions, such as pH, can affect C. difficile toxin production, spore formation, and cell survival. However, little is understood about the specific genes and pathways that facilitate environmental adaptation and lead to changes in C. difficile cell outcomes. In this study, we investigated two genes, CD2505 and CD2506, that are differentially regulated by pH to determine if they impact C. difficile growth and sporulation. Using deletion mutants, we examined the effects of both genes (herein smrR and smrT) on sporulation frequency, toxin production, and antimicrobial resistance. We determined that SmrR is a repressor of smrRT that responds to pH and suppresses sporulation and toxin production through regulation of the SmrT transporter. Further, we showed that SmrT confers resistance to erythromycin and lincomycin, establishing a connection between the regulation of sporulation and antimicrobial resistance.IMPORTANCEClostridioides difficile is a mammalian pathogen that colonizes the large intestine and produces toxins that lead to severe diarrheal disease. C. difficile is a major threat to public health due to its intrinsic resistance to antimicrobials and its ability to form dormant spores that are easily spread from host to host. In this study, we examined the contribution of two genes, smrR and smrT, on sporulation, toxin production, and antimicrobial resistance. Our results indicate that SmrR represses smrT expression, while production of SmrT increases spore and toxin production, as well as resistance to antibiotics.

The pH-responsive SmrR-SmrT system modulates C. difficile antimicrobial resistance, spore formation, and toxin production.

Clostridioides difficile is an anaerobic gastrointestinal pathogen that spreads through the environment as dormant spores. To survive, replicate, and sporulate in the host intestine, C. difficile must adapt to a variety of conditions in its environment, including changes in pH, the availability of metabolites, host immune factors, and a diverse array of other species. Prior studies showed that changes in intestinal conditions, such as pH, can affect C. difficile toxin production, spore formation, and cell survival. However, little is understood about the specific genes and pathways that facilitate environmental adaptation and lead to changes in C. difficile cell outcomes. In this study, we investigated two genes, CD2505 and CD2506, that are differentially regulated by pH to determine if they impact C. difficile growth and sporulation. Using deletion mutants, we examined the effects of both genes (herein smrR and smrT ) on sporulation frequency, toxin production, and antimicrobial resistance. We determined that SmrR is a repressor of smrRT that responds to pH and suppresses sporulation and toxin production through regulation of the SmrT transporter. Further, we showed that SmrT confers resistance to erythromycin and lincomycin, establishing a connection between the regulation of sporulation and antimicrobial resistance. IMPORTANCE: C. difficile is a mammalian pathogen that colonizes the large intestine and produces toxins that lead to severe diarrheal disease. C. difficile is a major threat to public health due to its intrinsic resistance to antimicrobials and its ability to form dormant spores that are easily spread from host to host. In this study, we examined the contribution of two genes, smrR and smrT on sporulation, toxin production, and antimicrobial resistance. Our results indicate that SmrR represses smrT expression, while production of SmrT increases spore and toxin production, as well as resistance to antibiotics.

The RgaS-RgaR two-component system promotes Clostridioides difficile sporulation through a small RNA and the Agr1 system.

The ability to form a dormant spore is essential for the survival of the anaerobic pathogen, Clostridioides difficile, outside of the mammalian gastrointestinal tract. The initiation of sporulation is governed by the master regulator of sporulation, Spo0A, which is activated by phosphorylation. Multiple sporulation factors control Spo0A phosphorylation; however, this regulatory pathway is not well defined in C. difficile. We discovered that RgaS and RgaR, a conserved orphan histidine kinase and orphan response regulator, function together as a cognate two-component regulatory system to directly activate transcription of several genes. One of these targets, agrB1D1, encodes gene products that synthesize and export a small quorum-sensing peptide, AgrD1, which positively influences expression of early sporulation genes. Another target, a small regulatory RNA now known as SpoZ, impacts later stages of sporulation through a small hypothetical protein and an additional, unknown regulatory mechanism(s). Unlike Agr systems in many organisms, AgrD1 does not activate the RgaS-RgaR two-component system, and thus, is not responsible for autoregulating its own production. Altogether, we demonstrate that C. difficile utilizes a conserved two-component system that is uncoupled from quorum-sensing to promote sporulation through two distinct regulatory pathways.

Glycine fermentation by C. difficile promotes virulence and spore formation, and is induced by host cathelicidin.

Clostridioides difficile is a leading cause of antibiotic-associated diarrheal disease. C. difficile colonization, growth, and toxin production in the intestine is strongly associated with its ability to use amino acids to generate energy, but little is known about the impact of specific amino acids on C. difficile pathogenesis. The amino acid glycine is enriched in the dysbiotic gut and is suspected to contribute to C. difficile infection. We hypothesized that the use of glycine as an energy source contributes to colonization of the intestine and pathogenesis of C. difficile. To test this hypothesis, we deleted the glycine reductase (GR) genes grdAB, rendering C. difficile unable to ferment glycine, and investigated the impact on growth and pathogenesis. Our data show that the grd pathway promotes growth, toxin production, and sporulation. Glycine fermentation also had a significant impact on toxin production and pathogenesis of C. difficile in the hamster model of disease. Furthermore, we determined that the grd locus is regulated by host cathelicidin (LL-37) and the cathelicidin-responsive regulator, ClnR, indicating that the host peptide signals to control glycine catabolism. The induction of glycine fermentation by LL-37 demonstrates a direct link between the host immune response and the bacterial reactions of toxin production and spore formation.

The predicted acetoin dehydrogenase pathway represses sporulation of Clostridioides difficile.

Clostridioides difficile is a major gastrointestinal pathogen that is transmitted as a dormant spore. As an intestinal pathogen, C. difficile must contend with variable environmental conditions, including fluctuations in pH and nutrient availability. Nutrition and pH both influence growth and spore formation, but how pH and nutrition jointly influence sporulation are not known. In this study, we investigated the dual impact of pH and pH-dependent metabolism on C. difficile sporulation. Specifically, we examined the impacts of pH and the metabolite acetoin on C. difficile growth and sporulation. We found that expression of the predicted acetoin dehydrogenase operon, acoRABCL , was pH-dependent and regulated by acetoin. Regulation of the C. difficile aco locus is distinct from other characterized systems and appears to involve a co-transcribed DeoR-family regulator rather than the sigma 54 -dependent activator. In addition, an acoA null mutant produced significantly more spores and initiated sporulation earlier than the parent strain. However, unlike other Firmicutes, growth and culture density of C. difficile was not increased by acetoin availability or disruption of the aco pathway. Together, these results indicate that acetoin, pH, and the aco pathway play important roles in nutritional repression of sporulation in C. difficile , but acetoin metabolism does not support cell growth as a stationary phase energy source. IMPORTANCE: Clostridioides difficile, or C. diff , is an anaerobic bacterium that lives within the gut of many mammals and causes infectious diarrhea. C. difficile is able to survive outside of the gut and transmit to new hosts by forming dormant spores. It is known that the pH of the intestine and the nutrients available both affect the growth and sporulation of C. diffiicile, but the specific conditions that result in sporulation in the host are not clear. In this study, we investigated how pH and the metabolite acetoin affect the ability of C. difficile to grow, proliferate, and form spores. We found that a mutant lacking the predicted acetoin metabolism pathway form more spores, but their growth is not impacted. These results show that C. difficile uses acetoin differently than many other species and that acetoin has an important role as an environmental metabolite that influences spore formation.

The RgaS-RgaR two-component system promotes Clostridioides difficile sporulation through a small RNA and the Agr1 system.

The ability to form a dormant spore is essential for the survival of the anaerobic, gastrointestinal pathogen Clostridioides difficile outside of the mammalian gastrointestinal tract. The initiation of sporulation is governed by the master regulator of sporulation, Spo0A, which is activated by phosphorylation. Multiple sporulation factors control Spo0A phosphorylation; however, this regulatory pathway is not well defined in C. difficile. We discovered that RgaS and RgaR, a conserved orphan histidine kinase and orphan response regulator, function together as a cognate two-component regulatory system to directly activate transcription of several genes. One of these targets, agrB1D1, encodes gene products that synthesize and export a small quorum-sensing peptide, AgrD1, which positively influences expression of early sporulation genes. Another target, a small regulatory RNA now known as SrsR, impacts later stages of sporulation through an unknown regulatory mechanism(s). Unlike Agr systems in many organisms, AgrD1 does not activate the RgaS-RgaR two-component system, and thus, is not responsible for autoregulating its own production. Altogether, we demonstrate that C. difficile utilizes a conserved two-component system that is uncoupled from quorum-sensing to promote sporulation through two distinct regulatory pathways.

A Conserved Switch Controls Virulence, Sporulation, and Motility in C. difficile.

Spore formation is required for environmental survival and transmission of the human enteropathogenic Clostridioides difficile . In all bacterial spore formers, sporulation is regulated through activation of the master response regulator, Spo0A. However, the factors and mechanisms that directly regulate C. difficile Spo0A activity are not defined. In the well-studied Bacillus species, Spo0A is directly inactivated by Spo0E, a small phosphatase. To understand Spo0E function in C. difficile , we created a null mutation of the spo0E ortholog and assessed sporulation and physiology. The spo0E mutant produced significantly more spores, demonstrating Spo0E represses C. difficile sporulation. Unexpectedly, the spo0E mutant also exhibited increased motility and toxin production, and enhanced virulence in animal infections. We uncovered that Spo0E interacts with both Spo0A and the toxin and motility regulator, RstA. Direct interactions between Spo0A, Spo0E, and RstA constitute a previously unknown molecular switch that coordinates sporulation with motility and toxin production. Reinvestigation of Spo0E function in B. subtilis revealed that Spo0E induced motility, demonstrating Spo0E regulation of motility and sporulation among divergent species. Further, we found that Spo0E orthologs are widespread among prokaryotes, suggesting that Spo0E performs conserved regulatory functions in diverse bacteria.

Identification of Functional Spo0A Residues Critical for Sporulation in Clostridioides difficile.

Clostridioides difficile is an anaerobic, Gram-positive pathogen that is responsible for C. difficile infection (CDI). To survive in the environment and spread to new hosts, C. difficile must form metabolically dormant spores. The formation of spores requires activation of the transcription factor Spo0A, which is the master regulator of sporulation in all endospore-forming bacteria. Though the sporulation initiation pathway has been delineated in the Bacilli, including the model spore-former Bacillus subtilis, the direct regulators of Spo0A in C. difficile remain undefined. C. difficile Spo0A shares highly conserved protein interaction regions with the B. subtilis sporulation proteins Spo0F and Spo0A, although many of the interacting factors present in B. subtilis are not encoded in C. difficile. To determine if comparable Spo0A residues are important for C. difficile sporulation initiation, site-directed mutagenesis was performed at conserved receiver domain residues and the effects on sporulation were examined. Mutation of residues important for homodimerization and interaction with positive and negative regulators of B. subtilis Spo0A and Spo0F impacted C. difficile Spo0A function. The data also demonstrated that mutation of many additional conserved residues altered C. difficile Spo0A activity, even when the corresponding Bacillus interacting proteins are not apparent in the C. difficile genome. Finally, the conserved aspartate residue at position 56 of C. difficile Spo0A was determined to be the phosphorylation site that is necessary for Spo0A activation. The finding that Spo0A interacting motifs maintain functionality suggests that C. difficile Spo0A interacts with yet unidentified proteins that regulate its activity and control spore formation.

Three Orphan Histidine Kinases Inhibit Clostridioides difficile Sporulation.

The ability of the anaerobic gastrointestinal pathogen Clostridioides difficile to survive outside the host relies on the formation of dormant endospores. Spore formation is contingent on the activation of a conserved transcription factor, Spo0A, by phosphorylation. Multiple kinases and phosphatases regulate Spo0A activity in other spore-forming organisms; however, these factors are not well conserved in C. difficile. Previously, we discovered that deletion of a predicted histidine kinase, CD1492, increases sporulation, indicating that CD1492 inhibits C. difficile spore formation. In this study, we investigate the functions of additional predicted orphan histidine kinases CD2492, CD1579, and CD1949, which are hypothesized to regulate Spo0A phosphorylation. Disruption of CD2492 also increased sporulation frequency, similarly to the CD1492 mutant and in contrast to a previous study. A CD1492 CD2492 mutant phenocopied the sporulation and gene expression patterns of the single mutants, suggesting that these proteins function in the same genetic pathway to repress sporulation. Deletion of CD1579 variably increased sporulation frequency; however, knockdown of CD1949 expression did not influence sporulation. We provide evidence that CD1492, CD2492, and CD1579 function as phosphatases, as mutation of the conserved histidine residue for phosphate transfer abolished CD2492 function, and expression of the CD1492 or CD2492 histidine site-directed mutants or the wild-type CD1579 allele in a parent strain resulted in a dominant-negative hypersporulation phenotype. Altogether, at least three predicted histidine kinases, CD1492, CD2492, and CD1579 (herein, PtpA, PtpB and PtpC), repress C. difficile sporulation initiation by regulating activity of Spo0A. IMPORTANCE The formation of inactive spores is critical for the long-term survival of the gastrointestinal pathogen Clostridioides difficile. The onset of sporulation is controlled by the master regulator of sporulation, Spo0A, which is activated by phosphorylation. Multiple kinases and phosphatases control Spo0A phosphorylation; however, this regulatory pathway is not defined in C. difficile. We show that two predicted histidine kinase proteins, CD1492 (PtpA) and CD2492 (PtpB), function in the same regulatory pathway to repress sporulation by preventing Spo0A phosphorylation. We show that another predicted histidine kinase protein, CD1579 (PtpC), also represses sporulation and present evidence that a fourth predicted histidine kinase protein, CD1949, does not impact sporulation. These results support the idea that C. difficile inhibits sporulation initiation through multiple phosphatases.

Genetic mechanisms governing sporulation initiation in Clostridioides difficile.

As an anaerobe, Clostridioides difficile relies on the formation of a dormant spore for survival outside of the mammalian host's gastrointestinal tract. The spore is recalcitrant to desiccation, numerous disinfectants, UV light, and antibiotics, permitting long-term survival against environmental insults and efficient transmission from host to host. Although the morphological stages of spore formation are similar between C. difficile and other well-studied endospore-forming bacteria, the C. difficile genome does not appear to encode many of the known, conserved regulatory factors that are necessary to initiate sporulation in other spore-forming bacteria. The absence of early sporulation-specific orthologs suggests that C. difficile has evolved to control sporulation initiation in response to its unique and specific ecological niche and environmental cues within the host. Here, we review our current understanding and highlight the recent discoveries that have begun to unravel the regulatory pathways and molecular mechanisms by which C. difficile induces spore formation.

c-di-GMP Inhibits Early Sporulation in Clostridioides difficile.

The formation of dormant spores is essential for the anaerobic pathogen Clostridioides difficile to survive outside the host gastrointestinal tract. The regulatory pathways and environmental signals that initiate C. difficile spore formation within the host are not well understood. One second-messenger signaling molecule, cyclic diguanylate (c-di-GMP), modulates several physiological processes important for C. difficile pathogenesis and colonization, but the impact of c-di-GMP on sporulation is unknown. In this study, we investigated the contribution of c-di-GMP to C. difficile sporulation. The overexpression of a gene encoding a diguanylate cyclase, dccA, decreased the sporulation frequency and early sporulation gene transcription in both the epidemic R20291 and historical 630Δerm strains. The expression of a dccA allele encoding a catalytically inactive DccA that is unable to synthesize c-di-GMP no longer inhibited sporulation, indicating that the accumulation of intracellular c-di-GMP reduces C. difficile sporulation. A null mutation in dccA slightly increased sporulation in R20291 and slightly decreased sporulation in 630Δerm, suggesting that DccA contributes to the intracellular pool of c-di-GMP in a strain-dependent manner. However, these data were highly variable, underscoring the complex regulation involved in modulating intracellular c-di-GMP concentrations. Finally, the overexpression of dccA in known sporulation mutants revealed that c-di-GMP is likely signaling through an unidentified regulatory pathway to control early sporulation events in C. difficile. c-di-GMP-dependent regulation of C. difficile sporulation may represent an unexplored avenue of potential environmental and intracellular signaling that contributes to the complex regulation of sporulation initiation. IMPORTANCE Many bacterial organisms utilize the small signaling molecule cyclic diguanylate (c-di-GMP) to regulate important physiological processes, including motility, toxin production, biofilm formation, and colonization. c-di-GMP inhibits motility and toxin production and promotes biofilm formation and colonization in the anaerobic, gastrointestinal pathogen Clostridioides difficile. However, the impact of c-di-GMP on C. difficile spore formation, a critical step in this pathogen's life cycle, is unknown. Here, we demonstrate that c-di-GMP negatively impacts sporulation in two clinically relevant C. difficile strains, the epidemic strain R20291 and the historical strain 630Δerm. The pathway through which c-di-GMP controls sporulation was investigated, and our results suggest that c-di-GMP is likely signaling through an unidentified regulatory pathway to control C. difficile sporulation. This work implicates c-di-GMP metabolism as a mechanism to integrate environmental and intracellular cues through c-di-GMP levels to influence C. difficile sporulation.

Strain-Dependent RstA Regulation of Clostridioides difficile Toxin Production and Sporulation.

The anaerobic spore former Clostridioides difficile causes significant diarrheal disease in humans and other mammals. Infection begins with the ingestion of dormant spores, which subsequently germinate within the host gastrointestinal tract. There, the vegetative cells proliferate and secrete two exotoxins, TcdA and TcdB, which cause disease symptoms. Although spore formation and toxin production are critical for C. difficile pathogenesis, the regulatory links between these two physiological processes are not well understood and are strain dependent. Previously, we identified a conserved C. difficile regulator, RstA, that promotes sporulation initiation through an unknown mechanism and directly and indirectly represses toxin and motility gene transcription in the historical isolate 630Δerm To test whether perceived strain-dependent differences in toxin production and sporulation are mediated by RstA, we created an rstA mutant in the epidemic ribotype 027 strain R20291. RstA affected sporulation and toxin gene expression similarly but more robustly in R20291 than in 630Δerm In contrast, no effect on motility gene expression was observed in R20291. Reporter assays measuring transcriptional regulation of tcdR, the sigma factor gene essential for toxin gene expression, identified sequence-dependent effects influencing repression by RstA and CodY, a global nutritional sensor, in four diverse C. difficile strains. Finally, sequence- and strain-dependent differences were evident in RstA negative autoregulation of rstA transcription. Altogether, our data suggest that strain-dependent differences in RstA regulation contribute to the sporulation and toxin phenotypes observed in R20291. Our data establish RstA as an important regulator of C. difficile virulence traits.IMPORTANCE Two critical traits of Clostridioides difficile pathogenesis are toxin production, which causes disease symptoms, and spore formation, which permits survival outside the gastrointestinal tract. The multifunctional regulator RstA promotes sporulation and prevents toxin production in the historical strain 630Δerm Here, we show that RstA exhibits stronger effects on these phenotypes in an epidemic isolate, R20291, and additional strain-specific effects on toxin and rstA expression are evident. Our data demonstrate that sequence-specific differences within the promoter for the toxin regulator TcdR contribute to the regulation of toxin production by RstA and CodY. These sequence differences account for some of the variability in toxin production among isolates and may allow strains to differentially control toxin production in response to a variety of signals.

Sporulation and Germination in Clostridial Pathogens.

As obligate anaerobes, clostridial pathogens depend on their metabolically dormant, oxygen-tolerant spore form to transmit disease. However, the molecular mechanisms by which those spores germinate to initiate infection and then form new spores to transmit infection remain poorly understood. While sporulation and germination have been well characterized in Bacillus subtilis and Bacillus anthracis, striking differences in the regulation of these processes have been observed between the bacilli and the clostridia, with even some conserved proteins exhibiting differences in their requirements and functions. Here, we review our current understanding of how clostridial pathogens, specifically Clostridium perfringens, Clostridium botulinum, and Clostridioides difficile, induce sporulation in response to environmental cues, assemble resistant spores, and germinate metabolically dormant spores in response to environmental cues. We also discuss the direct relationship between toxin production and spore formation in these pathogens.

Phase variation of a signal transduction system controls Clostridioides difficile colony morphology, motility, and virulence.

Recent work has revealed that Clostridioides difficile, a major cause of nosocomial diarrheal disease, exhibits phenotypic heterogeneity within a clonal population as a result of phase variation. Many C. difficile strains representing multiple ribotypes develop two colony morphotypes, termed rough and smooth, but the biological implications of this phenomenon have not been explored. Here, we examine the molecular basis and physiological relevance of the distinct colony morphotypes produced by this bacterium. We show that C. difficile reversibly differentiates into rough and smooth colony morphologies and that bacteria derived from the isolates display discrete motility behaviors. We identified an atypical phase-variable signal transduction system consisting of a histidine kinase and two response regulators, named herein colony morphology regulators RST (CmrRST), which mediates the switch in colony morphology and motility behaviors. The CmrRST-regulated surface motility is independent of flagella and type IV pili, suggesting a novel mechanism of cell migration in C. difficile. Microscopic analysis of cell and colony structure indicates that CmrRST promotes the formation of elongated bacteria arranged in bundled chains, which may contribute to bacterial migration on surfaces. In a hamster model of acute C. difficile disease, the CmrRST system is required for disease development. Furthermore, we provide evidence that CmrRST phase varies during infection, suggesting that the intestinal environment impacts the proportion of CmrRST-expressing C. difficile. Our findings indicate that C. difficile employs phase variation of the CmrRST signal transduction system to generate phenotypic heterogeneity during infection, with concomitant effects on bacterial physiology and pathogenesis.

Regulation and Anaerobic Function of the Clostridioides difficile ß-Lactamase.

Clostridioides difficile causes severe antibiotic-associated diarrhea and colitis. C. difficile is an anaerobic, Gram-positive sporeformer that is highly resistant to ß-lactams, the most commonly prescribed antibiotics. The resistance of C. difficile to ß-lactam antibiotics allows the pathogen to replicate and cause disease in antibiotic-treated patients. However, the mechanisms of ß-lactam resistance in C. difficile are not fully understood. Our data reinforce prior evidence that C. difficile produces a ß-lactamase, which is a common ß-lactam resistance mechanism found in other bacterial species. Here, we characterize the C. difficilebla operon that encodes a lipoprotein of unknown function and a ß-lactamase that was greatly induced in response to several classes of ß-lactam antibiotics. An in-frame deletion of the operon abolished ß-lactamase activity in C. difficile strain 630Δerm and resulted in decreased resistance to the ß-lactam ampicillin. We found that the activity of this ß-lactamase, BlaCDD, is dependent upon the redox state of the enzyme. In addition, we observed that transport of BlaCDD out of the cytosol and to the cell surface is facilitated by an N-terminal signal sequence. Our data demonstrate that a cotranscribed lipoprotein, BlaX, aids in BlaCDD activity. Further, we identified a conserved BlaRI regulatory system and demonstrated via insertional disruption that BlaRI controls transcription of the blaXCDD genes in response to ß-lactams. These results provide support for the function of a ß-lactamase in C. difficile antibiotic resistance and reveal the unique roles of a coregulated lipoprotein and reducing environment in C. difficile ß-lactamase activity.

RstA Is a Major Regulator of Clostridioides difficile Toxin Production and Motility.

Clostridioides difficile infection (CDI) is a toxin-mediated diarrheal disease. Several factors have been identified that influence the production of the two major C. difficile toxins, TcdA and TcdB, but prior published evidence suggested that additional unknown factors were involved in toxin regulation. Previously, we identified a C. difficile regulator, RstA, that promotes sporulation and represses motility and toxin production. We observed that the predicted DNA-binding domain of RstA was required for RstA-dependent repression of toxin genes, motility genes, and rstA transcription. In this study, we further investigated the regulation of toxin and motility gene expression by RstA. DNA pulldown assays confirmed that RstA directly binds the rstA promoter via the predicted DNA-binding domain. Through mutational analysis of the rstA promoter, we identified several nucleotides that are important for RstA-dependent transcriptional regulation. Further, we observed that RstA directly binds and regulates the promoters of the toxin genes tcdA and tcdB, as well as the promoters for the sigD and tcdR genes, which encode regulators of toxin gene expression. Complementation analyses with the Clostridium perfringens RstA ortholog and a multispecies chimeric RstA protein revealed that the C. difficile C-terminal domain is required for RstA DNA-binding activity, suggesting that species-specific signaling controls RstA function. Our data demonstrate that RstA is a transcriptional repressor that autoregulates its own expression and directly inhibits transcription of the two toxin genes and two positive toxin regulators, thereby acting at multiple regulatory points to control toxin production.IMPORTANCEClostridioides difficile is an anaerobic, gastrointestinal pathogen of humans and other mammals. C. difficile produces two major toxins, TcdA and TcdB, which cause the symptoms of the disease, and forms dormant endospores to survive the aerobic environment outside the host. A recently discovered regulatory factor, RstA, inhibits toxin production and positively influences spore formation. Herein, we determine that RstA directly binds its own promoter DNA to repress its own gene transcription. In addition, our data demonstrate that RstA directly represses toxin gene expression and gene expression of two toxin gene activators, TcdR and SigD, creating a complex regulatory network to tightly control toxin production. This study provides a novel regulatory link between C. difficile sporulation and toxin production. Further, our data suggest that C. difficile toxin production is regulated through a direct, species-specific sensing mechanism.

The C. difficile clnRAB operon initiates adaptations to the host environment in response to LL-37.

To cause disease, Clostridioides (Clostridium) difficile must resist killing by innate immune effectors in the intestine, including the host antimicrobial peptide, cathelicidin (LL-37). The mechanisms that enable C. difficile to adapt to the intestine in the presence of antimicrobial peptides are unknown. Expression analyses revealed an operon, CD630_16170-CD630_16190 (clnRAB), which is highly induced by LL-37 and is not expressed in response to other cell-surface active antimicrobials. This operon encodes a predicted transcriptional regulator (ClnR) and an ABC transporter system (ClnAB), all of which are required for function. Analyses of a clnR mutant indicate that ClnR is a pleiotropic regulator that directly binds to LL-37 and controls expression of numerous genes, including many involved in metabolism, cellular transport, signaling, gene regulation, and pathogenesis. The data suggest that ClnRAB is a novel regulatory mechanism that senses LL-37 as a host signal and regulates gene expression to adapt to the host intestinal environment during infection.

Determination of the in vitro Sporulation Frequency of Clostridium difficile.

The anaerobic, gastrointestinal pathogen, Clostridium difficile, persists within the environment and spreads from host-to-host via its infectious form, the spore. To effectively study spore formation, the physical differentiation of vegetative cells from spores is required to determine the proportion of spores within a population of C. difficile. This protocol describes a method to accurately enumerate both viable vegetative cells and spores separately and subsequently calculate a sporulation frequency of a mixed C. difficile population from various in vitro growth conditions (Edwards et al., 2016b).