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Bacillus subtilis spores are encased in two concentric shells: an outer proteinaceous “coat” and an inner peptidoglycan “cortex,” separated by a membrane. Cortex assembly depends on coat assembly initiation, but how cells achieve this coordination across the membrane is unclear. Here, we report that the protein SpoVID monitors the polymerization state of the coat basement layer via an extension to a functional intracellular LysM domain that arrests sporulation when coat assembly is initiated improperly. Whereas extracellular LysM domains bind mature peptidoglycan, SpoVID LysM binds to the membrane-bound lipid II peptidoglycan precursor. We propose that improper coat assembly exposes the SpoVID LysM domain, which then sequesters lipid II and prevents cortex assembly. SpoVID defines a widespread group of firmicute proteins with a characteristic N-terminal domain and C-terminal peptidoglycan-binding domains that might combine coat and cortex assembly roles to mediate a developmental checkpoint linking the morphogenesis of two spatially separated supramolecular structures.

Clostridioides difficile is a spore-forming bacterial pathogen that is the leading cause of hospital-acquired gastroenteritis. C. difficile infections begin when its spore form germinates in the vertebrate gut upon sensing bile acids. These germinants induce a proteolytic signaling cascade controlled by three members of the subtilisin-like serine protease family, CspA, CspB, and CspC. Notably, even though CspC and CspA are both pseudoproteases, they are nevertheless required to sense germinants and activate the protease, CspB. Thus, CspC and CspA are part of a growing list of pseudoenzymes that play important roles in regulating cellular processes. However, despite their importance, the structural properties of pseudoenzymes that allow them to function as regulators remain poorly understood. Our recently determined crystal structure of CspC revealed that its degenerate site residues align closely with the catalytic triad of CspB, so in this study we tested whether the ancestral protease activity of the CspC and CspA pseudoproteases could be “resurrected.” Restoring the catalytic triad to these pseudoproteases failed to resurrect their protease activity, although the mutations differentially affected the stability and function of these pseudoproteases. Degenerate site mutations destabilized CspC and impaired spore germination without impacting CspA stability or function. Thus, our results surprisingly reveal that the presence of a catalytic triad does not necessarily predict protease activity. Since close homologs of C. difficile CspA occasionally carry an intact catalytic triad, our results imply that bioinformatics predictions of enzyme activity may overlook pseudoenzymes in some cases.

Clostridioides difficile is a leading cause of healthcare-associated infections worldwide. C. difficile infections are transmitted by its metabolically dormant, aerotolerant spore form. Functional spore formation depends on the assembly of two protective layers: a thick layer of modified peptidoglycan known as the cortex layer and a multilayered proteinaceous meshwork known as the coat. We previously identified two spore morphogenetic proteins, SpoIVA and SipL, that are essential for recruiting coat proteins to the developing forespore and making functional spores. While SpoIVA and SipL directly interact, the identities of the proteins they recruit to the forespore remained unknown. We used mass spectrometry-based affinity proteomics to identify proteins that interact with the SpoIVA-SipL complex. These analyses identified the Peptostreptococcaceae family-specific, sporulation-induced bitopic membrane protein CD3457 (renamed SpoVQ) as a protein that interacts with SipL and SpoIVA. Loss of SpoVQ decreased heat-resistant spore formation by ∼5-fold and reduced cortex thickness∼2-fold; the thinner cortex layer of ΔspoVQ spores correlated with higher levels of spontaneous germination (i.e., in the absence of germinant). Notably, loss of SpoVQ in either spoIVA or sipL mutants prevented cortex synthesis altogether and greatly impaired the localization of a SipL-mCherry fusion protein around the forespore. Thus, SpoVQ is a novel regulator of C. difficile cortex synthesis that appears to link cortex and coat formation. The identification of SpoVQ as a spore morphogenetic protein further highlights how Peptostreptococcaceae family-specific mechanisms control spore formation in C. difficile.ImportanceThe Centers for Disease Control has designated Clostridioides difficile as an urgent threat because of its intrinsic antibiotic resistance. C. difficile persists in the presence of antibiotics in part because it makes metabolically dormant spores. While recent work has shown that preventing the formation of infectious spores can reduce C. difficile disease recurrence, more selective anti-sporulation therapies are needed. The identification of spore morphogenetic factors specific to C. difficile would facilitate the development of such therapies. In this study, we identified SpoVQ (CD3457) as a spore morphogenetic protein specific to the Peptostreptococcaceae family that regulates the formation of C. difficile’s protective spore cortex layer. SpoVQ acts in concert with the known spore coat morphogenetic factors, SpoIVA and SipL, to link formation of the protective coat and cortex layers. These data reveal a novel pathway that could be targeted to prevent the formation of infectious C. difficile spores.

The major pathogen Clostridioides difficile depends on its spore form to transmit disease. However, the mechanism by which C. difficile assembles spores remains poorly characterized. We previously showed that binding between the spore morphogenetic proteins SpoIVA and SipL regulates assembly of the protective coat layer around the forespore. In this study, we determined that mutations in the C. difficile SpoIVA ATPase motifs result in relatively minor defects in spore formation, in contrast with Bacillus subtilis. Nevertheless, our data suggest that SipL preferentially recognizes the ATP-bound form of SpoIVA and identify a specific residue in the SipL C-terminal LysM domain that is critical for recognizing the ATP-bound form of SpoIVA. These findings advance our understanding of how SpoIVA-SipL interactions regulate C. difficile spore assembly., The nosocomial pathogen Clostridioides difficile is a spore-forming obligate anaerobe that depends on its aerotolerant spore form to transmit infections. Functional spore formation depends on the assembly of a proteinaceous layer known as the coat around the developing spore. In C. difficile, coat assembly depends on the conserved spore protein SpoIVA and the clostridial-organism-specific spore protein SipL, which directly interact. Mutations that disrupt their interaction cause the coat to mislocalize and impair spore formation. In Bacillus subtilis, SpoIVA is an ATPase that uses ATP hydrolysis to drive its polymerization around the forespore. Loss of SpoIVA ATPase activity impairs B. subtilis SpoIVA encasement of the forespore and activates a quality control mechanism that eliminates these defective cells. Since this mechanism is lacking in C. difficile, we tested whether mutations in the C. difficile SpoIVA ATPase motifs impact functional spore formation. Disrupting C. difficile SpoIVA ATPase motifs resulted in phenotypes that were typically >104-fold less severe than the equivalent mutations in B. subtilis. Interestingly, mutation of ATPase motif residues predicted to abrogate SpoIVA binding to ATP decreased the SpoIVA-SipL interaction, whereas mutation of ATPase motif residues predicted to disrupt ATP hydrolysis but maintain ATP binding enhanced the SpoIVA-SipL interaction. When a sipL mutation known to reduce binding to SpoIVA was combined with a spoIVA mutation predicted to prevent SpoIVA binding to ATP, spore formation was severely exacerbated. Since this phenotype is allele specific, our data imply that SipL recognizes the ATP-bound form of SpoIVA and highlight the importance of this interaction for functional C. difficile spore formation. IMPORTANCE The major pathogen Clostridioides difficile depends on its spore form to transmit disease. However, the mechanism by which C. difficile assembles spores remains poorly characterized. We previously showed that binding between the spore morphogenetic proteins SpoIVA and SipL regulates assembly of the protective coat layer around the forespore. In this study, we determined that mutations in the C. difficile SpoIVA ATPase motifs result in relatively minor defects in spore formation, in contrast with Bacillus subtilis. Nevertheless, our data suggest that SipL preferentially recognizes the ATP-bound form of SpoIVA and identify a specific residue in the SipL C-terminal LysM domain that is critical for recognizing the ATP-bound form of SpoIVA. These findings advance our understanding of how SpoIVA-SipL interactions regulate C. difficile spore assembly.

Here we introduce plasmids for xylose-regulated expression and repression of genes in Clostridioides difficile. The xylose-inducible expression vector allows for ∼100-fold induction of an mCherryOpt reporter gene. Induction is titratable and uniform from cell to cell. The gene repression plasmid is a CRISPR interference (CRISPRi) system based on a nuclease-defective, codon-optimized allele of the Streptococcus pyogenes Cas9 protein (dCas9) that is targeted to a gene of interest by a constitutively expressed single guide RNA (sgRNA). Expression of dCas9 is induced by xylose, allowing investigators to control the timing and extent of gene silencing, as demonstrated here by dose-dependent repression of a chromosomal gene for a red fluorescent protein (maximum repression, ∼100-fold). To validate the utility of CRISPRi for deciphering gene function in C. difficile, we knocked down the expression of three genes involved in the biogenesis of the cell envelope: the cell division gene ftsZ, the S-layer protein gene slpA, and the peptidoglycan synthase gene pbp-0712. CRISPRi confirmed known or expected phenotypes associated with the loss of FtsZ and SlpA and revealed that the previously uncharacterized peptidoglycan synthase PBP-0712 is needed for proper elongation, cell division, and protection against lysis. IMPORTANCE Clostridioides difficile has become the leading cause of hospital-acquired diarrhea in developed countries. A better understanding of the basic biology of this devastating pathogen might lead to novel approaches for preventing or treating C. difficile infections. Here we introduce new plasmid vectors that allow for titratable induction (P(xyl)) or knockdown (CRISPRi) of gene expression. The CRISPRi plasmid allows for easy depletion of target proteins in C. difficile. Besides bypassing the lengthy process of mutant construction, CRISPRi can be used to study the function of essential genes, which are particularly important targets for antibiotic development.

This work analyzes the high-pressure (HP) germination of spores of the food-borne pathogen Clostridium perfringens (with inner membrane [IM] germinant receptors [GRs]) and the opportunistic pathogen Clostridium difficile (with no IM GRs), which has growing implications as an emerging food safety threat. In contrast to those of spores of Bacillus species, mechanisms of HP germination of clostridial spores have not been well studied. HP treatments trigger Bacillus spore germination through spores' IM GRs at ∼150 MPa or through SpoVA channels for release of spores' dipicolinic acid (DPA) at ≥400 MPa, and DPA-less spores have lower wet heat resistance than dormant spores. We found that C. difficile spores exhibited no germination events upon 150-MPa treatment and were not heat sensitized. In contrast, 150-MPa-treated unactivated C. perfringens spores released DPA and became heat sensitive, although most spores did not complete germination by fully rehydrating the spore core, but this treatment of heat-activated spores led to almost complete germination and greater heat sensitization. Spores of both clostridial organisms released DPA during 550-MPa treatment, but C. difficile spores did not complete germination and remained heat resistant. Heat-activated 550-MPa-HP-treated C. perfringens spores germinated almost completely and became heat sensitive. However, unactivated 550-MPa-treated C. perfringens spores did not germinate completely and were less heat sensitive than spores that completed germination. Since C. difficile and C. perfringens spores use different mechanisms for sensing germinants, our results may allow refinement of HP methods for their inactivation in foods and other applications and may guide the development of commercially sterile low-acid foods. IMPORTANCE Spores of various clostridial organisms cause human disease, sometimes due to food contamination by spores. Because of these spores' resistance to normal decontamination regimens, there is continued interest in ways to kill spores without compromising food quality. High hydrostatic pressure (HP) under appropriate conditions can inactivate bacterial spores. With growing use of HP for food pasteurization, advancement of HP for commercial production of sterile low-acid foods requires understanding of mechanisms of spores' interactions with HP. While much is known about HP germination and inactivation of spores of Bacillus species, how HP germinates and inactivates clostridial spores is less well understood. In this work we have tried to remedy this information deficit by examining germination of spores of Clostridium difficile and Clostridium perfringens by several HP and temperature levels. The results may give insight that could facilitate more efficient methods for spore eradication in food sterilization or pasteurization, biodecontamination, and health care.

The spore-forming obligate anaerobe Clostridium difficile is a leading cause of antibiotic-associated diarrhea around the world. In order for C. difficile to cause infection, its metabolically dormant spores must germinate in the gastrointestinal tract. During germination, spores degrade their protective cortex peptidoglycan layers, release dipicolinic acid (DPA), and hydrate their cores. In C. difficile , cortex hydrolysis is necessary for DPA release, whereas in Bacillus subtilis , DPA release is necessary for cortex hydrolysis. Given this difference, we tested whether DPA synthesis and/or release was required for C. difficile spore germination by constructing mutations in either spoVAC or dpaAB , which encode an ion channel predicted to transport DPA into the forespore and the enzyme complex predicted to synthesize DPA, respectively. C. difficile spoVAC and dpaAB mutant spores lacked DPA but could be stably purified and were more hydrated than wild-type spores; in contrast, B. subtilis spoVAC and dpaAB mutant spores were unstable. Although C. difficile spoVAC and dpaAB mutant spores exhibited wild-type germination responses, they were more readily killed by wet heat. Cortex hydrolysis was not affected by this treatment, indicating that wet heat inhibits a stage downstream of this event. Interestingly, C. difficile spoVAC mutant spores were significantly more sensitive to heat treatment than dpaAB mutant spores, indicating that SpoVAC plays additional roles in conferring heat resistance. Taken together, our results demonstrate that SpoVAC and DPA synthetase control C. difficile spore resistance and reveal differential requirements for these proteins among the Firmicutes . IMPORTANCE Clostridium difficile is a spore-forming obligate anaerobe that causes ∼500,000 infections per year in the United States. Although spore germination is essential for C. difficile to cause disease, the factors required for this process have been only partially characterized. This study describes the roles of two factors, DpaAB and SpoVAC, which control the synthesis and release of dipicolinic acid (DPA), respectively, from bacterial spores. Previous studies of these proteins in other spore-forming organisms indicated that they are differentially required for spore formation, germination, and resistance. We now show that the proteins are dispensable for C. difficile spore formation and germination but are necessary for heat resistance. Thus, our study further highlights the diverse functions of DpaAB and SpoVAC in spore-forming organisms.

The metabolically dormant spore form of the major nosocomial pathogen Clostridioides difficile is its major infectious particle. However, the mechanisms controlling the formation of this resistant cell type are not well understood, particularly with respect to its outermost layer, the spore coat. We previously identified two spore-morphogenetic proteins in C. difficile: SpoIVA, which is conserved in all spore-forming organisms, and SipL, which is conserved only in the clostridia. Both SpoIVA and SipL are essential for heat-resistant spore formation and directly interact through SipL’s C-terminal LysM domain. In this study, we demonstrate that the LysM domain is critical for SipL and SpoIVA function, likely by helping recruit SipL to the forespore during spore morphogenesis. We further identified residues within the LysM domain that are important for binding SpoIVA and, thus, functional spore formation. These findings provide important insight into the molecular mechanisms controlling the assembly of infectious C. difficile spores., Spores are the major infectious particle of the Gram-positive nosocomial pathogen Clostridioides difficile (formerly Clostridium difficile), but the molecular details of how this organism forms these metabolically dormant cells remain poorly characterized. The composition of the spore coat in C. difficile differs markedly from that defined in the well-studied organism Bacillus subtilis, with only 25% of the ∼70 spore coat proteins being conserved between the two organisms and with only 2 of 9 coat assembly (morphogenetic) proteins defined in B. subtilis having homologs in C. difficile. We previously identified SipL as a clostridium-specific coat protein essential for functional spore formation. Heterologous expression analyses in Escherichia coli revealed that SipL directly interacts with C. difficile SpoIVA, a coat-morphogenetic protein conserved in all spore-forming organisms, through SipL’s C-terminal LysM domain. In this study, we show that SpoIVA-SipL binding is essential for C. difficile spore formation and identify specific residues within the LysM domain that stabilize this interaction. Fluorescence microscopy analyses indicate that binding of SipL’s LysM domain to SpoIVA is required for SipL to localize to the forespore while SpoIVA requires SipL to promote encasement of SpoIVA around the forespore. Since we also show that clostridial LysM domains are functionally interchangeable at least in C. difficile, the basic mechanism for SipL-dependent assembly of clostridial spore coats may be conserved. IMPORTANCE The metabolically dormant spore form of the major nosocomial pathogen Clostridioides difficile is its major infectious particle. However, the mechanisms controlling the formation of this resistant cell type are not well understood, particularly with respect to its outermost layer, the spore coat. We previously identified two spore-morphogenetic proteins in C. difficile: SpoIVA, which is conserved in all spore-forming organisms, and SipL, which is conserved only in the clostridia. Both SpoIVA and SipL are essential for heat-resistant spore formation and directly interact through SipL’s C-terminal LysM domain. In this study, we demonstrate that the LysM domain is critical for SipL and SpoIVA function, likely by helping recruit SipL to the forespore during spore morphogenesis. We further identified residues within the LysM domain that are important for binding SpoIVA and, thus, functional spore formation. These findings provide important insight into the molecular mechanisms controlling the assembly of infectious C. difficile spores.

The gastrointestinal pathogen, Clostridioides difficile, initiates infection when its metabolically dormant spore form germinates in the mammalian gut. While most spore-forming bacteria use transmembrane germinant receptors to sense nutrient germinants, C. difficile is thought to use the soluble pseudoprotease, CspC, to detect bile acid germinants. To gain insight into CspC’s unique mechanism of action, we solved its crystal structure. Guided by this structure, we identified CspC mutations that confer either hypo- or hyper-sensitivity to bile acid germinant. Surprisingly, hyper-sensitive CspC variants exhibited bile acid-independent germination as well as increased sensitivity to amino acid and/or calcium co-germinants. Since mutations in specific residues altered CspC’s responsiveness to these different signals, CspC plays a critical role in regulating C. difficile spore germination in response to multiple environmental signals. Taken together, these studies implicate CspC as being intimately involved in the detection of distinct classes of co-germinants in addition to bile acids and thus raises the possibility that CspC functions as a signaling node rather than a ligand-binding receptor., Author summary The major nosocomial pathogen Clostridioides difficile depends on spore germination to initiate infection. Interestingly, C. difficile’s germinant sensing mechanism differs markedly from other spore-forming bacteria, since it uses bile acids to induce germination and lacks the transmembrane germinant receptors conserved in almost all spore-forming organisms. Instead, C. difficile is thought to use CspC, a soluble pseudoprotease, to sense these unique bile acid germinants. To gain insight into how a pseudoprotease senses germinant and propagates this signal, we solved the crystal structure of C. difficile CspC. Guided by this structure, we identified mutations that alter the sensitivity of C. difficile spores to not only bile acid germinant but also to amino acid and calcium co-germinants. Taken together, our study implicates CspC in either directly or indirectly sensing these diverse small molecules and thus raises new questions regarding how C. difficile spores physically detect bile acid germinants and co-germinants.

Clostridioides (formerly Clostridium) difficile is a leading cause of healthcare-associated infections. Although considerable progress has been made in the understanding of its genome, the epigenome of C. difficile and its functional impact has not been systematically explored. Here, we perform a comprehensive DNA methylome analysis of C. difficile using 36 human isolates and observe a high level of epigenomic diversity. We discovered an orphan DNA methyltransferase with a well-defined specificity, the corresponding gene of which is highly conserved across our dataset and in all of the approximately 300 global C. difficile genomes examined. Inactivation of the methyltransferase gene negatively impacts sporulation, a key step in C. difficile disease transmission, and these results are consistently supported by multiomics data, genetic experiments and a mouse colonization model. Further experimental and transcriptomic analyses suggest that epigenetic regulation is associated with cell length, biofilm formation and host colonization. These findings provide a unique epigenetic dimension to characterize medically relevant biological processes in this important pathogen. This study also provides a set of methods for comparative epigenomics and integrative analysis, which we expect to be broadly applicable to bacterial epigenomic studies. In this work, Fang et al. analyse the epigenetic landscape of Clostridioides difficile and identify a DNA methyltransferase present across C. difficile strains that is required for optimal sporulation and in vivo colonization and disease.

Sporulation allows bacteria to survive adverse conditions and is essential to the lifecycle of some obligate anaerobes. In Bacillus subtilis, the sporulation-specific sigma factors, σ(F), σ(E), σ(G), and σ(K), activate compartment-specific transcriptional programs that drive sporulation through its morphological stages. The regulation of these sigma factors was predicted to be conserved across the Firmicutes, since the regulatory proteins controlling their activation are largely conserved. However, recent studies in (Pepto)Clostridium difficile, Clostridium acetobutylicum, Clostridium perfringens, and Clostridium botulinum have revealed striking differences in the order, activation, and function of sporulation sigma factors. These studies indicate that gene conservation does not necessarily predict gene function and that new mechanisms for controlling cell fate determination remain to be discovered in the anaerobic Clostridia.

The ability of clonal bacterial populations to generate genomic and phenotypic heterogeneity is thought to be of great importance for many commensal and pathogenic bacteria. One common mechanism contributing to diversity formation relies on the inversion of small genomic DNA segments in a process commonly referred to as conservative site-specific recombination. This phenomenon is known to occur in several bacterial lineages, however it remains notoriously difficult to identify due to the lack of conserved features. Here, we report an easy-to-implement method based on high-throughput paired-end sequencing for genome-wide detection of conservative site-specific recombination on a single-nucleotide level. We demonstrate the effectiveness of the method by successfully detecting several novel inversion sites in an epidemic isolate of the enteric pathogen Clostridium difficile. Using an experimental approach, we validate the inversion potential of all detected sites in C. difficile and quantify their prevalence during exponential and stationary growth in vitro. In addition, we demonstrate that the master recombinase RecV is responsible for the inversion of some but not all invertible sites. Using a fluorescent gene-reporter system, we show that at least one gene from a two-component system located next to an invertible site is expressed in an on-off mode reminiscent of phase variation. We further demonstrate the applicability of our method by mining 209 publicly available sequencing datasets and show that conservative site-specific recombination is common in the bacterial realm but appears to be absent in some lineages. Finally, we show that the gene content associated with the inversion sites is diverse and goes beyond traditionally described surface components. Overall, our method provides a robust platform for detection of conservative site-specific recombination in bacteria and opens a new avenue for global exploration of this important phenomenon., Author summary Bacteria in many ecological niches experience a common challenge in the form of unpredictable environmental fluctuations. Rapid adaptation to challenging conditions is important for bacterial survival and successful proliferation. Altering gene expression through DNA inversion is a common mechanism adopted by many bacterial species that allows quick generation of distinct subpopulations with altered fitness. The characterization of these systems beyond a few classical cases is lagging due to the difficulties to accurately detect such inversion on a population level. In this study, we implement an easy-to-use method for detecting small genomic inversions in bacterial genomes. We successfully applied our approach to detect known and novel inversion sites in C. difficile. We further show that all detected sites undergo inversion and exist at different frequencies in vitro. The inversion of several sites seems dependent on the master recombinase RecV. We expand our analysis to a large collection of bacterial and archaeal strains and show that our method can be globally applied for detection of small genomic inversions. Taken together, this study advances the ability to characterize this important phenomenon.

Clostridium difficile causes considerable health care-associated gastrointestinal disease that is transmitted by its metabolically dormant spore form. Upon entering the gut, C. difficile spores germinate and outgrow to produce vegetative cells that release disease-causing toxins. C. difficile spore germination depends on the Csp family of (pseudo)proteases and the cortex hydrolase SleC. The CspC pseudoprotease functions as a bile salt germinant receptor that activates the protease CspB, which in turn proteolytically activates the SleC zymogen. Active SleC degrades the protective cortex layer, allowing spores to outgrow and resume metabolism. We previously showed that the CspA pseudoprotease domain, which is initially produced as a fusion to CspB, controls the incorporation of the CspC germinant receptor in mature spores. However, study of the individual Csp proteins has been complicated by the polar effects of TargeTron-based gene disruption on the cspBA-cspC operon. To overcome these limitations, we have used pyrE -based allelic exchange to create individual deletions of the regions encoding CspB, CspA, CspBA, and CspC in strain 630Δ erm . Our results indicate that stable CspA levels in sporulating cells depend on CspB and confirm that CspA maximizes CspC incorporation into spores. Interestingly, we observed that csp and sleC mutants spontaneously germinate more frequently in 630Δ erm than equivalent mutants in the JIR8094 and UK1 strain backgrounds. Analyses of this phenomenon suggest that only a subpopulation of C. difficile 630Δ erm spores can spontaneously germinate, in contrast with Bacillus subtilis spores. We also show that C. difficile clinical isolates that encode truncated CspBA variants have sequencing errors that actually produce full-length CspBA. IMPORTANCE Clostridium difficile is a leading cause of health care-associated infections. Initiation of C. difficile infection depends on spore germination, a process controlled by Csp family (pseudo)proteases. The CspC pseudoprotease is a germinant receptor that senses bile salts and activates the CspB protease, which activates a hydrolase required for germination. Previous work implicated the CspA pseudoprotease in controlling CspC incorporation into spores but relied on plasmid-based overexpression. Here we have used allelic exchange to study the functions of CspB and CspA. We determined that CspA production and/or stability depends on CspB and confirmed that CspA maximizes CspC incorporation into spores. Our data also suggest that a subpopulation of C. difficile spores spontaneously germinates in the absence of bile salt germinants and/or Csp proteins.

Clostridium difficile is a Gram-positive spore-forming obligate anaerobe that is a leading cause of antibiotic-associated diarrhea worldwide. In order for C. difficile to initiate infection, its aerotolerant spore form must germinate in the gut of mammalian hosts. While almost all spore-forming organisms use transmembrane germinant receptors to trigger germination, C. difficile uses the pseudoprotease CspC to sense bile salt germinants. CspC activates the related subtilisin-like protease CspB, which then proteolytically activates the cortex hydrolase SleC. Activated SleC degrades the protective spore cortex layer, a step that is essential for germination to proceed. Since CspC incorporation into spores also depends on CspA, a related pseudoprotease domain, Csp family proteins play a critical role in germination. However, how Csps are incorporated into spores remains unknown. In this study, we demonstrate that incorporation of the CspC, CspB, and CspA germination regulators into spores depends on CD0311 (renamed GerG), a previously uncharacterized hypothetical protein. The reduced levels of Csps in gerG spores correlate with reduced responsiveness to bile salt germinants and increased germination heterogeneity in single-spore germination assays. Interestingly, asparagine-rich repeat sequences in GerG’s central region facilitate spontaneous gel formation in vitro even though they are dispensable for GerG-mediated control of germination. Since GerG is found exclusively in C. difficile, our results suggest that exploiting GerG function could represent a promising avenue for developing C. difficile-specific anti-infective therapies., IMPORTANCE The spore-forming bacterium Clostridium difficile is a leading cause of health care-associated infections. While a subset of antibiotics can treat C. difficile infections (CDIs), the primary determinant of CDI disease susceptibility is prior antibiotic exposure, since it reduces the colonization resistance conferred by a diverse microflora. Thus, therapies that minimize perturbations to the gut microbiome should be more effective at reducing CDIs and their recurrence, the main source of disease complications. Given that spore germination is essential for C. difficile to initiate infection and that C. difficile uses a unique pathway to initiate germination, methods that inhibit distinct elements of germination could selectively prevent C. difficile disease recurrence. Here, we identify GerG as a C. difficile-specific protein that controls the incorporation of germinant signaling proteins into spores. Since gerG mutant spores exhibit germination defects and are less responsive to germinant, GerG may represent a promising target for developing therapeutics against CDI.

The Gram-positive nosocomial pathogen Clostridium difficile induces sporulation during growth in the gastrointestinal tract. Sporulation is necessary for this obligate anaerobe to form metabolically dormant spores that can resist antibiotic treatment, survive exit from the mammalian host, and transmit C. difficile infections. In this chapter, we describe a method for inducing C. difficile sporulation in vitro. This method can be used to study sporulation and maximize spore purification yields for a number of C. difficile strain backgrounds. We also describe procedures for visualizing spore formation using phase-contrast microscopy and for quantifying the efficiency of sporulation using heat resistance as a measure of functional spore formation.

An internal cysteine protease domain (CPD) autoproteolytically regulates Clostridium difficile glucosylating toxins by releasing a cytotoxic effector domain into target cells. CPD activity is itself allosterically regulated by the eukaryotic-specific molecule inositol hexakisphosphate (InsP6). Although allostery controls the function of most proteins, the molecular details underlying this regulatory mechanism are often difficult to characterize. Here we use chemical probes to show that apo-CPD is in dynamic equilibrium between active and inactive states. InsP6 dramatically shifts this equilibrium towards an active conformer that is further restrained upon binding a suicide substrate. Structural analyses combined with systematic mutational and disulfide bond engineering studies reveal that residues within a β-hairpin region functionally couple the InsP6 binding site to the active site. Collectively, our results identify an allosteric circuit that allows bacterial virulence factors to sense and respond to the eukaryotic environment.

The MogR transcriptional repressor of the intracellular pathogen Listeria monocytogenes recognizes AT-rich binding sites in promoters of flagellar genes to downregulate flagellar gene expression during infection. We describe here the 1.8 A resolution crystal structure of MogR bound to the recognition sequence 5' ATTTTTTAAAAAAAT 3' present within the flaA promoter region. Our structure shows that MogR binds as a dimer. Each half-site is recognized in the major groove by a helix-turn-helix motif and in the minor groove by a loop from the symmetry-related molecule, resulting in a "crossover" binding mode. This oversampling through minor groove interactions is important for specificity. The MogR binding site has structural features of A-tract DNA and is bent by approximately 52 degrees away from the dimer. The structure explains how MogR achieves binding specificity in the AT-rich genome of L. monocytogenes and explains the evolutionary conservation of A-tract sequence elements within promoter regions of MogR-regulated flagellar genes.

Vibrio cholerae RTX (repeats in toxin) is an actin-disrupting toxin that is autoprocessed by an internal cysteine protease domain (CPD). The RTX CPD is efficiently activated by the eukaryote-specific small molecule inositol hexakisphosphate (InsP 6 ), and we present the 2.1 angstrom structure of the RTX CPD in complex with InsP 6 . InsP 6 binds to a conserved basic cleft that is distant from the protease active site. Biochemical and kinetic analyses of CPD mutants indicate that InsP 6 binding induces an allosteric switch that leads to the autoprocessing and intracellular release of toxin-effector domains.

Sporulation is an ancient developmental process that involves the formation of a highly resistant endospore within a larger mother cell. In the model organism Bacillus subtilis, sporulation-specific sigma factors activate compartment-specific transcriptional programs that drive spore morphogenesis. σG activity in the forespore depends on the formation of a secretion complex, known as the “feeding tube,” that bridges the mother cell and forespore and maintains forespore integrity. Even though these channel components are conserved in all spore formers, recent studies in the major nosocomial pathogen Clostridium difficile suggested that these components are dispensable for σG activity. In this study, we investigated the requirements of the SpoIIQ and SpoIIIA proteins during C. difficile sporulation. C. difficile spoIIQ, spoIIIA, and spoIIIAH mutants exhibited defects in engulfment, tethering of coat to the forespore, and heat-resistant spore formation, even though they activate σG at wildtype levels. Although the spoIIQ, spoIIIA, and spoIIIAH mutants were defective in engulfment, metabolic labeling studies revealed that they nevertheless actively transformed the peptidoglycan at the leading edge of engulfment. In vitro pull-down assays further demonstrated that C. difficile SpoIIQ directly interacts with SpoIIIAH. Interestingly, mutation of the conserved Walker A ATP binding motif, but not the Walker B ATP hydrolysis motif, disrupted SpoIIIAA function during C. difficile spore formation. This finding contrasts with B. subtilis, which requires both Walker A and B motifs for SpoIIIAA function. Taken together, our findings suggest that inhibiting SpoIIQ, SpoIIIAA, or SpoIIIAH function could prevent the formation of infectious C. difficile spores and thus disease transmission., Author Summary The bacterial spore-forming pathogen Clostridium difficile is a leading cause of nosocomial infections in the United States and represents a significant threat to healthcare systems around the world. As an obligate anaerobe, C. difficile must form spores in order to survive exit from the gastrointestinal tract. Accordingly, spore formation is essential for C. difficile disease transmission. Since the mechanisms controlling this process remain poorly characterized, we analyzed the importance of highly conserved secretion channel components during C. difficile sporulation. In the model organism Bacillus subtilis, this channel had previously been shown to function as a “feeding tube” that allows the mother cell to nurture the developing forespore and sustain transcription in the forespore. We show here that conserved components of this structure in C. difficile are dispensable for forespore transcription, although they are important for completing forespore engulfment and retaining the protective spore coat around the forespore, in contrast with B. subtilis. The results of our study suggest that targeting these conserved proteins could prevent C. difficile spore formation and thus disease transmission.

Flagellar motility is an essential mechanism by which bacteria adapt to and survive in diverse environments. Although flagella confer an advantage to many bacterial pathogens for colonization during infection, bacterial flagellins also stimulate host innate immune responses. Consequently, many bacterial pathogens down-regulate flagella production following initial infection. Listeria monocytogenes is a facultative intracellular pathogen that represses transcription of flagellar motility genes at physiological temperatures (37°C and above). Temperature-dependent expression of flagellar motility genes is mediated by the opposing activities of MogR, a DNA-binding transcriptional repressor, and DegU, a response regulator that functions as an indirect antagonist of MogR. In this study, we identify an additional component of the molecular circuitry governing temperature-dependent flagellar gene expression. At low temperatures (30°C and below), MogR repression activity is specifically inhibited by an anti-repressor, GmaR. We demonstrate that GmaR forms a stable complex with MogR, preventing MogR from binding its DNA target sites. GmaR anti-repression activity is temperature dependent due to DegU-dependent transcriptional activation of gmaR at low temperatures. Thus, GmaR production represents the first committed step for flagella production in L. monocytogenes. Interestingly, GmaR also functions as a glycosyltransferase exhibiting O-linked N-acetylglucosamine transferase (OGT) activity for flagellin (FlaA). GmaR is the first OGT to be identified and characterized in prokaryotes that specifically β-O-GlcNAcylates a prokaryotic protein. Unlike the well-characterized, highly conserved OGT regulatory protein in eukaryotes, the catalytic activity of GmaR is functionally separable from its anti-repression function. These results establish GmaR as the first known example of a bifunctional protein that transcriptionally regulates expression of its enzymatic substrate.

Bacterial pathogens frequently use protein secretion to mediate interactions with their hosts. Here we found that a virulence locus (HSI-I) of Pseudomonas aeruginosa encodes a protein secretion apparatus. The apparatus assembled in discrete subcellular locations and exported Hcp1, a hexameric protein that forms rings with a 40 angstrom internal diameter. Regulatory patterns of HSI-I suggested that the apparatus functions during chronic infections. We detected Hcp1 in pulmonary secretions of cystic fibrosis (CF) patients and Hcp1-specific antibodies in their sera. Thus, HSI-I likely contributes to the pathogenesis of P. aeruginosa in CF patients. HSI-I–related loci are widely distributed among bacterial pathogens and may play a general role in mediating host interactions.

The spore-forming bacterial pathogen Clostridium difficile is a leading cause of health-care-associated diarrhea worldwide. Although C. difficile spore formation is essential for disease transmission, the regulatory pathways that control this developmental process have only been partially characterized. In the well-studied spore-former Bacillus subtilis, the highly conserved σ(E) , SpoIIID and σ(K) regulatory proteins control gene expression in the mother cell to ensure proper spore formation. To define the precise requirement for SpoIIID and σ(K) during C. difficile sporulation, we analyzed spoIIID and sigK mutants using heterologous expression systems and RNA-Seq transcriptional profiling. These analyses revealed that expression of sigK from a SpoIIID-independent promoter largely bypasses the need for SpoIIID to produce heat-resistant spores. We also observed that σ(K) is active upon translation, suggesting that SpoIIID primarily functions to activate sigK. SpoIIID nevertheless plays auxiliary roles during sporulation, as it enhances levels of the exosporium morphogenetic protein CdeC in a σ(K) -dependent manner. Analyses of purified spores further revealed that SpoIIID and σ(K) control the adherence of the CotB coat protein to C. difficile spores, indicating that these proteins regulate multiple stages of spore formation. Collectively, these results highlight that diverse mechanisms control spore formation in the Firmicutes.

Clostridium difficile is a major nosocomial pathogen whose infections are difficult to treat because of their frequent recurrence. The spores of C. difficile are responsible for these clinical features, as they resist common disinfectants and antibiotic treatment. Although spores are the major transmissive form of C. difficile, little is known about their composition or morphogenesis. Spore morphogenesis has been well characterized for Bacillus sp., but Bacillus sp. spore coat proteins are poorly conserved in Clostridium sp. Of the known spore morphogenetic proteins in Bacillus subtilis, SpoIVA is one of the mostly highly conserved in the Bacilli and the Clostridia. Using genetic analyses, we demonstrate that SpoIVA is required for proper spore morphogenesis in C. difficile. In particular, a spoIVA mutant exhibits defects in spore coat localization but not cortex formation. Our study also identifies SipL, a previously uncharacterized protein found in proteomic studies of C. difficile spores, as another critical spore morphogenetic protein, since a sipL mutant phenocopies a spoIVA mutant. Biochemical analyses and mutational analyses indicate that SpoIVA and SipL directly interact. This interaction depends on the Walker A ATP binding motif of SpoIVA and the LysM domain of SipL. Collectively, these results provide the first insights into spore morphogenesis in C. difficile.

Spores are the major transmissive form of the nosocomial pathogen Clostridium difficile, a leading cause of healthcare-associated diarrhea worldwide. Successful transmission of C. difficile requires that its hardy, resistant spores germinate into vegetative cells in the gastrointestinal tract. A critical step during this process is the degradation of the spore cortex, a thick layer of peptidoglycan surrounding the spore core. In Clostridium sp., cortex degradation depends on the proteolytic activation of the cortex hydrolase, SleC. Previous studies have implicated Csps as being necessary for SleC cleavage during germination; however, their mechanism of action has remained poorly characterized. In this study, we demonstrate that CspB is a subtilisin-like serine protease whose activity is essential for efficient SleC cleavage and C. difficile spore germination. By solving the first crystal structure of a Csp family member, CspB, to 1.6 Å, we identify key structural domains within CspB. In contrast with all previously solved structures of prokaryotic subtilases, the CspB prodomain remains tightly bound to the wildtype subtilase domain and sterically occludes a catalytically competent active site. The structure, combined with biochemical and genetic analyses, reveals that Csp proteases contain a unique jellyroll domain insertion critical for stabilizing the protease in vitro and in C. difficile. Collectively, our study provides the first molecular insight into CspB activity and function. These studies may inform the development of inhibitors that can prevent clostridial spore germination and thus disease transmission., Author Summary Clostridium difficile is the leading cause of health-care associated diarrhea worldwide. C. difficile infections begin when its spores transform into vegetative cells during a process called germination. In Clostridium sp., germination requires that the spore cortex, a thick, protective layer, be removed by the cortex hydrolase SleC. While previous studies have shown that SleC activity depends on a subtilisin-like protease, CspB, the mechanisms regulating CspB function have not been characterized. In this study, we solved the first crystal structure of the Csp family of proteases and identified its key functional regions. We determined that CspB carries a unique jellyroll domain required for stabilizing the protein both in vitro and in C. difficile and a prodomain required for proper folding of the protease. Unlike all other prokaryotic subtilisin-like proteases, the prodomain remains bound to CspB and inhibits its protease activity until the germination signal is sensed. Our study provides new insight into how germination is regulated in C. difficile and may inform the development of inhibitors that can prevent germination and thus C. difficile transmission.

TcdB, an intracellular bacterial toxin that inactivates small GTPases, is a major Clostridium difficile virulence factor. Recent studies have found that TcdB produced by emerging/hypervirulent strains of C. difficile is more potent than TcdB from historical strains, and in the current work, studies were performed to investigate the underlying mechanisms for this change in TcdB toxicity. Using a series of biochemical analyses we found that TcdB from a hypervirulent strain (TcdB(HV) ) was more efficient at autoprocessing than TcdB from a historical strain (TcdB(HIST) ). TcdB(HV) and TcdB(HIST) were activated by similar concentrations of IP6; however, the overall efficiency of processing was 20% higher for TcdB(HV) . Using an activity-based fluorescent probe (AWP19) an intermediate, activated but uncleaved, form of TcdB(HIST) was identified, while only a processed form of TcdB(HV) could be detected under the same conditions. Using a much higher concentration (200 µM) of the probe revealed an activated uncleaved form of TcdB(HV) , indicating a preferential and more efficient engagement of intramolecular substrate than TcdB(HIST) . Furthermore, a peptide-based inhibitor (Ac-GSL-AOMK) was found to block the cytotoxicity of TcdB(HIST) at a lower concentration than required to inhibit TcdB(HV) . These findings suggest that TcdB(HV) may cause increased cytotoxicity due to more efficient autoprocessing.

Clostridium difficile is a Gram-positive spore-forming pathogen and a leading cause of nosocomial diarrhea. C. difficile infections are transmitted when ingested spores germinate in the gastrointestinal tract and transform into vegetative cells. Germination begins when the germinant receptor CspC detects bile salts in the gut. CspC is a subtilisin-like serine pseudoprotease that activates the related CspB serine protease through an unknown mechanism. Activated CspB cleaves the pro-SleC zymogen, which allows the activated SleC cortex hydrolase to degrade the protective cortex layer. While these regulators are essential for C. difficile spores to outgrow and form toxin-secreting vegetative cells, the mechanisms controlling their function have only been partially characterized. In this study, we identify the lipoprotein GerS as a novel regulator of C. difficile spore germination using targeted mutagenesis. A gerS mutant has a severe germination defect and fails to degrade cortex even though it processes SleC at wildtype levels. Using complementation analyses, we demonstrate that GerS secretion, but not lipidation, is necessary for GerS to activate SleC. Importantly, loss of GerS attenuates the virulence of C. difficile in a hamster model of infection. Since GerS appears to be conserved exclusively in related Peptostreptococcaeace family members, our results contribute to a growing body of work indicating that C. difficile has evolved distinct mechanisms for controlling the exit from dormancy relative to B. subtilis and other spore-forming organisms., Author Summary Clostridium difficile is a spore-forming bacterium capable of causing severe diarrhea. The dormant spore-form of C. difficile is necessary to cause infection, since vegetative cells of this organism cannot survive in the presence of oxygen. Spores are difficult to eradicate because they can withstand extreme environmental conditions and chemical insults including antibiotics. However, since spores cannot grow, they must transform back into actively replicating cells once the appropriate environmental conditions are sensed through a process called germination. A key step during germination is the break-down of a specialized cell wall layer in the spore known as cortex by the SleC hydrolase. In this paper, we identify GerS as a novel lipid-modified protein that is important for C. difficile germination to occur. GerS is made at high levels during spore formation and gets packaged into mature spores. We show that GerS is required for the cortex hydrolase SleC to degrade the protective cortex layer, since a strain lacking GerS does not lose its cortex layer. Loss of GerS prevents C. difficile from causing infection in a hamster model of infection, suggesting that GerS is a novel target for drug development.

Cystatins are cysteine protease inhibitors that are at the front-line of defense against pathogens that secrete proteases as virulence factors. In this issue, Vincents et al. (2008) reveal how the bacterial protease IdeS from Streptococcus pyogenes hijacks normal cystatin C function to convert it into a cofactor that enhances proteolytic destruction of host-defense antibodies.

Clostridium difficile is a spore-forming obligate anaerobe that is a leading cause of healthcare-associated infections. C. difficile infections begin when its metabolically dormant spores germinate in the gut of susceptible individuals. Binding of bile salt germinants to the Csp family pseudoprotease CspC triggers a proteolytic signaling cascade consisting of the Csp family protease CspB and the cortex hydrolase SleC. Conserved across many of the Clostridia, Csp proteases are subtilisin-like serine proteases that activate pro-SleC by cleaving off its inhibitory pro-peptide. Active SleC degrades the protective cortex layer, allowing spores to resume metabolism and growth. This signaling pathway, however, is differentially regulated in C. difficile, since CspC functions both as a germinant receptor and regulator of CspB activity. CspB is also produced as a fusion to a catalytically inactive CspA domain that subsequently undergoes interdomain processing during spore formation. In this study, we investigated the role of the CspA pseudoprotease domain in regulating C. difficile spore germination. Mutational analyses revealed that the CspA domain controls CspC germinant receptor levels in mature spores and is required for optimal spore germination, particularly when CspA is fused to the CspB protease. During spore formation, the YabG protease separates these domains, although YabG itself is dispensable for germination. Bioinformatic analyses of Csp family members suggest that the CspC-regulated signaling pathway characterized in C. difficile is conserved in related Peptostreptococcaceae family members but not in the Clostridiaceae or Lachnospiraceae. Our results indicate that pseudoproteases play critical roles in regulating C. difficile spore germination and highlight that diverse mechanisms control spore germination in the Clostridia.

SummaryClostridium difficile is a leading cause of nosocomial infections. The major virulence factors of this pathogen are the multi-domain toxins TcdA and TcdB. These toxins contain a cysteine protease domain (CPD) that autoproteolytically releases a cytotoxic effector domain upon binding intracellular inositol hexakisphosphate. Currently, there are no known inhibitors of this protease. Here, we describe the rational design of covalent small molecule inhibitors of TcdB CPD. We identified compounds that inactivate TcdB holotoxin function in cells and solved the structure of inhibitor-bound protease to 2.0 Å. This structure reveals the molecular basis of CPD substrate recognition and informed the synthesis of activity-based probes for this enzyme. The inhibitors presented will guide the development of therapeutics targeting C. difficile, and the probes will serve as tools for studying the unique activation mechanism of bacterial toxin CPDs.

International audience; The strict anaerobe Clostridium difficile is the most common cause of antibiotic-associated diarrhoea. The oxygen-resistant C. difficile spores play a central role in the infectious cycle, contributing to transmission, infection and recurrence. The spore surface layers, the coat and exosporium, enable the spores to resist physical and chemical stress. However, little is known about the mechanisms of their assembly. In this study, we characterized a new spore protein, CotL, which is required for the assembly of the spore coat. The cotL gene was expressed in the mother cell compartment under the dual control of the RNA polymerase sigma factors, σE and σK . CotL was localized in the spore coat, and the spores of the cotL mutant had a major morphologic defect at the level of the coat/exosporium layers. Therefore, the mutant spores contained a reduced amount of several coat/exosporium proteins and a defect in their localization in sporulating cells. Finally, cotL mutant spores were more sensitive to lysozyme and were impaired in germination, a phenotype likely to be associated with the structurally altered coat. Collectively, these results strongly suggest that CotL is a morphogenetic protein essential for the assembly of the spore coat in C. difficile.

Clostridium difficile is the primary cause of nosocomial diarrhea and pseudomembranous colitis. It produces dormant spores, which serve as an infectious vehicle responsible for transmission of the disease and persistence of the organism in the environment. In Bacillus subtilis, the sin locus coding SinR (113 aa) and SinI (57 aa) is responsible for sporulation inhibition. In B. subtilis, SinR mainly acts as a repressor of its target genes to control sporulation, biofilm formation, and autolysis. SinI is an inhibitor of SinR, so their interaction determines whether SinR can inhibit its target gene expression. The C. difficile genome carries two sinR homologs in the operon that we named sinR and sinR’, coding for SinR (112 aa) and SinR’ (105 aa), respectively. In this study, we constructed and characterized sin locus mutants in two different C. difficile strains R20291 and JIR8094, to decipher the locus’s role in C. difficile physiology. Transcriptome analysis of the sinRR’ mutants revealed their pleiotropic roles in controlling several pathways including sporulation, toxin production, and motility in C. difficile. Through various genetic and biochemical experiments, we have shown that SinR can regulate transcription of key regulators in these pathways, which includes sigD, spo0A, and codY. We have found that SinR’ acts as an antagonist to SinR by blocking its repressor activity. Using a hamster model, we have also demonstrated that the sin locus is needed for successful C. difficile infection. This study reveals the sin locus as a central link that connects the gene regulatory networks of sporulation, toxin production, and motility; three key pathways that are important for C. difficile pathogenesis., Author summary In Bacillus subtilis, sporulation, competence and biofilm formation are regulated by a pleiotropic regulator called SinR. Two sinR homologs are present in C. difficile genome as an operon and henceforth labeled as sinR and sinR’. Our detailed investigation revealed that in C. difficile, the SinR and SinR’ are key master regulators needed for the regulation of several pathways including sporulation, toxin production, and motility.