Toxins TcdA Research Articles

Chemical genetic analysis of enoxolone inhibition of Clostridioides difficile toxin production reveals adenine deaminase and ATP synthase as antivirulence targets

Toxins TcdA and TcdB are the main virulence factors of Clostridioides difficile, a leading cause of hospital-acquired diarrhea. Despite their importance, there is a significant knowledge gap of druggable targets for inhibiting toxin production. To address this, we screened non-antibiotic phytochemicals to identify potential chemical genetic probes to discover anti-virulence drug targets. This led to the identification of 18β-glycyrrhetinic acid (enoxolone), a licorice metabolite, as an inhibitor of TcdA and TcdB biosynthesis. Using affinity-based proteomics, potential targets were identified as ATP synthase subunit alpha (AtpA) and adenine deaminase (Ade, which catalyzes conversion of adenine to hypoxanthine in the purine salvage pathway). To validate these targets, a multi-faceted approach was adopted. Gene silencing of ade and atpA inhibited toxin biosynthesis, while SPR and ITC molecular interaction analyses revealed direct binding of enoxolone to Ade. Metabolomics demonstrated enoxolone induced the accumulation of adenosine, while depleting hypoxanthine and ATP in C. difficile. Transcriptomics further revealed enoxolone dysregulated phosphate uptake genes, which correlated with reduced cellular phosphate levels. These findings suggest that enoxolone's cellular action is multi-targeted. Accordingly, supplementation with both hypoxanthine and triethyl phosphate (TEP), a phosphate source, was required to fully restore toxin production in the presence of enoxolone. In conclusion, through the characterization of enoxolone, we identified promising anti-virulence targets that interfere with nucleotide salvage and ATP synthesis, which may also block toxin biosynthesis.

Inhibition of Clostridioides difficile toxins TcdA and TcdB by the amiodarone derivative dronedarone.

The dreaded nosocomial pathogen Clostridioides difficile causes diarrhea and severe inflammation of the colon, especially after the use of certain antibiotics. The bacterium releases two deleterious toxins, TcdA and TcdB, into the gut, which are mainly responsible for the symptoms of C.difficile-associated diseases (CDADs). Both toxins are capable of entering independently into various host cells, e.g., intestinal epithelial cells, where they mono-O-glucosylate and inactivate Rho and/or Ras GTPases, important molecular switches for various cellular functions. We have shown recently that the cellular uptake of the Clostridioides difficile toxins TcdA and TcdB (TcdA/B) is inhibited by the licensed class III antiarrhythmic drug amiodarone (Schumacher et al. in Gut Microbes 15(2):2256695, 2023). Mechanistically, amiodarone delays the cellular uptake of both toxins into target cells most likely by lowering membrane cholesterol levels and by interfering with membrane insertion and/or pore formation of TcdA/B. However, serious side effects, such as thyroid dysfunction and severe pulmonary fibrosis, limit the clinical use of amiodarone in patients with C. difficile infection (CDI). For that reason, we aimed to test whether dronedarone, an amiodarone derivative with a more favorable side effect profile, is also capable of inhibiting TcdA/B. To this end, we tested in vitro with various methods the impact of dronedarone on the intoxication of Vero and CaCo-2 cells with TcdA/B. Importantly, preincubation of both cell lines with dronedarone for 1h at concentrations in the low micromolar range rendered the cells less sensitive toward TcdA/B-induced Rac1 glucosylation, collapse of the actin cytoskeleton, cell rounding, and cytopathic effects, respectively. Our study points toward the possibility of repurposing the already approved drug dronedarone as the preferable safer-to-use alternative to amiodarone for inhibiting TcdA/B in the (supportive) therapy of CDADs.

Formation of the pyruvoyl-dependent proline reductase Prd from Clostridioides difficile requires the maturation enzyme PrdH.

Stickland fermentation, the coupled oxidation and reduction of amino acid pairs, is a major pathway for obtaining energy in the nosocomial bacterium Clostridioides difficile. D-proline is the preferred substrate for the reductive path, making it not only a key component of the general metabolism but also impacting on the expression of the clostridial toxins TcdA and TcdB. D-proline reduction is catalyzed by the proline reductase Prd, which belongs to the pyruvoyl-dependent enzymes. These enzymes are translated as inactive proenzymes and require subsequent processing to install the covalently bound pyruvate. Whereas pyruvoyl formation by intramolecular serinolysis has been studied in unrelated enzymes, details about pyruvoyl generation by cysteinolysis as in Prd are lacking. Here, we show that Prd maturation requires a small dimeric protein that we have named PrdH. PrdH (CD630_32430) is co-encoded with the PrdA and PrdB subunits of Prd and also found in species producing similar reductases. By producing stable variants of PrdA and PrdB, we demonstrate that PrdH-mediated cleavage and pyruvoyl formation in the PrdA subunit requires PrdB, which can be harnessed to produce active recombinant Prd for subsequent analyses. We further created PrdA- and PrdH-mutants to get insight into the interaction of the components and into the processing reaction itself. Finally, we show that deletion of prdH renders C. difficile insensitive to proline concentrations in culture media, suggesting that this processing factor is essential for proline utilization. Due to the link between Stickland fermentation and pathogenesis, we suggest PrdH may be an attractive target for drug development.

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

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.

Exploring the Pathogenesis of Clostridium difficile Infection Based on Weighted Gene Co-expression Network Analysis

Clostridioides difficile, a Gram-positive, spore-forming, anaerobic bacterium, is a leading cause of antibiotic-associated diarrhea and colitis. Its infections, often linked to antibiotic use in healthcare settings, range from mild diarrhea to pseudomembranous colitis. This study explores the pathogenic and toxin action mechanisms of Clostridioides difficile, particularly its toxins TcdA and TcdB, which disrupt host cells through glycosyltransferase activity, affecting cell structure and intestinal integrity, while also triggering inflammation and immune responses. Treatment strategies for Clostridioides difficile infection continue to evolve, encompassing antibiotics, microbiota replacement, monoclonal antibodies, and emerging therapies. The study employs Weighted Gene Co-expression Network Analysis (WGCNA)to analyze the GSE29008 dataset, categorizing samples into normal, toxin A, and toxin B groups. Notably, the 'midnightblue' module shows a strong positive correlation with toxin B, indicating its significant role in severe inflammation and tissue damage, emphasizing the importance of understanding the toxin action mechanisms for developing effective treatments and public health policies.

A Colonic Organoid Model Challenged with the Large Toxins of Clostridioides difficile TcdA and TcdB Exhibit Deregulated Tight Junction Proteins.

Clostridioides difficile toxins TcdA and TcdB are responsible for diarrhea and colitis. Lack of functional studies in organoid models of the gut prompted us to elucidate the toxin's effects on epithelial barrier function and the molecular mechanisms for diarrhea and inflammation. Human adult colon organoids were cultured on membrane inserts. Tight junction (TJ) proteins and actin cytoskeleton were analyzed for expression via Western blotting and via confocal laser-scanning microscopy for subcellular localization. Polarized intestinal organoid monolayers were established from stem cell-containing colon organoids to apply toxins from the apical side and to perform functional measurements in the organoid model. The toxins caused a reduction in transepithelial electrical resistance in human colonic organoid monolayers with sublethal concentrations. Concomitantly, we detected increased paracellular permeability fluorescein and FITC-dextran-4000. Human colonic organoid monolayers exposed to the toxins exhibited redistribution of barrier-forming TJ proteins claudin-1, -4 and tricellulin, whereas channel-forming claudin-2 expression was increased. Perijunctional F-actin cytoskeleton organization was affected. Adult stem cell-derived human colonic organoid monolayers were applicable as a colon infection model for electrophysiological measurements. The TJ changes noted can explain the epithelial barrier dysfunction and diarrhea in patients, as well as increased entry of luminal antigens triggering inflammation.

Exploring the inhibitory potential of the antiarrhythmic drug amiodarone against Clostridioides difficile toxins TcdA and TcdB

ABSTRACT The intestinal pathogen Clostridioides difficile is the leading cause of antibiotic-associated diarrhea and pseudomembranous colitis in humans. The symptoms of C. difficile-associated diseases (CDADs) are directly associated with the pathogen’s toxins TcdA and TcdB, which enter host cells and inactivate Rho and/or Ras GTPases by glucosylation. Membrane cholesterol is crucial during the intoxication process of TcdA and TcdB, and likely involved during pore formation of both toxins in endosomal membranes, a key step after cellular uptake for the translocation of the glucosyltransferase domain of both toxins from endosomes into the host cell cytosol. The licensed drug amiodarone, a multichannel blocker commonly used in the treatment of cardiac dysrhythmias, is also capable of inhibiting endosomal acidification and, as shown recently, cholesterol biosynthesis. Thus, we were keen to investigate in vitro with cultured cells and human intestinal organoids, whether amiodarone preincubation protects from TcdA and/or TcdB intoxication. Amiodarone conferred protection against both toxins independently and in combination as well as against toxin variants from the clinically relevant, epidemic C. difficile strain NAP1/027. Further mechanistic studies suggested that amiodarone’s mode-of-inhibition involves also interference with the translocation pore of both toxins. Our study opens the possibility of repurposing the licensed drug amiodarone as a novel pan-variant antitoxin therapeutic in the context of CDADs.

Biofilm Formation of Clostridioides difficile, Toxin Production and Alternatives to Conventional Antibiotics in the Treatment of CDI.

Clostridioides difficile is considered a nosocomial pathogen that flares up in patients exposed to antibiotic treatment. However, four out of ten patients diagnosed with C. difficile infection (CDI) acquired the infection from non-hospitalized individuals, many of whom have not been treated with antibiotics. Treatment of recurrent CDI (rCDI) with antibiotics, especially vancomycin (VAN) and metronidazole (MNZ), increases the risk of experiencing a relapse by as much as 70%. Fidaxomicin, on the other hand, proved more effective than VAN and MNZ by preventing the initial transcription of RNA toxin genes. Alternative forms of treatment include quorum quenching (QQ) that blocks toxin synthesis, binding of small anion molecules such as tolevamer to toxins, monoclonal antibodies, such as bezlotoxumab and actoxumab, bacteriophage therapy, probiotics, and fecal microbial transplants (FMTs). This review summarizes factors that affect the colonization of C. difficile and the pathogenicity of toxins TcdA and TcdB. The different approaches experimented with in the destruction of C. difficile and treatment of CDI are evaluated.

Prevalence of Clostridioides difficile in dogs (Canis familiaris) with gastrointestinal disorders in Rio de Janeiro

Clostridioides difficile infections (CDI) have a high morbidity and mortality rate and have always been considered a nosocomial disease. Nonetheless, the number of cases of community-acquired CDI is increasing, and new evidence suggests additional C. difficile reservoirs exist. Pathogenic C. difficile strains have been found in livestock, domestic animals, and meat, so a zoonotic transmission has been proposed. ObjectiveThe goal of this study was to isolate C. difficile strains in dogs at a veterinary clinic in Rio de Janeiro, Brazil, and characterize clinical and pathological findings associated with lower gastrointestinal tract disorders. MethodsFifty stool samples and biopsy fragments from dogs were obtained and cultured in the CDBA selective medium. All suggestive C. difficile colonies were confirmed by MALDI-TOF MS and PCR (tpi gene). Vancomycin, metronidazole, moxifloxacin, erythromycin, and rifampicin were tested for antibiotic susceptibility. Biofilm, motility assays, and a PCR for the toxins (tcdA, tcdB, and cdtB), as well as ribotyping, were also performed. ResultsBlood samples and colonic biopsy fragments were examined in C. difficile positive dogs. Ten animals (20%) tested positive for C. difficile by using stool samples, but not from biopsy fragments. Most C. difficile strains were toxigenic: six were A+B+ belonging to RT106; two were A+B+ belonging to RT014/020; and two were A-B- belonging to RT010. All strains were biofilm producers. In the motility test, 40% of strains were as motile as the positive control, CD630 (RT012). In the disc diffusion test, two strains (RT010) were resistant to erythromycin and metronidazole; and another to metronidazole (RT014/020). In terms of C. difficile clinicopathological correlations, no statistically significant morphological changes, such as pseudomembranous and "volcano" lesions, were observed. Regarding hematological data, dogs positive for C. difficile had leucopenia (p = 0.02) and lymphopenia (p = 0.03). There was a significant correlation between senility and the presence of C. difficile in the dogs studied (p = 0,02). ConclusionsAlthough C. difficile has not been linked to canine diarrheal disorders, it appears to be more common in dogs with intestinal dysfunctions. The isolation of ribotypes frequently involved in human CDI outbreaks around the world supports the theory of C. difficile zoonotic transmission.

Network analysis of toxin production in Clostridioides difficile identifies key metabolic dependencies.

Clostridioides difficile pathogenesis is mediated through its two toxin proteins, TcdA and TcdB, which induce intestinal epithelial cell death and inflammation. It is possible to alter C. difficile toxin production by changing various metabolite concentrations within the extracellular environment. However, it is unknown which intracellular metabolic pathways are involved and how they regulate toxin production. To investigate the response of intracellular metabolic pathways to diverse nutritional environments and toxin production states, we use previously published genome-scale metabolic models of C. difficile strains CD630 and CDR20291 (iCdG709 and iCdR703). We integrated publicly available transcriptomic data with the models using the RIPTiDe algorithm to create 16 unique contextualized C. difficile models representing a range of nutritional environments and toxin states. We used Random Forest with flux sampling and shadow pricing analyses to identify metabolic patterns correlated with toxin states and environment. Specifically, we found that arginine and ornithine uptake is particularly active in low toxin states. Additionally, uptake of arginine and ornithine is highly dependent on intracellular fatty acid and large polymer metabolite pools. We also applied the metabolic transformation algorithm (MTA) to identify model perturbations that shift metabolism from a high toxin state to a low toxin state. This analysis expands our understanding of toxin production in C. difficile and identifies metabolic dependencies that could be leveraged to mitigate disease severity.

Structural Basis for Binding of Neutralizing Antibodies to Clostridioides difficile Binary Toxin.

Clostridioides difficile is a Gram-positive opportunistic human pathogen that causes 15,000 deaths annually in the United States, prompting a need for vaccine development. In addition to the important toxins TcdA and TcdB, binary toxin (CDT) plays a significant role in the pathogenesis of certain C. difficile ribotypes by catalyzing the ADP-ribosylation of actin in host cells. However, the mechanisms of CDT neutralization by antibodies have not been studied, limiting our understanding of key epitopes for CDT antigen design. Therefore, we isolated neutralizing monoclonal antibodies against CDT and characterized their mechanisms of neutralization structurally and biochemically. Here, 2.5-Å and 2.6-Å resolution X-ray crystal structures of the antibodies BINTOXB/22 and BINTOXB/9, respectively, in complex with CDTb-the CDT subunit that forms a heptameric pore for the delivery of toxic CDTa enzyme into the host cytosol-showed that both antibodies sterically clash with adjacent protomers in the assembled heptamer. Assessment of trypsin-induced oligomerization of the purified CDTb protoxin in vitro showed that BINTOXB/22 and BINTOXB/9 prevented the assembly of di-heptamers upon prodomain cleavage. This work suggests that the CDT oligomerization process can be effectively targeted by antibodies, which will aid in the development of C. difficile vaccines and therapeutics. IMPORTANCE Clostridioides difficile strains associated with worse clinical outcomes have been found to secrete a toxin called CDT (or binary toxin). As blocking the function of this toxin could help mitigate C. difficile infections, we sought to determine the molecular basis for the inhibition of CDT by monoclonal antibodies. We isolated monoclonal antibodies targeting the B-component of CDT (CDTb) and selected two with neutralizing activity for detailed structural and biochemical characterization. High-resolution crystal structures of each antibody bound to CDTb showed that their presence would preclude the assembly of a CDTb oligomer required for activity. Oligomerization of CDTb in vitro was shown to be blocked in the presence of the neutralizing antibodies, but not a control antibody.

Different bile acids have versatile effects on sporulation, toxin levels and biofilm formation of different Clostridioides difficile strains

Clostridioides difficile infection develops following ingestion of virulent stains by a susceptible host. Once germinated, toxins TcdA and TcdB, and in some of the strains binary toxin, are secreted, eliciting disease. Bile acids play a significant role in the process of spore germination and outgrowth, with cholate and its derivative enhancing colony formation, while chenodeoxycholate inhibit germination and outgrowth. This work investigated bile acids' impact on spore germination, toxin levels and biofilm formation in various strain types (STs). Thirty C. difficile isolates (A+ B+ CDT-\\+) of different STs were exposed to increasing concentrations of the bile acids, cholic acid (CA), taurocholic acid (TCA) and chenodeoxycholic acid (CDCA). Following treatments, spore germination was determined. Toxin concentrations were semi-quantified using the C. Diff Tox A/B II™ kit. Biofilm formation was detected by the microplate assay with crystal violet. SYTO® 9 and propidium iodide staining were used for live and dead cell detection, respectively, inside the biofilm.Toxins levels were increased by 1.5–28-fold in response to CA and by 1.5–20-fold in response to TCA, and decreased by 1–37-fold due to CDCA exposure. CA had a concentration-dependent effect on biofilm formation, with the low concentration (0.1%) inducing- and the higher concentrations inhibiting biofilm formation, while CDCA significantly reduced biofilm production at all concentrations. There were no differences in the bile acids effects on different STs. Further investigation might identify a specific bile acids' combination with inhibitory effects on C. difficile toxin and biofilm production, which could modulate toxin formation to reduce the likelihood of developing CDI.

Neutralising Effects of Different Antibodies on Clostridioides difficile Toxins TcdA and TcdB in a Translational Approach

Given the high prevalence of intestinal disease in humans and animals, there is a strong need for clinically relevant models recapitulating gastrointestinal systems, ideally replacing in vivo models in accordance with the principles of the 3R. We established a canine organoid system and analysed the neutralising effects of recombinant versus natural antibodies on Clostridioides difficile toxins A and B in this in vitro system. Sulforhodamine B cytotoxicity assays in 2D and FITC-dextran barrier integrity assays on basal-out and apical-out organoids revealed that recombinant, but not natural antibodies, effectively neutralised C. difficile toxins. Our findings emphasise that canine intestinal organoids can be used to test different components and suggest that they can be further refined to also mirror complex interactions between the intestinal epithelium and other cells.

A leaky human colon model reveals uncoupled apical/basal cytotoxicity in early Clostridioides difficile toxin exposure.

Clostridioides difficile (C. difficile) toxins A (TcdA) and B (TcdB) cause antibiotic-associated colitis in part by disrupting epithelial barrier function. Accurate in vitro models are necessary to detect early toxicity kinetics, investigate disease etiology, and develop preclinical models for new therapies. Properties of cancer cell lines and organoids inherently limit these efforts. We developed adult stem cell-derived monolayers of differentiated human colonic epithelium (hCE) with barrier function, investigated the impact of toxins on apical/basal aspects of monolayers, and evaluated whether a leaky epithelial barrier enhances toxicity. Single-cell RNA-sequencing (scRNAseq) mapped C. difficile-relevant genes to human lineages. Transcriptomics compared hCE to Caco-2, informed timing of in vitro stem cell differentiation, and revealed transcriptional responses to TcdA. Transepithelial electrical resistance (TEER) and fluorescent permeability assays measured cytotoxicity. Contribution of TcdB toxicity was evaluated in a diclofenac-induced leaky gut model. scRNAseq demonstrated broad and variable toxin receptor expression. Absorptive colonocytes in vivo displayed increased toxin receptor, Rho GTPase, and cell junction gene expression. Advanced TcdA toxicity generally decreased cytokine/chemokine and increased tight junction and death receptor genes. Differentiated Caco-2 cells remained immature whereas hCE monolayers were similar to mature colonocytes in vivo. Basal exposure of TcdA/B caused greater toxicity and apoptosis than apical exposure. Apical exposure to toxins was enhanced by diclofenac. Apical/basal toxicities are uncoupled with more rapid onset and increased magnitude postbasal toxin exposure. Leaky junctions enhance toxicity of apical TcdB exposure. hCE monolayers represent a physiologically relevant and sensitive system to evaluate the impact of microbial toxins on gut epithelium.NEW & NOTEWORTHY Novel human colonocyte monolayer cultures, benchmarked by transcriptomics for physiological relevance, detect early cytopathic impacts of Clostridioides difficile toxins TcdA and TcdB. A fluorescent ZO-1 reporter in primary human colonocytes is used to track epithelial barrier disruption in response to TcdA. Basal TcdA/B exposure generally caused more rapid onset and cytotoxicity than apical exposure. Transcriptomics demonstrate changes in tight junction, chemokine, and cytokine receptor gene expression post-TcdA exposure. Diclofenac-induced leaky epithelium enhanced apical exposure toxicity.

Adenosine receptors differentially mediate enteric glial cell death induced by Clostridioides difficile Toxins A and B.

Increased risk of intestinal dysfunction has been reported in patients after Clostridioides difficile infection (CDI). Enteric glial cells (EGCs), a component of the enteric nervous system (ENS), contribute to gut homeostasis. Previous studies showed that adenosine receptors, A2A and A2B, modulate inflammation during CDI. However, it is unknown how these receptors can modulate the EGC response to the C. difficile toxins (TcdA and TcdB). We investigated the effects of these toxins on the expression of adenosine receptors in EGCs and the role of these receptors on toxin-induced EGC death. Rat EGCs line were incubated with TcdA or TcdB alone or in combination with adenosine analogues 1h prior to toxins challenge. After incubation, EGCs were collected to evaluate gene expression (adenosine receptors and proinflammatory markers) and cell death. In vivo, WT, A2A, and A2B KO mice were infected with C. difficile, euthanized on day 3 post-infection, and cecum tissue was processed. TcdA and TcdB increased A2A and A3 transcripts, as well as decreased A2B. A2A agonist, but not A2A antagonist, decreased apoptosis induced by TcdA and TcdB in EGCs. A2B blocker, but not A2B agonist, diminished apoptosis in EGCs challenged with both toxins. A3 agonist, but not A3 blocker, reduced apoptosis in EGCs challenged with TcdA and TcdB. Inhibition of protein kinase A (PKA) and CREB, both involved in the main signaling pathway driven by activation of adenosine receptors, decreased EGC apoptosis induced by both toxins. A2A agonist and A2B antagonist decreased S100B upregulation induced by C. difficile toxins in EGCs. In vivo, infected A2B KO mice, but not A2A, exhibited a decrease in cell death, including EGCs and enteric neuron loss, compared to infected WT mice, reduced intestinal damage and decreased IL-6 and S100B levels in cecum. Our findings indicate that upregulation of A2A and A3 and downregulation of A2B in EGCs and downregulation of A2B in intestinal tissues elicit a protective response against C. difficile toxins. Adenosine receptors appear to play a regulatory role in EGCs death and proinflammatory response induced by TcdA and TcdB, and thus may be potential targets of intervention to prevent post-CDI intestinal dysmotility.

2'-Fucosyllactose inhibits proliferation of Clostridioides difficile ATCC 43599 in the CDi-screen, an in vitro model simulating Clostridioides difficile infection.

Clostridioides difficile is a Gram-positive anaerobic bacterium that can produce the toxins TcdA and/or TcdB and is considered an opportunistic pathogen. C. difficile is mainly transmitted as endospores, which germinate to produce the pathogenic vegetative cells under suitable conditions in the gut. To efficiently screen novel therapeutic- interventions against the proliferation of C. difficile within a complex microbial community, platforms are needed that facilitate parallel experimentation. In order to allow for screening of novel interventions a medium-to-high throughput in vitro system is desirable. To this end, we have developed the 96-well CDi-screen platform that employs an adapted simulated ileal effluent medium (CDi-SIEM) and allows for culturing of pathogenic C. difficile. C. difficile strain ATCC 43599 was inoculated in the form of vegetative cells and spores into the CDi-screen in the presence and absence of a cultured fecal microbiota and incubated for 48h. To demonstrate its utility, we investigated the effect of the human milk oligosaccharide 2'-Fucosyllactose (2'-FL) at 4 and 8 mg/mL on C. difficile outgrowth and toxin production in the CDi-screen. The test conditions were sampled after 24 and 48 hours. C. difficile -specific primers were used to monitor C. difficile growth via qPCR and barcoded 16S rRNA gene amplicon sequencing facilitated the in-depth analysis of gut microbial community dynamics. C. difficile ATCC 43599 proliferated in CDi-SIEM, both when inoculated as spores and as vegetative cells. The strain reached cell numbers expressed as C. difficile genome equivalents of up to 10 8 cells per mL after 24h of incubation. 2'-FL significantly inhibited the outgrowth of the ATTC 43599 strain within a complex human gut microbial community in the CDi-screen. In addition, a dose-dependent modulation of the gut microbial community composition by 2'-FL supplementation was detected, with a significant increase in the relative abundance of the genus Blautia in the presence of 2'-FL. The CDi-screen is suitable for studying C. difficile proliferation in a complex gut ecosystem and for screening for anti-pathogenic interventions that target C. difficile directly and/or indirectly through interactions with the gut microbiota. Different doses of compounds such as in this study the dose of the human milk oligosaccharide 2'-FL can be screened for efficacy in the inhibition of C. difficile proliferation.

Single domain antibodies against enteric pathogen virulence factors are active as curli fiber fusions on probiotic E. coli Nissle 1917.

Enteric microbial pathogens, including Escherichia coli, Shigella and Cryptosporidium species, take a particularly heavy toll in low-income countries and are highly associated with infant mortality. We describe here a means to display anti-infective agents on the surface of a probiotic bacterium. Because of their stability and versatility, VHHs, the variable domains of camelid heavy-chain-only antibodies, have potential as components of novel agents to treat or prevent enteric infectious disease. We isolated and characterized VHHs targeting several enteropathogenic E. coli (EPEC) virulence factors: flagellin (Fla), which is required for bacterial motility and promotes colonization; both intimin and the translocated intimin receptor (Tir), which together play key roles in attachment to enterocytes; and E. coli secreted protein A (EspA), an essential component of the type III secretion system (T3SS) that is required for virulence. Several VHHs that recognize Fla, intimin, or Tir blocked function in vitro. The probiotic strain E. coli Nissle 1917 (EcN) produces on the bacterial surface curli fibers, which are the major proteinaceous component of E. coli biofilms. A subset of Fla-, intimin-, or Tir-binding VHHs, as well as VHHs that recognize either a T3SS of another important bacterial pathogen (Shigella flexneri), a soluble bacterial toxin (Shiga toxin or Clostridioides difficile toxin TcdA), or a major surface antigen of an important eukaryotic pathogen (Cryptosporidium parvum) were fused to CsgA, the major curli fiber subunit. Scanning electron micrographs indicated CsgA-VHH fusions were assembled into curli fibers on the EcN surface, and Congo Red binding indicated that these recombinant curli fibers were produced at high levels. Ectopic production of these VHHs conferred on EcN the cognate binding activity and, in the case of anti-Shiga toxin, was neutralizing. Taken together, these results demonstrate the potential of the curli-based pathogen sequestration strategy described herein and contribute to the development of novel VHH-based gut therapeutics.

Bezlotoxumab prevents extraintestinal organ damage induced by Clostridioides difficile infection

ABSTRACT Clostridioides difficile is the most common cause of infectious antibiotic-associated diarrhea, with disease mediated by two major toxins TcdA and TcdB. In severe cases, systemic disease complications may arise, resulting in fatal disease. Systemic disease in animal models has been described, with thymic damage an observable consequence of severe disease in mice. Using a mouse model of C. difficile infection, we examined this disease phenotype, focussing on the thymus and serum markers of systemic disease. The efficacy of bezlotoxumab, a monoclonal TcdB therapeutic, to prevent toxin mediated systemic disease complications was also examined. C. difficile infection causes toxin-dependent thymic damage and CD4+CD8+ thymocyte depletion in mice. These systemic complications coincide with changes in biochemical markers of liver and kidney function, including increased serum urea and creatinine, and hypoglycemia. Administration of bezlotoxumab during C. difficile infection prevents systemic disease and thymic atrophy, without blocking gut damage, suggesting the leakage of gut contents into circulation may influence systemic disease. As the thymus has such a crucial role in T cell production and immune system development, these findings may have important implications in relapse of C. difficile disease and impaired immunity during C. difficile infection. The prevention of thymic atrophy and reduced systemic response following bezlotoxumab treatment, without altering colonic damage, highlights the importance of systemic disease in C. difficile infection, and provides new insights into the mechanism of action for this therapeutic. Abbreviations: Acute kidney injury (AKI); Alanine Transaminase (ALT); Aspartate Aminotransferase (AST); C. difficile infection (CDI); chronic kidney disease (CKD); combined repetitive oligo-peptides (CROPS); cardiovascular disease (CVD); Double positive (DP); hematoxylin and eosin (H&E); immunohistochemical (IHC); multiple organ dysfunction syndrome (MODS); phosphate buffered saline (PBS); standard error of the mean (SEM); surface layer proteins (SLP); Single positive (SP); wild-type (WT).

Clostridioides difficile TcdB Toxin Glucosylates Rho GTPase by an SNi Mechanism and Ion Pair Transition State.

Toxins TcdA and TcdB from Clostridioides difficile glucosylate human colon Rho GTPases. TcdA and TcdB glucosylation of RhoGTPases results in cytoskeletal changes, causing cell rounding and loss of intestinal integrity. Clostridial toxins TcdA and TcdB are proposed to catalyze glucosylation of Rho GTPases with retention of stereochemistry from UDP-glucose. We used kinetic isotope effects to analyze the mechanisms and transition-state structures of the glucohydrolase and glucosyltransferase activities of TcdB. TcdB catalyzes Rho GTPase glucosylation with retention of stereochemistry, while hydrolysis of UDP-glucose by TcdB causes inversion of stereochemistry. Kinetic analysis revealed TcdB glucosylation via the formation of a ternary complex with no intermediate, supporting an SNi mechanism with nucleophilic attack and leaving group departure occurring on the same face of the glucose ring. Kinetic isotope effects combined with quantum mechanical calculations revealed that the transition states of both glucohydrolase and glucosyltransferase activities of TcdB are highly dissociative. Specifically, the TcdB glucosyltransferase reaction proceeds via an SNi mechanism with the formation of a distinct oxocarbenium phosphate ion pair transition state where the glycosidic bond to the UDP leaving group breaks prior to attack of the threonine nucleophile from Rho GTPase.

Host humoral immune suppression by Clostridioides difficile Toxin A and Toxin B

Abstract Gram-positive Clostridioides difficile is the greatest cause of nosocomial enteric disease in the US and is associated with a high rate of morbidity and mortality. During C. difficile infection (CDI), two secreted toxins (TcdA and TcdB) drive pathogenesis. Although T-dependent humoral immunity, specifically high affinity TcdA- and TcdB-neutralizing IgG, is the best correlation for protection against CDI recurrence, this response is commonly lacking following CDI in both mice and patients, leading to a high chance of recurrence. We previously showed that in murine CDI, there was a lack of T follicular helper (Tfh) cell differentiation, reduced IgG class-switching, poor toxin neutralization, and lack of resistance to reinfection. Preliminary data also suggested that TcdB inhibited IgG responses following vaccination against C. difficile. We therefore hypothesize that TcdA and/or TcdB suppress C. difficile-specific T-dependent humoral immunity. Following either CDI or intraperitoneal injection with TcdA or TcdB, immune cells from mesenteric lymph nodes were analyzed for changes in gene expression. Changes in dendritic cell maturation markers were observed in the presence of TcdB but not TcdA. Multi-targeting mRNA profiling showed altered expression of antigen presentation and CD4+ T cell activation genes. Only one of the known TcdB cell entry receptors was expressed by immune cells, specifically by CD4+ T cells and B cells. Efforts are underway to measure the impact of TcdA and TcdB treatment on CD4+ T cell differentiation, Tfh proliferation, and T-B cell interactions. Our data indicate that toxins may hamper neutralizing IgG production by affecting multiple aspects of T-dependent humoral immunity. This work was supported by NIH grants AI134719 (M.L.L.) and AI119048 (J.D.B.).