TFPI is a colonic crypt receptor for TcdB from hypervirulent clade 2 C. difficile.

The emergence of hypervirulent clade 2 Clostridioides difficile is associated with severe symptoms and accounts for >20% of global infections. TcdB is a dominant virulence factor of C. difficile, and clade 2 strains exclusively express two TcdB variants (TcdB2 and TcdB4) that use unknown receptors distinct from the classic TcdB. Here, we performed CRISPR/Cas9 screens for TcdB4 and identified tissue factor pathway inhibitor (TFPI) as its receptor. Using cryo-EM, we determined a complex structure of the full-length TcdB4 with TFPI, defining a common receptor-binding region for TcdB. Residue variations within this region divide major TcdB variants into 2 classes: one recognizes Frizzled (FZD), and the other recognizes TFPI. TFPI is highly expressed in the intestinal glands, and recombinant TFPI protects the colonic epithelium from TcdB2/4. These findings establish TFPI as a colonic crypt receptor for TcdB from clade 2 C. difficile and reveal new mechanisms for CDI pathogenesis.

A network of small RNAs regulates sporulation initiation in Clostridioides difficile.

The obligate anaerobic, enteric pathogen Clostridioides difficile persists in the intestinal tract by forming antibiotic-resistant endospores that contribute to relapsing and recurrent infections. Despite the importance of sporulation for C. difficile pathogenesis, environmental cues and molecular mechanisms that regulate sporulation initiation remain ill-defined. Here, by using RIL-seq to globally capture the Hfq-dependent RNA-RNA interactome, we discovered a network of small RNAs that bind to mRNAs encoding sporulation-related genes. We show that two of these small RNAs, SpoX and SpoY, regulate translation of the master regulator of sporulation, Spo0A, in an opposing manner, which ultimately leads to altered sporulation rates. Infection of antibiotic-treated mice with SpoX and SpoY deletion mutants revealed a global effect on gut colonization and intestinal sporulation. Our work uncovers an elaborate RNA-RNA interactome controlling the physiology and virulence of C. difficile and identifies a complex post-transcriptional layer in the regulation of spore formation in this important human pathogen.

Deficiency of IL-22-binding protein enhances the ability of the gut microbiota to protect against enteric pathogens.

Interleukin 22 (IL-22) promotes intestinal barrier integrity, stimulating epithelial cells to enact defense mechanisms against enteric infections, including the production of antimicrobial peptides. IL-22 binding protein (IL-22BP) is a soluble decoy encoded by the Il22ra2 gene that decreases IL-22 bioavailability, attenuating IL-22 signaling. The impact of IL-22BP on gut microbiota composition and functioning is poorly understood. We found that Il22ra2-/- mice are better protected against Clostridioides difficile and Citrobacter rodentium infections. This protection relied on IL-22-induced antimicrobial mechanisms before the infection occurred, rather than during the infection itself. Indeed, the gut microbiota of Il22ra2-/- mice mitigated infection of wild-type (WT) mice when transferred via cohousing or by cecal microbiota transplantation. Indicator species analysis of WT and Il22ra2-/- mice with and without cohousing disclosed that IL22BP deficiency yields a gut bacterial composition distinct from that of WT mice. Manipulation of dietary fiber content, measurements of intestinal short-chain fatty acids and oral treatment with acetate disclosed that resistance to C. difficile infection is related to increased production of acetate by Il22ra2-/--associated microbiota. Together, these findings suggest that IL-22BP represents a potential therapeutic target for those at risk for or with already manifest infection with this and perhaps other enteropathogens.

A dual-action antibiotic that kills Clostridioides difficile vegetative cells and inhibits spore germination.

Clostridioides difficile infection (CDI) is the most lethal of the five CDC urgent public health treats, resulting in 12,800 annual deaths in the United States alone [Antibiotic Resistance Threats in the United States, 2019 (2019), www.cdc.gov/DrugResistance/Biggest-Threats.html]. The high recurrence rate and the inability of antibiotics to treat such infections mandate discovery of new therapeutics. A major challenge with CDI is the production of spores, leading to multiple recurrences of infection in 25% of patients [C. P. Kelly, J. T. LaMont, N. Engl. J. Med. 359, 1932-1940 (2008)], with potentially lethal consequence. Herein, we describe the discovery of an oxadiazole as a bactericidal anti-C. difficile agent that inhibits both cell-wall peptidoglycan biosynthesis and spore germination. We document that the oxadiazole binds to the lytic transglycosylase SleC and the pseudoprotease CspC for prevention of spore germination. SleC degrades the cortex peptidoglycan, a critical step in the initiation of spore germination. CspC senses germinants and cogerminants. Binding to SleC is with higher affinity than that to CspC. Prevention of spore germination breaks the nefarious cycles of CDI recurrence in the face of the antibiotic challenge, which is a primary cause of therapeutic failure. The oxadiazole exhibits efficacy in a mouse model of recurrent CDI and holds promise in clinical treatment of CDI.

Clostridioides difficile infection: history, epidemiology, risk factors, prevention, clinical manifestations, treatment, and future options.

SUMMARYClostridioides difficile infection (CDI) is one of the major issues in nosocomial infections. This bacterium is constantly evolving and poses complex challenges for clinicians, often encountered in real-life scenarios. In the face of CDI, we are increasingly equipped with new therapeutic strategies, such as monoclonal antibodies and live biotherapeutic products, which need to be thoroughly understood to fully harness their benefits. Moreover, interesting options are currently under study for the future, including bacteriophages, vaccines, and antibiotic inhibitors. Surveillance and prevention strategies continue to play a pivotal role in limiting the spread of the infection. In this review, we aim to provide the reader with a comprehensive overview of epidemiological aspects, predisposing factors, clinical manifestations, diagnostic tools, and current and future prophylactic and therapeutic options for C. difficile infection.

Fecal microbiota transplantation: current challenges and future landscapes.

SUMMARYGiven the importance of gut microbial homeostasis in maintaining health, there has been considerable interest in developing innovative therapeutic strategies for restoring gut microbiota. One such approach, fecal microbiota transplantation (FMT), is the main "whole gut microbiome replacement" strategy and has been integrated into clinical practice guidelines for treating recurrent Clostridioides difficile infection (rCDI). Furthermore, the potential application of FMT in other indications such as inflammatory bowel disease (IBD), metabolic syndrome, and solid tumor malignancies is an area of intense interest and active research. However, the complex and variable nature of FMT makes it challenging to address its precise functionality and to assess clinical efficacy and safety in different disease contexts. In this review, we outline clinical applications, efficacy, durability, and safety of FMT and provide a comprehensive assessment of its procedural and administration aspects. The clinical applications of FMT in children and cancer immunotherapy are also described. We focus on data from human studies in IBD in contrast with rCDI to delineate the putative mechanisms of this treatment in IBD as a model, including colonization resistance and functional restoration through bacterial engraftment, modulating effects of virome/phageome, gut metabolome and host interactions, and immunoregulatory actions of FMT. Furthermore, we comprehensively review omics technologies, metagenomic approaches, and bioinformatics pipelines to characterize complex microbial communities and discuss their limitations. FMT regulatory challenges, ethical considerations, and pharmacomicrobiomics are also highlighted to shed light on future development of tailored microbiome-based therapeutics.

Clostridioides difficile canonical L,D-transpeptidases catalyze a novel type of peptidoglycan cross-links and are not required for beta-lactam resistance.

Clostridioides difficile is the leading cause of antibiotic-associated diarrhea worldwide with significant morbidity and mortality. This organism is naturally resistant to several beta-lactam antibiotics that inhibit the polymerization of peptidoglycan, an essential component of the bacteria cell envelope. Previous work has revealed that C. difficile peptidoglycan has an unusual composition. It mostly contains 3-3 cross-links, catalyzed by enzymes called L,D-transpeptidases (Ldts) that are poorly inhibited by beta-lactams. It was therefore hypothesized that peptidoglycan polymerization by these enzymes could underpin antibiotic resistance. Here, we investigated the catalytic activity of the three canonical Ldts encoded by C. difficile (LdtCd1, LdtCd2, and LdtCd3) in vitro and explored their contribution to growth and antibiotic resistance. We show that two of these enzymes catalyze the formation of novel types of peptidoglycan cross-links using meso-diaminopimelic acid both as a donor and an acceptor, also observed in peptidoglycan sacculi. We demonstrate that the simultaneous deletion of these three genes only has a minor impact on both peptidoglycan structure and resistance to beta-lactams. This unexpected result therefore implies that the formation of 3-3 peptidoglycan cross-links in C. difficile is catalyzed by as yet unidentified noncanonical Ldt enzymes.

Tyrosine-modifying glycosylation by Yersinia effectors.

Mono-O-glycosylation of target proteins by bacterial toxins or effector proteins is a well-known mechanism by which bacteria interfere with essential functions of host cells. The respective glycosyltransferases are important virulence factors such as the Clostridioides difficile toxins A and B. Here, we describe two glycosyltransferases of Yersinia species that have a high sequence identity: YeGT from the zoonotic pathogen Yersinia enterocolitica and YkGT from the murine pathogen Yersinia kristensenii. We show that both modify Rho family proteins by attachment of GlcNAc at tyrosine residues (Tyr-34 in RhoA). Notably, the enzymes differed in their target protein specificity. While YeGT modified RhoA, B, and C, YkGT possessed a broader substrate spectrum and glycosylated not only Rho but also Rac and Cdc42 subfamily proteins. Mutagenesis studies indicated that residue 177 is important for this broader target spectrum. We determined the crystal structure of YeGT shortened by 16 residues N terminally (sYeGT) in the ligand-free state and bound to UDP, the product of substrate hydrolysis. The structure assigns sYeGT to the GT-A family. It shares high structural similarity to glycosyltransferase domains from toxins. We also demonstrated that the 16 most N-terminal residues of YeGT and YkGT are important for the mediated translocation into the host cell using the pore-forming protective antigen of anthrax toxin. Mediated introduction into HeLa cells or ectopic expression of YeGT and YkGT caused morphological changes and redistribution of the actin cytoskeleton. The data suggest that YeGT and YkGT are likely bacterial effectors belonging to the family of tyrosine glycosylating bacterial glycosyltransferases.

Proteolytic activity of surface-exposed HtrA determines its expression level and is needed to survive acidic conditions in Clostridioides difficile.

To survive in the host, pathogenic bacteria need to be able to react to the unfavorable conditions that they encounter, like low pH, elevated temperatures, antimicrobial peptides and many more. These conditions may lead to unfolding of envelope proteins and this may be lethal. One of the mechanisms through which bacteria are able to survive these conditions is through the protease/foldase activity of the high temperature requirement A (HtrA) protein. The gut pathogen Clostridioides difficile encodes one HtrA homolog that is predicted to contain a membrane anchor and a single PDZ domain. The function of HtrA in C. difficile is hitherto unknown but previous work has shown that an insertional mutant of htrA displayed elevated toxin levels, less sporulation and decreased binding to target cells. Here, we show that HtrA is membrane associated and localized on the surface of C. difficile and characterize the requirements for proteolytic activity of recombinant soluble HtrA. In addition, we show that the level of HtrA in the bacteria heavily depends on its proteolytic activity. Finally, we show that proteolytic activity of HtrA is required for survival under acidic conditions.

Histidine kinase-mediated cross-regulation of the vancomycin-resistance operon in Clostridioides difficile.

The dipeptide D-Ala-D-Ala is an essential component of peptidoglycan and the target of vancomycin. Most Clostridioides difficile strains possess the vanG operon responsible for the synthesis of D-Ala-D-Ser, which can replace D-Ala-D-Ala in peptidoglycan. The C. difficile vanG operon is regulated by a two-component system, VanRS, but is not induced sufficiently by vancomycin to confer resistance to this antibiotic. Surprisingly, in the absence of the VanS histidine kinase (HK), the vanG operon is still induced by vancomycin and also by another antibiotic, ramoplanin, in a VanR-dependent manner. This suggested the cross-regulation of VanR by another HK or kinases that are activated in the presence of certain lipid II-targeting antibiotics. We identified these HKs as CD35990 and CD22880. However, mutations in either or both HKs did not affect the regulation of the vanG operon in wild-type cells suggesting that intact VanS prevents the cross-activation of VanR by non-cognate HKs. Overproduction of VanR in the absence of VanS, CD35990, and CD22880 led to high expression of the vanG operon indicating that VanR can potentially utilize at least one more phosphate donor for its activation. Candidate targets of CD35990- and CD22880-mediated regulation in the presence of vancomycin or ramoplanin were identified by RNA-Seq.

Systems biology approach to functionally assess the Clostridioides difficile pangenome reveals genetic diversity with discriminatory power.

Combatting Clostridioides difficile infections, a dominant cause of hospital-associated infections with incidence and resulting deaths increasing worldwide, is complicated by the frequent emergence of new virulent strains. Here, we employ whole-genome sequencing, high-throughput phenotypic screenings, and genome-scale models of metabolism to evaluate the genetic diversity of 451 strains of C. difficile. Constructing the C. difficile pangenome based on this set revealed 9,924 distinct gene clusters, of which 2,899 (29%) are defined as core, 2,968 (30%) are defined as unique, and the remaining 4,057 (41%) are defined as accessory. We develop a strain typing method, sequence typing by accessory genome (STAG), that identifies 176 genetically distinct groups of strains and allows for explicit interrogation of accessory gene content. Thirty-five strains representative of the overall set were experimentally profiled on 95 different nutrient sources, revealing 26 distinct growth profiles and unique nutrient preferences; 451 strain-specific genome scale models of metabolism were constructed, allowing us to computationally probe phenotypic diversity in 28,864 unique conditions. The models create a mechanistic link between the observed phenotypes and strain-specific genetic differences and exhibit an ability to correctly predict growth in 76% of measured cases. The typing and model predictions are used to identify and contextualize discriminating genetic features and phenotypes that may contribute to the emergence of new problematic strains.

The clinical effectiveness of Fidaxomicin compared to Vancomycin in the treatment of Clostridioides difficile infection, a single center real-world experience.

BACKGROUND: The use of fidaxomicin is recommended as first line therapy for all patients with Clostridioides difficile infection (CDI). However, real-world studies have shown conflicting evidence of superiority. METHODS: We conducted a retrospective single center study of patients diagnosed with CDI between 2011-2021. A primary composite outcome of clinical failure, 30-day relapse or CDI-related death was used. A multivariable cause specific Cox proportional hazards model was used to evaluate fidaxomicin compared to vancomycin in preventing the composite outcome. A separate model was fit on a subset of patients with C. difficile ribotypes adjusting for ribotype. RESULTS: There were 598 patients included, of whom 84 received fidaxomicin. The primary outcome occurred in 8 (9.5%) in the fidaxomicin group compared to 111 (21.6%) in the vancomycin group. The adjusted multivariable model showed fidaxomicin was associated with 63% reduction in the risk of the composite outcome compared to vancomycin (HR = 0.37, 95% CI 0.17-0.80). In the 337 patients with ribotype data after adjusting for ribotype 027, the results showing superiority of fidaxomicin were maintained (HR = 0.19, 95% CI 0.05-0.77). CONCLUSION: In the treatment of CDI, we showed that real-world use of fidaxomicin is associated with lower risk of a composite endpoint of treatment failure.

Fecal Pharmacokinetics and Gut Microbiome Effects of Oral Omadacycline Versus Vancomycin in Healthy Volunteers.

BACKGROUND: Clostridioides difficile infection (CDI) is a common healthcare-associated infection with limited treatment options. Omadacycline, an aminomethylcycline tetracycline, has potent in vitro activity against C difficile and a low propensity to cause CDI in clinical trials. We aimed to assess fecal pharmacokinetics and gut microbiome effects of oral omadacycline compared to oral vancomycin in healthy adults. METHODS: This was a phase 1, nonblinded, randomized clinical trial conducted in healthy volunteers aged 18-40 years. Subjects received a 10-day course of omadacycline or vancomycin. Stool samples were collected at baseline, daily during therapy, and at follow-up visits. Omadacycline and vancomycin stool concentrations were assessed, and microbiome changes were compared. RESULTS: Sixteen healthy volunteers with a mean age of 26 (standard deviation [SD], 5) years were enrolled; 62.5% were male, and participants' mean body mass index was 23.5 (SD, 4.0) kg/m2. Omadacycline was well tolerated with no safety signal differences between the 2 antibiotics. A rapid initial increase in fecal concentrations of omadacycline was observed compared to vancomycin, with maximum concentrations achieved within 48 hours. A significant difference in alpha diversity was observed following therapy in both the omadacycline and vancomycin groups (P < .05). Bacterial abundance and beta diversity analysis showed differing microbiome changes in subjects who received omadacycline versus vancomycin. CONCLUSIONS: Subjects given omadacycline had high fecal concentrations with a distinct microbiome profile compared to vancomycin. CLINICAL TRIALS REGISTRATION: NCT06030219.

A Phase 2 Extension Study Evaluating the Immunogenicity, Safety, and Tolerability of 3 or 4 Doses of a Clostridioides difficile Vaccine in Healthy US Adults Aged 65 to 85 Years.

BACKGROUND: This phase 2 extension explored the long-term antibody persistence of an investigational Clostridioides difficile vaccine and the safety, tolerability, and immunogenicity of dose 4 approximately 12 months post-dose 3. METHODS: One year post-dose 3, healthy US 65- to 85-year-olds (N = 300) were randomized to dose 4 of vaccine at previously received antigen levels (100 or 200 µg) or placebo. Assessments included safety and percentages of participants achieving neutralizing antibody titers above prespecified thresholds (≥219 and ≥2586 neutralization units/mL for toxins A and B, respectively). RESULTS: In participants previously given three 200-µg doses and placebo in the extension, toxin A and B neutralizing antibodies were above prevaccination levels 48 months post-dose 3 (36 months after placebo); 24.0% and 26.0% had toxin A and B antibodies at or above prespecified thresholds, respectively. Neutralizing antibodies increased post-dose 4 (12 months post-dose 3) and persisted to 36 months post-dose 4. Thirty days post-dose 4, all participants had toxin A and 86.5% to 100% had toxin B titers at or above prespecified thresholds. Local reactions were more frequent in vaccine recipients. Systemic and adverse event frequencies were similar across groups. CONCLUSIONS: C difficile vaccine immune responses persisted 48 months post-dose 3. Dose 4 was immunogenic and well tolerated, supporting continued development. Clinical Trials Registration. ClinicalTrials.gov NCT02561195.

MicroRNA miR-27a-5p reduces intestinal inflammation induced by Clostridioides difficile flagella by regulating the NF-κB signaling pathway.

BACKGROUND: Clostridioides difficile is a major cause of nosocomial post-antibiotic infections, often resulting in severe inflammation and watery diarrhea. Previous studies have highlighted the role of C. difficile flagellin FliC in activating the TLR5 receptor and triggering NF-κB cell signaling, leading to the release of pro-inflammatory cytokines. However, the microRNAs (miRNAs) mediated regulatory mechanisms underlying the FliC-induced inflammatory response remain unclear. METHODS: miRNA expression levels were analyzed in Caco-2 intestinal epithelial cells following FliC stimulation, infection with the epidemic C. difficile R20291 strain, or its unflagellated mutant by RT-qPCR. Chemical inhibitors were used to block NF-κB signaling, and their impact on miR-27a-5p expression was assessed. Knockdown and overexpression experiments with miRNA inhibitor and mimic were conducted to elucidate miR-27a-5p's functional role in FliC-induced inflammatory responses. Additionally, a mouse model of C. difficile infection was treated with miR-27a-5p to evaluate its therapeutic potential in vivo. RESULTS: miR-27a-5p showed significant FliC-dependent overexpression in Caco-2 cells. Inhibition of NF-κB signaling suppressed miR-27a-5p overexpression. Knockdown of miR-27a-5p increased NF-κB activation and TNF-α and IL-8 cytokine production, while its overexpression had the opposite effect. Moreover, miR-27a-5p was overexpressed in the caeca of C. difficile-infected mice, correlating with intestinal IL-8 levels. Treatment of infected mice with miR-27a-5p mimic reduced disease severity and intestinal inflammation. CONCLUSION: miR-27a-5p plays a crucial role in regulating C. difficile-induced inflammation, suggesting its potential as a therapeutic target for controlling severe infection. These findings offer valuable insights into potential therapeutic strategies for managing C. difficile infection and associated inflammatory complications.

Microbiome and Metabolome Restoration After Administration of Fecal Microbiota, Live-jslm (REBYOTA®) for Preventing Recurrent Clostridioides difficile Infection.

BACKGROUND: Microbiota-based treatments are effective in preventing recurrent Clostridioides difficile infection (rCDI). Fecal microbiota, live-jslm (REBYOTA®; RBL, previously RBX2660) was shown to prevent rCDI in a phase 3, randomized, double-blinded placebo controlled clinical trial (PUNCH™ CD3). METHODS: Stool samples from participants in PUNCH™ CD3 who received a single blinded dose of rectally administered RBL or placebo were sequenced to determine microbial community composition and calculate the Microbiome Health Index for post-antibiotic dysbiosis (MHI-A). The composition of bile acids (BAs) in the same samples was quantified by liquid chromatography mass spectrometry. Relationships between BA composition and microbiota community structure and correlations with treatment outcomes were assessed. RESULTS: Before administration, Gammaproteobacteria and Bacilli dominated the microbiota community and primary BAs were more prevalent than secondary BAs. Clinical success after administration correlated with shifts to predominantly Bacteroidia and Clostridia, a significant increase in MHI-A, and a shift from primary to secondary BAs. Several microbiota and BA changes were more extensive in RBL-treated responders compared to placebo-treated responders, and microbiota changes correlated with BA changes. CONCLUSIONS: Clinical response and RBL administration were associated with significant restoration of microbiota and BA composition. CLINICAL TRIALS REGISTRATION: NCT03244644.

Predicting Increased Incidence of Common Antibiotic-Resistant and Antibiotic-Associated Pathogens Using Ensemble Species Distribution Modeling.

The Centers for Disease Control estimates antibiotic-associated pathogens result in 2.8 million infections and 38,000 deaths annually in the United States. This study applies species distribution modeling to elucidate the impact of environmental determinants of human infectious disease in an era of rapid global change. We modeled methicillin-resistant Staphylococcus aureus and Clostridioides difficile using 31 publicly accessible bioclimatic, healthcare, and sociodemographic variables. Ensemble models were created from 8 unique statistical and machine learning algorithms. Using International Classification of Diseases, 10th Edition codes, we identified 305,528 diagnoses of methicillin-resistant S.aureus and 302,001 diagnoses of C.difficile presence. Three environmental factors - average maximum temperature, specific humidity, and agricultural land density - emerged as major predictors of increased methicillin-resistant S.aureus and C.difficile presence; variables representing healthcare availability were less important. Species distribution modeling may be a powerful tool for identifying areas at increased risk for disease presence and have important implications for disease surveillance systems.

Effect of Leptin Receptor Q223R Polymorphism on Clostridioides difficile Infection-Induced Macrophage Migration Inhibitory Factor Production.

Proinflammatory cytokine levels and host genetic makeup are key determinants of Clostridioides difficile infection (CDI) outcomes. We previously reported that blocking the inflammatory cytokine macrophage migration inhibitory factor (MIF) ameliorates CDI. Here, we determined kinetics of MIF production and its association with a common genetic variant in leptin receptor (LEPR) using blood from patients with CDI. We found highest plasma MIF early after C difficile exposure and in individuals who express mutant/derived LEPR. Our data suggest that early-phase CDI provides a possible window of opportunity in which MIF targeting, potentially in combination with LEPR genotype, could have therapeutic utility.

A sequence invariable region in TcdB2 is required for toxin escape from Clostridioides difficile.

Sequence differences among the subtypes of Clostridioides difficile toxin TcdB (2,366 amino acids) are broadly distributed across the entire protein, with the notable exception of 76 residues at the protein's carboxy terminus. This sequence invariable region (SIR) is identical at the DNA and protein level among the TcdB variants, suggesting this string of amino acids has undergone selective pressure to prevent alterations. The functional role of the SIR domain in TcdB has not been determined. Analysis of a recombinantly constructed TcdB mutant lacking the SIR domain did not identify changes in TcdB's enzymatic or cytopathic activities. To further assess the SIR region, we constructed a C. difficile strain with the final 228 bp deleted from the tcdB gene, resulting in the production of a truncated form of TcdB lacking the SIR (TcdB2∆2291-2366). Using a combination of approaches, we found in the absence of the SIR sequence TcdB2∆2291-2366 retained cytotoxic activity but was not secreted from C. difficile. TcdB2∆2291-2366 was not released from the cell under autolytic conditions, indicating the SIR is involved in a more discrete step in toxin escape from the bacterium. Fractionation experiments combined with antibody detection found that TcdB2∆2291-2366 accumulates at the cell membrane but is unable to complete steps in secretion beyond this point. These data suggest conservation of the SIR domain across variants of TcdB could be influenced by the sequence's role in efficient escape of the toxin from C. difficile. IMPORTANCE: Clostridioides difficile is a leading cause of antibiotic associated disease in the United States. The primary virulence factors produced by C. difficile are two large glucosylating toxins TcdA and TcdB. To date, several sequence variants of TcdB have been identified that differ in various functional properties. Here, we identified a highly conserved region among TcdB subtypes that is required for release of the toxin from C. difficile. This study reveals a putative role for the longest stretch of invariable sequence among TcdB subtypes and provides new details regarding toxin release into the extracellular environment. Improving our understanding of the functional roles of the conserved regions of TcdB variants aids in the development of new, broadly applicable strategies to treat CDI.

Clostridioides difficile superoxide reductase mitigates oxygen sensitivity.

Clostridioides difficile causes a serious diarrheal disease and is a common healthcare-associated bacterial pathogen. Although it has a major impact on human health, the mechanistic details of C. difficile intestinal colonization remain undefined. C. difficile is highly sensitive to oxygen and requires anaerobic conditions for in vitro growth. However, the mammalian gut is not devoid of oxygen, and C. difficile tolerates moderate oxidative stress in vivo. The C. difficile genome encodes several antioxidant proteins, including a predicted superoxide reductase (SOR) that is upregulated upon exposure to antimicrobial peptides. The goal of this study was to establish SOR enzymatic activity and assess its role in protecting C. difficile against oxygen exposure. Insertional inactivation of sor rendered C. difficile more sensitive to superoxide, indicating that SOR contributes to antioxidant defense. Heterologous C. difficile sor expression in Escherichia coli conferred protection against superoxide-dependent growth inhibition, and the corresponding cell lysates showed superoxide scavenging activity. Finally, a C. difficile SOR mutant exhibited global proteome changes under oxygen stress when compared to the parent strain. Collectively, our data establish the enzymatic activity of C. difficile SOR, confirm its role in protection against oxidative stress, and demonstrate SOR's broader impacts on the C. difficile vegetative cell proteome.IMPORTANCEClostridioides difficile is an important pathogen strongly associated with healthcare settings and capable of causing severe diarrheal disease. While considered a strict anaerobe in vitro, C. difficile has been shown to tolerate low levels of oxygen in the mammalian host. Among other well-characterized antioxidant proteins, the C. difficile genome encodes a predicted superoxide reductase (SOR), an understudied component of antioxidant defense in pathogens. The significance of the research reported herein is the characterization of SOR's enzymatic activity, including confirmation of its role in protecting C. difficile against oxidative stress. This furthers our understanding of C. difficile pathogenesis and presents a potential new avenue for targeted therapies.