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Background: Clostridioides difficile binary toxin (CDT) defines the hypervirulence of strains in nosocomial antibiotic-induced colitis with the highest mortality. The objective of our study was to investigate the impact of CDT on the intestinal epithelial barrier and to enlighten the underlying molecular mechanisms. Methods: Functional measurements of epithelial barrier function by macromolecular permeability and electrophysiology were performed in human intestinal HT-29/B6 cell monolayers. Molecular analysis of the spatial distribution of tight junction protein and cytoskeleton was performed by super-resolution STED microscopy. Results: Sublethal concentrations of CDT-induced barrier dysfunction with decreased TER and increased permeability for 332 Da fluorescein and 4 kDa FITC-dextran. The molecular correlate to the functional barrier defect by CDT was found to be a tight junction protein subcellular redistribution with tricellulin, occludin, and claudin-4 off the tight junction domain. This redistribution was shown to be MLCK-dependent. Conclusions: CDT compromised epithelial barrier function in a human intestinal colonic cell model, even in sublethal concentrations, pointing to barrier dysfunction in the intestine and leak flux induction as a diarrheal mechanism. However, this cannot be attributed to the appearance of apoptosis and necrosis, but rather to an opening of the paracellular leak pathway as the result of epithelial tight junction alterations.

Toxin B (TcdB) is a major exotoxin responsible for diseases associated with Clostridioides difficile infection. Its sequence variations among clinical isolates may contribute to the difficulty in developing effective therapeutics. Here, we investigate receptor-binding specificity of major TcdB subtypes (TcdB1 to TcdB12). We find that representative members of subtypes 2, 4, 7, 10, 11, and 12 do not recognize the established host receptor, frizzled proteins (FZDs). Using a genome-wide CRISPR-Cas9-mediated screen, we identify tissue factor pathway inhibitor (TFPI) as a host receptor for TcdB4. TFPI is recognized by a region in TcdB4 that is homologous to the FZD-binding site in TcdB1. Analysis of 206 TcdB variant sequences reveals a set of six residues within this receptor-binding site that defines a TFPI binding-associated haplotype (designated B4/B7) that is present in all TcdB4 members, a subset of TcdB7, and one member of TcdB2. Intragenic micro-recombination (IR) events have occurred around this receptor-binding region in TcdB7 and TcdB2 members, resulting in either TFPI- or FZD-binding capabilities. Introduction of B4/B7-haplotype residues into TcdB1 enables dual recognition of TFPI and FZDs. Finally, TcdB10 also recognizes TFPI, although it does not belong to the B4/B7 haplotype, and shows species selectivity: it recognizes TFPI of chicken and to a lesser degree mouse, but not human, dog, or cattle versions. These findings identify TFPI as a TcdB receptor and reveal IR-driven changes on receptor-specificity among TcdB variants.

C. difficile is a major cause of antibiotic-associated gastrointestinal infections. Two C. difficile exotoxins (TcdA and TcdB) are major virulence factors associated with these infections, and chondroitin sulfate proteoglycan 4 (CSPG4) is a potential receptor for TcdB, but its pathophysiological relevance and the molecular details that govern recognition remain unknown. Here, we determine the cryo-EM structure of a TcdB–CSPG4 complex, revealing a unique binding site spatially composed of multiple discontinuous regions across TcdB. Mutations that selectively disrupt CSPG4 binding reduce TcdB toxicity in mice, while CSPG4-knockout mice show reduced damage to colonic tissues during C. difficile infections. We further show that bezlotoxumab, the only FDA approved anti-TcdB antibody, blocks CSPG4 binding via an allosteric mechanism, but it displays low neutralizing potency on many TcdB variants from epidemic hypervirulent strains due to sequence variations in its epitopes. In contrast, a CSPG4-mimicking decoy neutralizes major TcdB variants, suggesting a strategy to develop broad-spectrum therapeutics against TcdB., Chondroitin sulfate proteoglycan 4 (CSPG4) is a potential receptor for C. difficile toxin B (TcdB) during C. difficile infections (CDIs). Here, the cryo-EM structure of a TcdB–CSPG4 complex and CDI mouse models offer insights into CSPG4 role in CDIs and suggest a therapeutic strategy targeting TcdB.

Purpose: To report the outcomes of bypass grafting (BG) vs endovascular therapy (EVT) in patients with non-dialysis-dependent chronic kidney disease (CKD) and chronic limb-threatening ischemia (CLTI). Materials and Methods: The CRITISCH Registry is a prospective, national, interdisciplinary, multicenter registry evaluating the current practice of all available treatment options in 1200 consecutive CLTI patients. For the purposes of this analysis, only the 337 patients with non-dialysis-dependent CKD treated by either BG (n=86; median 78 years, 48 men) or EVT (n=251; median age 80 years, 135 men) were analyzed. The primary composite outcome was amputation-free survival (AFS); secondary outcomes were overall survival (OS) and amputation-free time (AFT). All outcomes were evaluated in Cox proportional hazards models; the results are reported as the hazard ratio (HR) and 95% confidence interval (CI). Results: The Cox regression analysis revealed a significantly greater hazard of amputation or death after BG (HR 1.78, 95% CI 1.05 to 3.03, p=0.028). The models for AFT and overall survival also suggested a higher hazard for BG, but the differences were not significant (AFT: HR 1.66, 95% CI 0.78 to 3.53, p=0.188; OS: HR 1.41, 95% CI 0.80 to 2.47, p=0.348). The absence of runoff vessels (HR 1.73, 95% CI 1.15 to 2.60, p=0.008) was associated with a decreased AFS. The likelihood of amputation was higher in male patients (HR 2.21, 95% CI 1.10 to 4.45, p=0.027) and was associated with a lack of runoff vessels (HR 1.95, 95% CI 0.96 to 3.95, p=0.065) and myocardial infarction (HR 3.74, 95% CI 1.23 to 11.35, p=0.020). Death was more likely in patients without runoff vessels (HR 1.76, 95% CI 1.11 to 2.80, p=0.016) and those with a higher risk score (HR 1.73, 95% CI 1.03 to 2.91, p=0.038). Conclusion: This analysis suggested that BG was associated with poorer AFS than EVT in patients with non-dialysis-dependent CKD and CLTI. Male sex, previous myocardial infarction, and the absence of runoff vessels were additionally identified as predictors of poorer outcomes.

TcdB is a potent cytotoxin produced by pathogenic Clostridioides difficile that inhibits Rho GTPases by mono-glucosylation. TcdB enters cells via receptor-mediated endocytosis. The pathogenic glucosyltransferase domain (GTD) egresses endosomes by pH-mediated conformational changes, and is subsequently released in an autoproteolytic manner. We here investigated the uptake, localization and degradation of TcdB. TcdB colocalized with lysosomal marker protein LAMP1, verifying the endosomal-lysosomal route of the toxin. In pulse assays endocytosed TcdB declined to a limit of detection within 2 hr, whereas the released GTD accumulated for up to 8 hr. We observed that autoproteolytic deficient TcdB NXN C698S was degraded significantly faster than wildtype TcdB, suggesting interference of TcdB with lysosomal degradation process. In fact, TcdB reduced lysosomal degradation of endosome cargo as tested with DQ-Green BSA. Lysosomal dysfunction was accompanied by perinuclear accumulation of LAMP1 and a weaker detection in immunoblots. Galectin-8 or galectin-3 was not recruited to lysosomes speaking against lysosome membrane damage. Changes in the autophagosomal marker LC3B suggested additional indirect effect of lysosomal dysfunction on the autophagic flux. In contrast to necrotic signaling induced in by TcdB, lysosomal dysfunction was not abolished by calcium channel blocker nifedipin, indicating separate cytopathogenic effects induced by TcdB during endo-lysosomal trafficking.

Clostridioides difficile is the major cause of antibiotic-associated colitis (CDAC) with increasing prevalence in morbidity and mortality. Severity of CDAC has been attributed to hypervirulent C. difficile strains, which in addition to toxin A and B (TcdA, TcdB) produce the binary toxin C. difficile transferase (CDT). However, the link between these toxins and host immune responses as potential drivers of immunopathology are still incompletely understood. Here, we provide first experimental evidence that C. difficile toxins efficiently activate human mucosal-associated invariant T (MAIT) cells. Among the tested toxins, CDT and more specifically, the substrate binding and pore-forming subunit CDTb provoked significant MAIT cell activation resulting in selective MAIT cell degranulation of the lytic granule components perforin and granzyme B. CDT-induced MAIT cell responses required accessory immune cells, and we suggest monocytes as a potential CDT target cell population. Within the peripheral blood mononuclear cell fraction, we found increased IL-18 levels following CDT stimulation and MAIT cell response was indeed partly dependent on this cytokine. Surprisingly, CDT-induced MAIT cell activation was found to be partially MR1-dependent, although bacterial-derived metabolite antigens were absent. However, the role of antigen presentation in this process was not analyzed here and needs to be validated in future studies. Thus, MR1-dependent induction of MAIT cell cytotoxicity might be instrumental for hypervirulent C. difficile to overcome cellular barriers and may contribute to pathophysiology of CDAC.

Clostridioides difficile is a major cause of nosocomial infection worldwide causing antibiotic-associated diarrhea and some cases are leading to pseudomembranous colitis. The main virulence factors are toxin A and toxin B. Hypervirulent strains of C. difficile are linked to higher mortality rates and most of these strains produce additionally the C. difficile binary toxin (CDT) that possesses two subunits, CDTa and CDTb. The latter is responsible for binding and transfer of CDTa into the cytoplasm of target cells; CDTa is an ADP ribosyltransferase catalyzing the modification of actin fibers that disturbs the actin vs microtubule balance and induces microtubule-based protrusions of the cell membrane increasing the adherence of C. difficile. The underlying mechanisms remain elusive. Thus, we performed a screening experiment using MS-based proteomics and phosphoproteomics techniques. Epithelial Hep-2 cells were treated with CDTa and CDTb in a multiplexed study for 4 and 8 h. Phosphopeptide enrichment was performed using affinity chromatography with TiO2 and Fe-NTA; for quantification, a TMT-based approach and DDA measurements were used. More than 4,300 proteins and 5,600 phosphosites were identified and quantified at all time points. Although only moderate changes were observed on proteome level, the phosphorylation level of nearly 1,100 phosphosites responded to toxin treatment. The data suggested that CSNK2A1 might act as an effector kinase after treatment with CDT. Additionally, we confirmed ADP-ribosylation on Arg-177 of actin and the kinetic of this modification for the first time.

Pyrin is a cytosolic immune sensor that forms an inflammasome when bacterial virulence factors inhibit RhoA, triggering the release of inflammatory cytokines, including IL-1β. Gain of function mutations in the MEFV gene encoding Pyrin cause auto-inflammatory disorders, such as familial Mediterranean fever (FMF) and Pyrin associated auto-inflammation with Neutrophilic Dermatosis (PAAND). To precisely define the role of Pyrin in detecting pathogen virulence factors in relevant human immune cells, we investigated how the Pyrin inflammasome response was initiated and regulated in monocyte-derived macrophages (hMDM) compared to human monocytes. Unlike monocytes and murine macrophages, we determined that hMDM failed to activate Pyrin in response to known Pyrin activators Clostridioides difficile (C. difficile) toxins A or B (TcdA or TcdB). In contrast, TcdB activated the NLRP3 inflammasome in hMDM. Notably, we ascertained that the Pyrin inflammasome response could be re-enabled in hMDM by prolonged priming with either LPS or type I or II interferons, and required an increase in Pyrin expression. These data demonstrate an unexpected redundancy in detecting these toxins by inflammasome sensors.

Clostridium difficile toxin A (TcdA) is a major exotoxin contributing to disruption of the colonic epithelium during C. difficile infection. TcdA contains a carbohydrate-binding combined repetitive oligopeptides (CROPs) domain that mediates its attachment to cell surfaces, but recent data suggest the existence of CROPs-independent receptors. Here, we carried out genome-wide clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9)-mediated screens using a truncated TcdA lacking the CROPs, and identified sulfated glycosaminoglycans (sGAGs) and low-density lipoprotein receptor (LDLR) as host factors contributing to binding and entry of TcdA. TcdA recognizes the sulfation group in sGAGs. Blocking sulfation and glycosaminoglycan synthesis reduces TcdA binding and entry into cells. Binding of TcdA to the colonic epithelium can be reduced by surfen, a small molecule that masks sGAGs, by GM-1111, a sulfated heparan sulfate analogue, and by sulfated cyclodextrin, a sulfated small molecule. Cells lacking LDLR also show reduced sensitivity to TcdA, although binding between LDLR and TcdA are not detected, suggesting that LDLR may facilitate endocytosis of TcdA. Finally, GM-1111 reduces TcdA-induced fluid accumulation and tissue damage in the colon in a mouse model in which TcdA is injected into the caecum. These data demonstrate in vivo and pathological relevance of TcdA-sGAGs interactions, and reveal a potential therapeutic approach of protecting colonic tissues by blocking these interactions.

Pyrin is a cytosolic immune sensor that nucleates an inflammasome in response to inhibition of RhoA by bacterial virulence factors, triggering the release of inflammatory cytokines, including IL-1β. Gain-of-function mutations in the MEFV gene encoding Pyrin cause autoinflammatory disorders, such as familial Mediterranean fever (FMF) and Pyrin-associated autoinflammation with neutrophilic dermatosis (PAAND). To precisely define the role of Pyrin in pathogen detection in human immune cells, we compared initiation and regulation of the Pyrin inflammasome response in monocyte-derived macrophages (hMDM). Unlike human monocytes and murine macrophages, we determined that hMDM failed to activate Pyrin in response to known Pyrin activators Clostridioides difficile (C. difficile) toxins A or B (TcdA or TcdB), as well as the bile acid analogue BAA-473. The Pyrin inflammasome response was enabled in hMDM by prolonged priming with either LPS or type I or II interferons and required an increase in Pyrin expression. Notably, FMF mutations lifted the requirement for prolonged priming for Pyrin activation in hMDM, enabling Pyrin activation in the absence of additional inflammatory signals. Unexpectedly, in the absence of a Pyrin response, we found that TcdB activated the NLRP3 inflammasome in hMDM. These data demonstrate that regulation of Pyrin activation in hMDM diverges from monocytes and highlights its dysregulation in FMF.

A Clostridioides difficile infection (CDI) is the most common nosocomial infection worldwide. The main virulence factors of pathogenic C. difficile are TcdA and TcdB, which inhibit small Rho-GTPases. The inhibition of small Rho-GTPases leads to the so-called cytopathic effect, a reorganization of the actin cytoskeleton, an impairment of the colon epithelium barrier function and inflammation. Additionally, TcdB induces a necrotic cell death termed pyknosis in vitro independently from its glucosyltransferases, which are characterized by chromatin condensation and ROS production. To understand the underlying mechanism of this pyknotic effect, we conducted a large-scale phosphoproteomic study. We included the analysis of alterations in the phosphoproteome after treatment with TcdA, which was investigated for the first time. TcdA exhibited no glucosyltransferase-independent necrotic effect and was, thus, a good control to elucidate the underlying mechanism of the glucosyltransferase-independent effect of TcdB. We found RAS to be a central upstream regulator of the glucosyltransferase-independent effect of TcdB. The inhibition of RAS led to a 68% reduction in necrosis. Further analysis revealed apolipoprotein C-III (APOC3) as a possible crucial factor of CDI-induced inflammation in vivo.

Toxin B (TcdB) produced by Clostridioides difficile is a main pathogenicity factor that affects a variety of different cell types within the colonic mucosa. TcdB is known to utilize frizzled-1,2,7 and chondroitin sulfate proteoglycan-4 (CSPG4) as protein receptors. By using human cervical cancer cell line HeLa CSPG4 knockout (CSPG4&minus, /&minus, ) cells as well as TcdB mutants which do not bind to either CSPG4 or frizzled-1,2,7, or both, we evaluated the impact of the individual receptors for cytopathic and cytotoxic effects of TcdB. We compared TcdB from the reference strain VPI10463 (TcdBVPI) and the endemic strain R20291 (TcdBR20) which does not interact with frizzled-1,2,7. TcdBVPI devoid of CSPG4 binding (TcdBVPI &Delta, CROP) shows identical cytopathic potency as full-length TcdB in HeLa CSPG4&minus, cells, indicating that interaction with frizzled proteins is not affected in the presence of the C-terminal CROP domain. We validated CSPG4 as cellular receptor for both TcdB toxinotypes in HeLa and HEp-2 cells. By exchange of a single phenylalanine residue, 1597 with serine, we generated a mutated TcdBVPI variant (TcdBVPI F1597S) that in accordance with TcdBR20 lacks binding to frizzled-1,2,7 and showed identical potency as TcdBR20 on HeLa cells. This enabled us to estimate the respective share of CSPG4 and frizzled-1,2,7 in the cytotoxic and cytopathic effect induced by TcdB. Our data reveal that binding to frizzled-1,2,7 and to CSPG4 occurs independently and in an additive manner.

Summary The exotoxin TcsL is a major virulence factor in Paeniclostridium (Clostridium) sordellii and responsible for the high lethality rate associated with P. sordellii infection. Here, we present a genome-wide CRISPR-Cas9-mediated screen using a human lung carcinoma cell line and identify semaphorin (SEMA) 6A and 6B as receptors for TcsL. Disrupting SEMA6A/6B expression in several distinct human cell lines and primary human endothelial cells results in reduced TcsL sensitivity, while SEMA6A/6B over-expression increases their sensitivity. TcsL recognizes the extracellular domain (ECD) of SEMA6A/6B via a region homologous to the receptor-binding site in Clostridioides difficile toxin B (TcdB), which binds the human receptor Frizzled. Exchanging the receptor-binding interfaces between TcsL and TcdB switches their receptor-binding specificity. Finally, administration of SEMA6A-ECD proteins protects human cells from TcsL toxicity and reduces TcsL-induced damage to lung tissues and the lethality rate in mice. These findings establish SEMA6A and 6B as pathophysiologically relevant receptors for TcsL.

Objectives The most effective first-line treatment between endovascular therapy and bypass surgery for patients with critical limb ischemia (CLI) is still not well defined. The primary aim of the interim analysis of CRITISCH (Registry of First-Line Treatments in Patients With Critical Limb Ischemia) was to compare both treatment options in a prospective confirmatory manner. Background Only 1 randomized controlled trial between endovascular therapy and bypass surgery has been published yet. Several retrospective studies showed comparable outcomes between the 2 treatment strategies, but in the majority of them, current endovascular technologies have not been included. Methods Between January 2013 and September 2014, 1,200 CLI patients (Rutherford 4 to 6) from 27 vascular centers were enrolled. The selection of the first-line treatment was left completely to the discretion of the responsible physician. The primary composite endpoint was amputation-free survival (AFS), that is, time to major amputation and/or death from any cause. A pre-specified interim analysis aimed at showing noninferiority of the endovascular therapy versus bypass surgery as to the hazard ratio (HR) of AFS (noninferiority bound = 1.33; interim α = 0.0058). Time-to-event analyses of major amputation, death, and the composite endpoint of reintervention and/or above-ankle amputation were also conducted. Results Endovascular therapy was applied to 642 (54%) and bypass surgery to 284 (24%) patients. Median follow-up time was 12 months in both groups. One-year AFS was 75% and 72%, respectively. The noninferiority of endovascular therapy versus bypass surgery for AFS was confirmed (HR: 0.91; upper bound of 1-sided (1 − 0.0058) confidence interval [CI]: 1.29; p = 0.003). An impact of the treatment strategy on time until death (HR: 1.14; 95% CI: 0.80 to 1.63; p = 0.453), major amputation (HR: 0.86; 95% CI:0.56 to 1.30; p = 0.463), and reintervention and/or above-ankle amputation (HR: 0.89; 95% CI: 0.70 to 1.14; p = 0.348) was not observed. Conclusions The interim analysis confirmed that when physicians are free to individualize therapy to CLI patients, the endovascular-first approach achieved a noninferior AFS rate compared with bypass surgery. (Registry of First-Line Treatments in Patients With Critical Limb Ischemia [CRITISCH]; NCT01877252)

Clostridioides difficile toxin A (TcdA) and Toxin B (TcdB) trigger inflammasome activation with caspase-1 activation in cultured cells, which in turn induce the release of IL-6, IFN-γ, and IL-8. Release of these proinflammatory responses is positively regulated by Ras-GTPases, which leads to the hypothesis that Ras glucosylation by glucosylating toxins results in (at least) reduced proinflammatory responses. Against this background, data on toxin-catalyzed Ras glucosylation are required to estimate of pro-inflammatory effect of the glucosylating toxins. In this study, a quantitative evaluation of the GTPase substrate profiles glucosylated in human colonic (Caco-2) cells treated with either TcdA, TcdB, or the related Clostridium sordellii lethal toxin (TcsL) was performed using multiple reaction monitoring (MRM) mass spectrometry. (H/K/N)Ras are presented to be glucosylated by TcsL and TcdA but by neither TcdB isoform tested. Furthermore, the glucosylation of (H/K/N)Ras was detected in TcdA-(not TcdB)-treated cells, as analyzed exploiting immunoblot analysis using the Ras glucosylation-sensitive 27H5 antibody. Furthermore, [14C]glucosylation of substrate GTPase was found to be increased in a cell-free system complemented with Caco-2 lysates. Under these conditions, (H/K/N)Ras glucosylation by TcdA was detected. In contrast, TcdB-catalyzed (H/K/N)Ras glucosylation was detected by neither MRM analysis, immunoblot analysis nor [14C]glucosylation in a cell-free system. The observation that TcdA (not TcdB) glucosylates Ras subtype GTPases correlates with the fact that TcdB (not TcdA) is primarily responsible for inflammatory responses in CDI. Finally, TcsL more efficaciously glucosylated Ras subtype GTPase as compared with TcdA, reinforcing the paradigm that TcsL is the prototype of a Ras glucosylating toxin.

Clostridioides difficile is the causative bacterium in 15–20% of all antibiotic associated diarrheas. The symptoms associated with C. difficile infection (CDI) are primarily induced by the two large exotoxins TcdA and TcdB. Both toxins enter target cells by receptor-mediated endocytosis. Although different toxin receptors have been identified, it is no valid therapeutic option to prevent receptor endocytosis. Therapeutics, such as neutralizing antibodies, directly targeting both toxins are in development. Interestingly, only the anti-TcdB antibody bezlotoxumab but not the anti-TcdA antibody actoxumab prevented recurrence of CDI in clinical trials. In this work, 31 human antibody fragments against TcdB were selected by antibody phage display from the human naive antibody gene libraries HAL9/10. These antibody fragments were further characterized by in vitro neutralization assays. The epitopes of the neutralizing and non-neutralizing antibody fragments were analyzed by domain mapping, TcdB fragment phage display, and peptide arrays, to identify neutralizing and non-neutralizing epitopes. A new neutralizing epitope within the glucosyltransferase domain of TcdB was identified, providing new insights into the relevance of different toxin regions in respect of neutralization and toxicity.

The large clostridial glucosylating toxin B (TcdB) is a major virulence factor of the nosocomial pathogen Clostridioides difficile. TcdB inhibits small GTPases by glucosylation leading to impaired downstream signaling. TcdB also possesses a glucosyltransferase independent effect described as pyknosis. To elucidate the impact of TcdB and its glucosylation-inactive mutant TcdBNXN on the kinome of human cells, SILAC labeled HEp-2 cells were treated with 2 nM TcdB for 8 h. Phosphopeptides were enriched using SCX chromatography, IMAC and TiO2 followed shotgun mass spectrometry analysis. Overall 4,197 phosphopeptides were identified; more than 1,200 phosphosites responded to treatment with TcdB or TcdBNXN. The data suggested that predominantly stress-activated MAPK-dependent signaling pathways were triggered by toxin B treatment.

Clostridioides difficile toxins TcdA and TcdB are large clostridial glucosyltransferases which are the main pathogenicity factors in C. difficile-associated diseases. Four highly conserved cysteines are present in all large clostridial glucosyltransferases. In this study we focused on the conserved cysteine 2232 within the combined repetitive oligopeptide domain of TcdB from reference strain VPI10463 (clade I). Cysteine 2232 is not present in TcdB from hypervirulent strain R20291 (clade II), where a tyrosine is found instead. Replacement of cysteine 2232 by tyrosine in TcdBV PI10463 reduced binding to the soluble fragments of the two known TcdB receptors, frizzled-2 (FZD2) and poliovirus receptor-like protein-3/nectin-3 (PVRL3). In line with this, TcdBR20291 showed weak binding to PVRL3 in pull-down assays which was increased when tyrosine 2232 was exchanged for cysteine. Surprisingly, we did not observe binding of TcdBR20291 to FZD2, indicating that this receptor is less important for this toxinotype. Competition assay with the receptor binding fragments (aa 1101–1836) of TcdBV PI10463 and TcdBR20291, as well as antibodies newly developed by antibody phage display, revealed different characteristics of the yet poorly described delivery domain of TcdB harboring the second receptor binding region. In summary, we found that conserved Cys-2232 in TcdB indirectly contributes to toxin–receptor interaction.

The pathogenicity locus (PaLoc) of Clostridioides difficile usually comprises five genes (tcdR, tcdB, tcdE, tcdA, tcdC). While the proteins TcdA and TcdB represent the main toxins of this pathogen, TcdR and TcdC are involved in the regulation of their production. TcdE is a holin family protein, members of which are usually involved in the transport of cell wall-degrading enzymes (endolysins) for phage-induced lysis. In the past, TcdE has been shown to contribute to the release of TcdA and TcdB, but it is unclear whether it mediates a specific transport or rather a lysis of cells. TcdE of C. difficile strains analyzed so far can be produced in three isoforms that are initiated from distinct N-terminal ATG codons. When produced in Escherichia coli, we found that the longest TcdE isoform had a moderate effect on cell growth, whereas the shortest isoform strongly induced lysis. The effect of the longest isoform was inhibitory for cell lysis, implying a regulatory function of the N-terminal 24 residues. We analyzed the PaLoc sequence of 44 C. difficile isolates and found that four of these apparently encode only the short TcdE isoforms, and the most closely related holins from C. difficile phages only possess one of these initiation codons, indicating that an N-terminal extension of TcdE evolved in C. difficile. All PaLoc sequences comprised also a conserved gene encoding a short fragment of an endolysin remnant of a phage holin/endolysin pair. We could produce this peptide, which we named TcdL, and demonstrated by bacterial two-hybrid analysis a self-interaction and an interaction with TcdB that might serve to mediate TcdE-dependent transport.

Clostridioides difficile is the causative bacterium in 15–20% of all antibiotic associated diarrheas. The symptoms associated with C. difficile infection (CDI) are primarily induced by the two large exotoxins TcdA and TcdB. Both toxins enter target cells by receptor-mediated endocytosis. Although different toxin receptors have been identified, it is no valid therapeutic option to prevent receptor endocytosis. Therapeutics, such as neutralizing antibodies, directly targeting both toxins are in development. Interestingly, only the anti-TcdB antibody bezlotoxumab but not the anti-TcdA antibody actoxumab prevented recurrence of CDI in clinical trials. In this work, 31 human antibody fragments against TcdB were selected by antibody phage display from the human naive antibody gene libraries HAL9/10. These antibody fragments were further characterized by in vitro neutralization assays. The epitopes of the neutralizing and non-neutralizing antibody fragments were analyzed by domain mapping, TcdB fragment phage display, and peptide arrays, to identify neutralizing and non-neutralizing epitopes. A new neutralizing epitope within the glucosyltransferase domain of TcdB was identified, providing new insights into the relevance of different toxin regions in respect of neutralization and toxicity.

Purpose: To examine the outcomes of all first-line strategies for the treatment of critical limb ischemia (CLI), identify factors that influenced the treatment choice, and determine the risk of amputation or death after each treatment. Methods: CRITISCH ( ClinicalTrials.gov identifier NCT01877252) is a multicenter, national, prospective registry evaluating all available treatment strategies applied in 1200 consecutive CLI patients in 27 vascular centers in Germany. The recruitment started in January 2013 and was completed in September 2014. Treatment options were endovascular revascularization (642, 53.5%), bypass surgery (284, 23.7%), femoral artery patchplasty (126, 10.5%) with or without concomitant peripheral intervention, conservative treatment (118, 9.8%), and primary major amputation (30, 2.5%). The primary endpoint of this study was amputation-free survival (AFS). The Society of Vascular Surgery’s suggested objective performance goal (OPG) for AFS (71%) was used as the effectiveness criterion. Multivariable regression methods were employed to identify variables that influenced the treatment selection and AFS after each treatment; results are presented as the hazard ratio (HR) and 95% confidence interval (CI). Results: The 12-month AFS estimates following endovascular therapy, bypass grafting, femoral patchplasty, and conservative treatment were 75%, 72%, 73%, and 72%, respectively. Factors influencing treatment choice were age, chronic kidney disease (CKD), diabetes, smoking, prior vascular procedures in the index leg, TransAtlantic Inter-Society Consensus II C/D lesions, and absence of runoff vessels. Cox regression analysis identified CKD (HR 2.07, 95% CI 1.26 to 3.41, p=0.004), the use of a prosthetic bypass conduit (HR 1.97, 95% CI 1.23 to 3.14, p=0.004), and previous vascular intervention in the index limb (HR 1.52, 95% CI 0.94 to 2.43, p=0.085) as independent risk factors for diminished AFS after bypass surgery. CKD (HR 1.47, 95% CI 1.09 to 1.99, p=0.012) and Rutherford category 6 (HR 1.81, 95% CI 1.30 to 2.52, p

The pathogenicity locus (PaLoc) of Clostridioides difficile usually comprises five genes (tcdR, tcdB, tcdE, tcdA, tcdC). While the proteins TcdA and TcdB represent the main toxins of this pathogen, TcdR and TcdC are involved in the regulation of their production. TcdE is a holin family protein, members of which are usually involved in the transport of cell wall-degrading enzymes (endolysins) for phage-induced lysis. In the past, TcdE has been shown to contribute to the release of TcdA and TcdB, but it is unclear whether it mediates a specific transport or rather a lysis of cells. TcdE of C. difficile strains analyzed so far can be produced in three isoforms that are initiated from distinct N-terminal ATG codons. When produced in Escherichia coli, we found that the longest TcdE isoform had a moderate effect on cell growth, whereas the shortest isoform strongly induced lysis. The effect of the longest isoform was inhibitory for cell lysis, implying a regulatory function of the N-terminal 24 residues. We analyzed the PaLoc sequence of 44 C. difficile isolates and found that four of these apparently encode only the short TcdE isoforms, and the most closely related holins from C. difficile phages only possess one of these initiation codons, indicating that an N-terminal extension of TcdE evolved in C. difficile. All PaLoc sequences comprised also a conserved gene encoding a short fragment of an endolysin remnant of a phage holin/endolysin pair. We could produce this peptide, which we named TcdL, and demonstrated by bacterial two-hybrid analysis a self-interaction and an interaction with TcdB that might serve to mediate TcdE-dependent transport.

Clostridioides difficile toxin A (TcdA) and Toxin B (TcdB) trigger inflammasome activation with caspase-1 activation in cultured cells, which in turn induce the release of IL-6, IFN-γ, and IL-8. Release of these proinflammatory responses is positively regulated by Ras-GTPases, which leads to the hypothesis that Ras glucosylation by glucosylating toxins results in (at least) reduced proinflammatory responses. Against this background, data on toxin-catalyzed Ras glucosylation are required to estimate of pro-inflammatory effect of the glucosylating toxins. In this study, a quantitative evaluation of the GTPase substrate profiles glucosylated in human colonic (Caco-2) cells treated with either TcdA, TcdB, or the related Clostridium sordellii lethal toxin (TcsL) was performed using multiple reaction monitoring (MRM) mass spectrometry. (H/K/N)Ras are presented to be glucosylated by TcsL and TcdA but by neither TcdB isoform tested. Furthermore, the glucosylation of (H/K/N)Ras was detected in TcdA-(not TcdB)-treated cells, as analyzed exploiting immunoblot analysis using the Ras glucosylation-sensitive 27H5 antibody. Furthermore, [14C]glucosylation of substrate GTPase was found to be increased in a cell-free system complemented with Caco-2 lysates. Under these conditions, (H/K/N)Ras glucosylation by TcdA was detected. In contrast, TcdB-catalyzed (H/K/N)Ras glucosylation was detected by neither MRM analysis, immunoblot analysis nor [14C]glucosylation in a cell-free system. The observation that TcdA (not TcdB) glucosylates Ras subtype GTPases correlates with the fact that TcdB (not TcdA) is primarily responsible for inflammatory responses in CDI. Finally, TcsL more efficaciously glucosylated Ras subtype GTPase as compared with TcdA, reinforcing the paradigm that TcsL is the prototype of a Ras glucosylating toxin.

Binary toxins are produced by several pathogenic bacteria. Examples are the C2 toxin from Clostridium botulinum, the iota toxin from Clostridium perfringens, and the CDT from Clostridium difficile. All these binary toxins have ADP-ribosyltransferases (ADPRT) as their enzymatically active component that modify monomeric actin in their target cells. The binary C2 toxin was intensively described as a tool for intracellular delivery of allogenic ADPRTs. Here, we firstly describe the binary toxin CDT from C. difficile as an effective tool for heterologous intracellular delivery. Even 60 kDa glucosyltransferase domains of large clostridial glucosyltransferases can be delivered into cells. The glucosyltransferase domains of five tested large clostridial glucosyltransferases were successfully introduced into cells as chimeric fusions to the CDTa adapter domain (CDTaN). Cell uptake was demonstrated by the analysis of cell morphology, cytoskeleton staining, and intracellular substrate glucosylation. The fusion toxins were functional only when the adapter domain of CDTa was N-terminally located, according to its native orientation. Thus, like other binary toxins, the CDTaN/b system can be used for standardized delivery systems not only for bacterial ADPRTs but also for a variety of bacterial glucosyltransferase domains.

Toxin A and Toxin B (TcdA/TcdB) are large glucosyltransferases produced by Clostridium difficile. TcdB but not TcdA induces reactive oxygen species-mediated early cell death (ECD) when applied at high concentrations. We found that nonglucosylated Rac1 is essential for induction of ECD since inhibition of Rac1 impedes this effect. ECD only occurs when TcdB is rapidly endocytosed. This was shown by generation of chimeras using the trunk of TcdB from a hypervirulent strain. TcdB from hypervirulent strain has been described to translocate from endosomes at higher pH values and thus, meaning faster than reference type TcdB. Accordingly, intracellular delivery of the glucosyltransferase domain of reference TcdB by the trunk of TcdB from hypervirulent strain increased ECD. Furthermore, proton transporters such as sodium/proton exchanger (NHE) or the ClC-5 anion/proton exchanger, both of which contribute to endosomal acidification, also affected cytotoxic potency of TcdB: Specific inhibition of NHE reduced cytotoxicity, whereas transfection of cells with the endosomal anion/proton exchanger ClC-5 increased cytotoxicity of TcdB. Our data suggest that both the uptake rate of TcdB into the cytosol and the status of nonglucosylated Rac1 are key determinants that are decisive for whether ECD or delayed apoptosis is triggered.

Clostridium difficile toxins A and B (TcdA and TcdB) belong to the class of large clostridial cytotoxins and inactivate by glucosylation some low molecular mass GTPases of the Rho-family (predominantly Rho, Rac and Cdc42), known as regulators of the actin cytoskeleton. TcdA and B also represent the main virulence factors of the anaerobic gram-positive bacterium that is the causal agent of pseudomembranous colitis. In our study, TcdB was chosen instead of TcdA for the well-known higher cytotoxic potency. Inactivation of Rho-family GTPases by this toxin in our experimental conditions induced morphological changes and reduction of electron-dense mast cell-specific granules in human mast cell line-1 (HMC-1) cells, but not cell death or permeabilisation of plasma-membranes. Previously reported patch-clamp dialysis experiments revealed that high intracellular free-Ca(2+) and GTPγS concentrations are capable of inducing exocytosis as indicated by significant membrane capacitance (Cm) increases in HMC-1 cells. In this study, we investigated the direct effects of TcdB upon HMC-1 cell "stimulated" Cm increase, as well as on "constitutive" secretion of hexosaminidase and interleukin-16 (IL-16). Compared to untreated control cells, HMC-1 cells incubated with TcdB for 3-24h exhibited a significant reduction of the mean absolute and relative Cm increase in response to free-Ca(2+) and GTPγS suggesting an inhibition of secretory processes by TcdB. In conclusion, the HMC-1 cell line represents a suitable model for the study of direct effects of C. difficile toxins on human mast cell secretory activity.

An analysis was conducted of intermediate outcomes and possible influencing factors in patients with end-stage renal disease (ESRD) and critical limb ischemia after lower limb revascularization compared with patients with regular renal function (non-ESRD).Data collection was performed by inquiry of the German multicenter registry of First-Line Treatments in Patients with Critical Limb Ischemia (CRITISCH); 102 ESRD patients and 674 non-ESRD patients were included. Four different therapy modalities were analysed: bypass surgery, endovascular therapy (EVT), femoral artery endarterectomy, and no vascular intervention (conservative treatment or primary major amputation). Predefined end points were amputation-free survival (AFS), death, major amputation, and reintervention. Cox regression models were built to analyze independent risk factors for outcome parameters.ESRD patients showed inferior results at 2 years in the rate of AFS (ESRD, 35.4%; non-ESRD, 67.2%; P .001). Similarly, death rate (ESRD, 55.0%; non-ESRD, 20.7%; P .001) and major amputation rate (ESRD, 24.5%; non-ESRD, 15.8%; P = .029) were significantly elevated for ESRD patients. The choice of therapeutic approach in ESRD did not influence the incidence of the investigated end points (death or major amputation: EVT, 56.9% vs bypass, 76.9% [P = .225]; death: EVT, 46.2% vs bypass, 61.5% [P = .372]; amputation: EVT, 15.4% vs bypass, 15.4% [P = 1.000]; reintervention: EVT, 32.3% vs bypass, 15.4% [P = .324]). Cox regression analysis indicated that dialysis patients carry a twofold increased hazard of death or major amputation (hazard ratio, 2.27; 95% confidence interval, 1.67-3.10; P .001), and open surgical treatment (all patients combined) was associated with reduced risk of death compared with EVT (hazard ratio, 0.58; 95% confidence interval, 0.37-0.91; P = .017). Comorbidities were not found to have a noticeable impact on AFS, survival, reintervention, or major amputation.Two-year AFS, overall survival, and freedom from major amputation were decreased in ESRD patients compared with non-ESRD patients with critical limb ischemia. Cardiovascular comorbidities were without significant impact on outcome parameters, whereas choice of treatment modality within the ESRD group did not influence AFS. Decision-making in ESRD as to choice of therapeutic approach in dialysis patients should notably account for the individual's lesion characteristics and vascular disease; surgical revascularization and EVT may be used as complementary options.

Secondary prevention in patients with critical limb ischemia (CLI) is crucial for the reduction of cardiovascular morbidity and mortality. Nonetheless, current recommendations are extrapolated from other high-risk populations because of the lack of CLI-dedicated trials. The aim of this explorative study was to evaluate the association of statin therapy with the outcomes of CLI patients.The First-Line Treatments in Patients With Critical Limb Ischemia (CRITISCH) registry is a prospective multicenter registry analyzing the effectiveness of all available treatment strategies in 1200 CLI patients. For the purposes of this analysis, patients were divided into two groups based on statin administration. Treatment crossovers and nonadherent patients were excluded from analysis. The primary composite end point of this study was the amputation-free survival (AFS). Major adverse cardiovascular and cerebral events (MACCEs), time to death, and time to major amputation were also analyzed.Statin therapy was applied in 445 individuals (37%), 371 (31%) patients received no statins, and 384 subjects were excluded from analysis (treatment crossovers). Patients receiving statins were more likely to be younger (P .001) and to have a history of coronary heart disease (P .001) or previous intervention at index limb (P .001). Patients receiving statin therapy had a lower hazard regarding AFS (hazard ratio [HR], 0.45; 95% confidence interval [CI], 0.34-0.63; P .001) and death (HR, 0.40; 95% CI, 0.24-0.66; P .001) as well as lower odds of MACCE (odds ratio, 0.41; 95% CI, 0.23-0.69; P = .001). However, statin therapy was not associated with reduced amputation rates (HR, 1.02; 95% CI, 0.67-1.56; P = .922). Statin effect on AFS was consistent among diabetics (HR, 0.47; 95% CI, 0.31-0.70; P .001), patients with chronic kidney disease (HR, 0.53; 95% CI, 0.32-0.87; P = .012), and patients older than 75 years (HR, 0.40; 95% CI, 0.26-0.60; P .001). Statin administration was also associated with an improved AFS in patients with antiplatelet medication (HR, 0.64; 95% CI, 0.41-0.99; P = .049) and without antiplatelet medication (HR, 0.26; 95% CI, 0.12-0.57; P = .001) and after both endovascular therapy (HR, 0.51; 95% CI, 0.34-0.76; P = .001) and bypass revascularization (HR, 0.38; 95% CI, 0.21-0.68; P = .001).Statin therapy in CLI patients is associated with an increased AFS and lower rates of mortality and MACCEs without improving, however, the salvage rates of the affected limb.

Summary TcdA and TcdB are the main pathogenicity factors of Clostridium difficile-associated diseases. Both toxins inhibit Rho GTPases, and consequently, apoptosis is induced in the affected cells. We found that TcdB at higher concentrations exhibits cytotoxic effects that are independent on Rho glucosylation. TcdB and the glucosyltransferase-deficient mutant TcdB D286/288N induced pyknotic cell death which was associated with chromatin condensation and reduced H3 phosphorylation. Affected cells showed ballooning of the nuclear envelope and loss of the integrity of the plasma membrane. Furthermore, pyknotic cells were positively stained with dihydroethidium indicating production of reactive oxygen species. In line with this, pyknosis was reduced by apocynin, an inhibitor of the NADPH oxidase. Bafilomycin A1 prevented cytotoxic effects showing that the newly observed pyknosis depends on intracellular action of TcdB rather than on a receptor-mediated effect. Blister formation and chromatin condensation was specifically induced by the glucosyltransferase domain of TcdB from strain VPI10473 since neither TcdBF from cdi1470 nor the chimera of TcdB harbouring the glucosyltransferase domain of TcdBF was able to induce these effects. In summary, TcdB induces two different and independent phenotypes: (i) cell rounding due to glucosylation of Rho GTPases and (ii) shrinkage of cells and nuclear blister induced by the high concentrations of TcdB independent of Rho glucosylation.

Toxin A (TcdA) and B (TcdB) from Clostridium difficile enter host cells by receptor-mediated endocytosis. A prerequisite for proper toxin action is the intracellular release of the glucosyltransferase domain by an inherent cysteine protease, which is allosterically activated by inositol hexaphosphate (IP6). We found that in in vitro assays, the C-terminally-truncated TcdA1–1065 was more efficient at IP6-induced cleavage compared with full-length TcdA. We hypothesized that the C-terminally-located combined repetitive oligopeptides (CROPs) interact with the N-terminal part of the toxin, thereby preventing autoproteolysis. Glutathione-S-transferase (GST) pull-down assays and microscale thermophoresis confirmed binding between the CROPs and the glucosyltransferase (TcdA1–542) or intermediate (TcdA1102–1847) domain of TcdA, respectively. This interaction between the N- and C-terminus was not found for TcdB. Functional assays revealed that TcdB was more susceptible to inactivation by extracellular IP6-induced cleavage. In vitro autoprocessing and inactivation of TcdA, however, significantly increased, either by acidification of the surrounding milieu or following exchange of its CROP domain by the homologous CROP domain of TcdB. Thus, TcdA CROPs contribute to the stabilization and protection of toxin conformation in addition to function as the main receptor binding domain.

An increase in cytosolic free calcium concentration [Ca2+]i initiates the exocytotic activity in various types of secretory cells. The guanosine 5′-O-[3-thio]triphosphate (GTPγS), a non-hydrolysable analogue of GTP (guanosine 5′-triphosphate), is an effective secretagogue for different cell types of different species, like mast cells, neutrophils or eosinophils. Consequently, the internal administration of GTPγS causes degranulation of mouse and rat mast cells. Regarding rat mast cells, it is proved that Ca2+ can cooperate with GTP or GTPγS in accelerating and increasing amplitude of the secretory response. All the previous studies with respect to capacitance recordings and mast cells were performed using mouse or rat mast cells, usually derived from peritoneum or the rat basophilic leukaemia cell line RBL. In this study, we applied the capacitance measurement technique to the human mast cell line-1 (HMC-1) cells, an immature cell line established from a patient with mast cell leukaemia. Patch-clamp dialysis experiments revealed that high intracellular free Ca2+ and GTPγS concentrations are both required for considerable capacitance increases in HMC-1 cells. During degranulation of HMC-1 cells, the total membrane capacitance (Cm) increase appeared continuously and, in some cases, as a discrete capacitance change, developing in a stepwise manner. Then, we tested the effect of latrunculin B upon HMC-1 cell capacitance increase as well as of some classic mast cell stimulators like PMA, A23187 and IL-1β in hexosaminidase release. Finally, we could conclude that the HMC-1 cell line represents a suitable model for the study of human mast cell degranulation.

Clostridium difficile is the major cause of intestinal infections in hospitals. The major virulence factors are toxin A (TcdA) and toxin B (TcdB), which belong to the group of clostridial glucosylating toxins (CGT) that inactivate small GTPases. After a 24 h incubation period with TcdA or a glucosyltransferase-deficient mutant TcdA (gdTcdA), quantitative changes in the proteome of colonic cells (Caco-2) were analyzed using high-resolution LC-MS/MS and the SILAC technique. The changes in abundance of more than 5100 proteins were quantified. Nearly 800 toxin-responsive proteins were identified that were involved in cell cycle, cell structure, and adhesion as well as metabolic processes. Several proteins localized to mitochondria or involved in lipid metabolism were consistently of higher abundance after TcdA treatment. All changes of protein abundance depended on the glucosyltransferase activity of TcdA. Glucosylation of the known targets of TcdA such as RhoA, RhoC, RhoG was detected by LC-MS/MS. In addition, an almost complete glucosylation of Rap1(A/B), Rap2(A/B/C) and a partial glucosylation of Ral(A/B) and (H/K/N)Ras were detected. The glucosylation pattern of TcdA was compared to that of other CGT like TcdB, the variant TcdB from C. difficile strain VPI 1470 (TcdBF), and lethal toxin from C. sordellii (TcsL).

Toxin B (TcdB) of the nosocomial pathogen C. difficile has been reported to exhibit a glucosyltransferase-dependent and -independent effect on treated HEp-2 cells at toxin concentration above 0.3 nM. In order to investigate and further characterize both effects epithelial cells were treated with wild type TcdB and glucosyltransferase-deficient TcdBNXN and their proteomes were analyzed by LC-MS. Triplex SILAC labeling was used for quantification. Identification of 5212 and quantification of 4712 protein groups was achieved. Out of these 257 were affected by TcdB treatment, 92 by TcdBNXN treatment and 49 by both. TcdB mainly led to changes in proteins that are related to "GTPase mediated signaling" and the "cytoskeleton" while "chromatin" and "cell cycle" related proteins were altered by both, TcdB and TcdBNXN . The obtained dataset of HEp-2 cell proteome helps us to better understand glucosyltransferase-dependent and -independent mechanisms of TcdB and TcdBNXN , particularly those involved in pyknotic cell death. All proteomics data have been deposited in the ProteomeXchange with the dataset identifier PXD006658 (https://proteomecentral.proteomexchange.org/dataset/PXD006658).

Virulent C. difficile toxins TcdA and TcdB invade host intestinal epithelia by endocytosis and use the acidic environment of intracellular vesicles for further processing and activation. We investigated the role of ClC-5, a chloride/proton exchanger expressed in the endosomes of gastrointestinal epithelial cells, in the activation and processing of C. difficile toxins. Enhanced intoxication by TcdA and TcdB was observed in cells expressing ClC-5 but not ClC-4, another chloride/proton exchanger with similar function but different localization. In accordance with the established physiological function of ClC-5, its expression lowered the endosomal pH in HEK293T cells by approximately 0.6 units and enhanced approximately 5-fold the internalization of TcdA. In colon HT29 cells, 34% of internalized TcdA localized to ClC-5-containing vesicles defined by colocalization with Rab5, Rab4a, and Rab7 as early and early-to-late of endosomes but not as Rab11-containing recycling endosomes. Impairing the cellular uptake of TcdA by deleting the toxin CROPs domain did not abolish the effects of ClC-5. In addition, the transport-incompetent mutant ClC-5 E268Q similarly enhanced both endosomal acidification and intoxication by TcdA but facilitated the internalization of the toxin to a lower extent. These data suggest that ClC-5 enhances the cytotoxic action of C. difficile toxins by accelerating the acidification and maturation of vesicles of the early and early-to-late endosomal system. The dispensable role of electrogenic ion transport suggests that the voltage-dependent nonlinear capacitances of mammalian CLC transporters serve important physiological functions. Our data shed light on the intersection between the endocytotic cascade of host epithelial cells and the internalization pathway of the large virulence C. difficile toxins. Identifying ClC-5 as a potential specific host ion transporter hijacked by toxins produced by pathogenic bacteria widens the horizon of possibilities for novel therapies of life-threatening gastrointestinal infections.

Two characteristics of toxins A and B from C. difficile (TcdA, TcdB) are important for the understanding of the pathogenic effect of these homologous toxins. First, these toxins are huge single-chain but multidomain proteins that display their action intracellularly within the cytosol of host cells. And second, albeit various cell types highly differ in their sensitivity toward these toxins, no toxin-resistant cell type has been described yet. Investigation of receptor-mediated uptake of these toxins is very ambitious. It demands discrimination between cell surface binding, interaction with more than one functional receptor responsible for uptake as well as other functional receptors that recognize bacterial pathogens and are not necessarily related with endocytosis. The current understanding of a complex uptake process is that TcdB interacts with at least two facultative receptors that mediate entry into host cells by redundant endocytotic pathways. Although both homologous toxins do obviously not share the same receptors, this principle of redundant binding domains found for TcdB does also account for TcdA.

Clostridium difficile toxin B (TcdB) is a critical virulence factor that causes diseases associated with C. difficile infection. Here we carried out CRISPR-Cas9-mediated genome-wide screens and identified the members of the Wnt receptor frizzled family (FZDs) as TcdB receptors. TcdB binds to the conserved Wnt-binding site known as the cysteine-rich domain (CRD), with the highest affinity towards FZD1, 2 and 7. TcdB competes with Wnt for binding to FZDs, and its binding blocks Wnt signalling. FZD1/2/7 triple-knockout cells are highly resistant to TcdB, and recombinant FZD2-CRD prevented TcdB binding to the colonic epithelium. Colonic organoids cultured from FZD7-knockout mice, combined with knockdown of FZD1 and 2, showed increased resistance to TcdB. The colonic epithelium in FZD7-knockout mice was less susceptible to TcdB-induced tissue damage in vivo. These findings establish FZDs as physiologically relevant receptors for TcdB in the colonic epithelium.

PURPOSE This study was carried out to investigate the impact of high concentrations of Clostridium difficile toxin A (TcdA) on the proteome of human cells. It should also be examined whether a catalytically deficient mutant (TcdANXN ) has an effect on target cells. EXPERIMENTAL DESIGN Proteome changes were investigated after treatment of HEp-2 cells with 20 nM TcdA for 8 h using a triplex SILAC labeling method and shotgun proteomics. Proteins from differently labeled and treated cells were combined for analysis using an HPLC coupled to an Orbitrap mass spectrometer. RESULTS Nearly 4000 proteins were identified in each replicate and 3500 could be quantified by SILAC triplicate analysis. 51 proteins exhibited an altered abundance with 29 up-regulated and 22 down-regulated proteins. In contrast, TcdANXN had no provable impact on the protein profile of HEp-2 cells. Data analysis of regulated proteins revealed that mainly plasma membrane, cell death, cell proliferation and actin cytoskeleton proteins were affected by TcdA treatment. CONCLUSIONS AND CLINICAL RELEVANCE This proteome analysis showed novel insights of TcdA impact onepithelial cells. Comparison with long-term treatment studies reveals distinctions in affected cellular processes that will improve the understanding of TcdA functions and might help to find new tools for diagnosis and treatment of CDI.

Clostridium difficile infections can induce mild to severe diarrhoea and the often associated characteristic pseudomembranous colitis. Two protein toxins, the large glucosyltransferases TcdA and TcdB, are the main pathogenicity factors that can induce all clinical symptoms in animal models. The classical molecular mode of action of these homologous toxins is the inhibition of Rho GTPases by mono-glucosylation. Rho-inhibition leads to breakdown of the actin cytoskeleton, induces stress-activated and pro-inflammatory signaling and eventually results in apoptosis of the affected cells. An increasing number of reports, however, have documented further qualities of TcdA and TcdB, including the production of reactive oxygen species (ROS) by target cells. This review summarizes observations dealing with the production of ROS induced by TcdA and TcdB, dissects pathways that contribute to this phenomenon and speculates about ROS in mediating pathogenesis. In conclusion, ROS have to be considered as a discrete, glucosyltransferase-independent quality of at least TcdB, triggered by different mechanisms.

Spinal muscular atrophy (SMA), a frequent neurodegenerative disease, is caused by reduced levels of functional survival of motoneuron (SMN) protein. SMN is involved in multiple pathways, including RNA metabolism and splicing as well as motoneuron development and function. Here we provide evidence for a major contribution of the Rho-kinase (ROCK) pathway in SMA pathogenesis. Using an in vivo protein interaction system based on SUMOylation of proteins, we found that SMN is directly interacting with profilin2a. Profilin2a binds to a stretch of proline residues in SMN, which is heavily impaired by a novel SMN2 missense mutation (S230L) derived from a SMA patient. In different SMA models, we identified differential phosphorylation of the ROCK-downstream targets cofilin, myosin-light chain phosphatase and profilin2a. We suggest that hyper-phosphorylation of profilin2a is the molecular link between SMN and the ROCK pathway repressing neurite outgrowth in neuronal cells. Finally, we found a neuron-specific increase in the F-/G-actin ratio that further support the role of actin dynamics in SMA pathogenesis.

Toxin A (TcdA) and toxin B (TcdB) are the major virulence factors of Clostridium difficile and are the causative agents for clinical symptoms, such as secretory diarrhoea and pseudomembranous colitis. Mast cells are essentially involved in the toxin-induced colonic inflammatory processes. To study the direct effects of these toxins on the expression of inflammatory genes, a DNA microarray containing evaluated probes of 90 selected inflammatory genes was applied to the immature mast cell line HMC-1. TcdA and TcdB induced up-regulation of only a limited number of genes within the early phase of cell treatment. Interleukin-8 (IL-8), transcription factor c-jun and heme oxygenase-1 messenger RNA (mRNA) increased more than 2-fold. In contrast, IL-16, known as a CD4(+) T-cell chemoattractant factor and the chemokine receptor cKit were down-regulated. Stimulation of HMC-1 cells with IL-8 had no effect on IL-16 mRNA level, indicating that both cytokines were independently affected by the toxins. Regulation of both cytokines, however, depended on glucosylation of Rho GTPases as tested by application of enzyme-deficient TcdA or TcdB. Down-regulation of total and secreted IL-16 protein was checked by enzyme-linked immunosorbent assay. The data implicate that TcdA and TcdB affect lymphocyte migration by modulating release of the chemoattractant factor IL-16 from mast cells. In addition, this is the first report showing that Rho GTPases are involved in the regulation of IL-16 expression.

Summary Clostridium difficile toxin A and B (TcdA/TcdB) are glucosyltransferases that glucosylate GTPases of the Rho family. The epidermal growth factor (EGF) positively modulates C. difficile toxin-induced disturbance of the intestinal barrier function by an unknown mechanism. We found that EGF-treated CaCo-2 monolayers were less susceptible to TcdA-catalysed glucosylation of Rac1 but not of RhoA, which correlated with phosphorylation of Rac1 at Ser-71. Phospho-Rac1/phospho-Cdc42 (Ser-71) still bound to the PAK-CRIB domain indicating an active state. A more detailed characterization of phospho-Rac1 was performed using the phosphomimetic mutant Rac1 S71E. Ectopic expression of Rac1 S71E induced a specific phenotype of cells showing an increase in filopodial structures that were also induced by EGF. Rac1 S71E (and Cdc42 S71E) but not Rac1 S71A was at least fivefold weaker substrate for TcdA-catalysed glucosylation compared with wild type Rac1. The protective effect was checked in transfection experiments where Rac1 S71E and, to a lesser extent, Cdc42 S71E reduced the TcdA-induced cytopathic effect. Thus, Ser-71 phosphorylation of Rac1 might be interesting for modulation of microbial pathogenesis where Rho GTPases, especially Rac1 and Cdc42, are involved. In addition, this is the first description of a specific functional outcome of Rac1 phosphorylation at Ser-71.

Clostridium sordellii lethal toxin (TcsL) belongs to the family of clostridial glucosylating toxins. TcsL exhibits glucosyltransferase activity to inactivate Rho and Ras proteins. On cultured cells, TcsL causes actin reorganization ("cytopathic effect") and apoptotic cell death ("cytotoxic effect"). This study is based on the concept that the cytotoxic effects of TcsL depend on the glucosylation of critical substrate proteins rather than on the glucosyltransferase activity per se. The cytotoxic effects of TcsL depend on the glucosyltransferase activity of TcsL, as neither chemically inactivated TcsL nor a glucosyltransferase-deficient mutant version of TcsL caused it. The TcsL homologous toxin B from Clostridium difficile serotype F strain 1470 (TcdBF) also failed to cause cytotoxic effects. Correlation of the toxins' respective protein substrate specificities highlighted (H/K/N)Ras as critical substrate proteins for the cytotoxic effects. (H/K/N)Ras are critical upstream regulators of phosphatidylinositide 3'-OH kinase (PI3K)/Akt survival signaling. Tauroursodeoxycholic acid (TUDCA) classified to activate PI3K/Akt signaling downstream of apoptosis-inducing stimuli prevented the cytotoxic effects of TcsL. In conclusion, (H/K/N)Ras glucosylation and subsequent inhibition of PI3K/Akt signaling are critical for the cytotoxic effects of TcsL.

The intestinal epithelial cell line HT-29 was used to study the apoptotic effect of Clostridium difficile toxin A (TcdA). TcdA is a 300 kDa single-chain protein, which glucosylates and thereby inactivates small GTPases of the Rho family (Rho, Rac and Cdc42). The effect of TcdA-catalysed glucosylation of the Rho GTPases is well known: reorganization of the actin cytoskeleton with accompanying morphological changes in cells, leading to complete rounding of cells and destruction of the intestinal barrier function. Less is known about the mechanism by which apoptosis is induced in TcdA-treated cells. In this study, TcdA induced the activation of caspase-3, -8 and -9. Apoptosis, as estimated by the DNA content of cells, started as early as 24 h after the addition of TcdA. The impact of Rho glucosylation was obvious when mutant TcdA with reduced or deficient glucosyltransferase activity was applied. TcdA mutant W101A, with 50-fold reduced glucosyltransferase activity, induced apoptosis only at an equipotent concentration compared with wild-type TcdA at a 50 % effective concentration of 0.2 nM. The enzyme-deficient mutant TcdA D285/287N was not able to induce apoptosis. Apoptosis induced by TcdA strictly depended on the activation of caspases, and was completely blocked by the pan-caspase inhibitor z-VAD-fmk. Destruction of the actin cytoskeleton by latrunculin B was not sufficient to induce apoptosis, indicating that apoptosis induced by TcdA must be due to another mechanism. In summary, TcdA-induced apoptosis (cytotoxic effect) depends on the glucosylation of Rho GTPases, but is not triggered by destruction of the actin cytoskeleton (cytopathic effect).

Background Neurofibromatosis type 1 (NF1) is one of the most frequently inherited diseases. Vascular manifestations have been reported, mostly concerning stenosis of the renal artery or spontaneous rupture of single arteries. Also, venous aneurysms have been reported. Methods We present clinical and pathological findings of a young patient with NF1 with lethal bleeding due to spontaneous rupture of a lumbal and renal artery. Results High fragility of several arteries as well as veins in the abdominal and pelvic arterial and venous system resulted in a destroyed vessel structure caused by invading neurogen fibres. Conclusions In rare cases, NF1 is associated with a severe systemic vasculopathy concerning the arterial and venous vascular system.

Glucosylation of RhoA, Rac1, and Cdc42 byClostridium difficiletoxin B from strain VPI 10463 (TcdB) results in actin reorganization (cytopathic effect) and apoptosis (cytotoxic effect). Toxin B from variantC. difficilestrain 1470 serotype F (TcdBF) differs from TcdB with regard to substrate proteins, as it glucosylates Rac1 and R-Ras but not RhoA and Cdc42. In this study, we addressed the question of whether the cellular effects of the toxins depend on their protein substrate specificity. Rat basophilic leukemia (RBL) cells were synchronized using the thymidine double-block technique. We show that cells were most sensitive to the cytotoxic effect of TcdB in S phase, as analyzed in terms of phosphatidyl serine externalization, fragmentation of nuclei, and activation of caspase-3; in contrast, TcdBF induced only a marginal cytotoxic effect, suggesting that inactivation of RhoA (but not of Rac1) was required for the cytotoxic effect. The glucosylation of Rac1 was correlated to the cytopathic effect of either toxin, suggesting a close connection of the two effects. The cytotoxic effect of TcdB was executed by caspase-3, as it was responsive to inhibition by acetyl-Asp-Met-Gln-Asp-aldehyde (Ac-DMQD-CHO), an inhibitor of caspase-3. The viability of TcdB-treated RBL cells was reduced, whereas the viability of TcdBF-treated cells was unchanged, further confirming that inactivation of RhoA is required for the cytotoxic effect. In conclusion, the protein substrate specificity of the glucosylating toxins determines their biological activity.

Objective Analysis of in-hospital outcomes in patients treated for critical limb ischemia (CLI) and end-stage renal disease (ESRD) compared to CLI patients with normal renal function. Methods A subgroup analysis of the German CRITISCH registry, a prospective multicenter registry, assessing the first-line treatment strategies in CLI patients in 27 vascular centers in Germany was performed. The study cohort was divided into ESRD patients (n = 102) and patients with normal renal function (n = 674; glomerular filtration rate >60/mL/min/1.73 m 2 ). The following first-line treatment strategies were assessed: endovascular therapy (EVT), bypass surgery, patch plasty, and no vascular intervention (conservative treatment, primary amputation). Uni- and multivariate analyses were performed to identify differences between groups as to six end points: amputation or death (composite end point), amputation, death, hemodynamic failure, major adverse cardiac and cerebrovascular events, and reintervention. Results Differences between the ESRD and non-ESRD group were found regarding the applied first-line therapy ( P = .016): The first-line treatment strategies in ESRD patients were EVT in 64% (n = 65), bypass surgery in 13% (n = 13), patch plasty in 11% (n = 11), and no vascular intervention in 13% (n = 13). In non-ESRD patients, EVT was applied in 48% (n = 326), bypass surgery in 27% (n = 185), patch plasty in 13% (n = 86), and no vascular intervention in 11% (n = 77). For ESRD patients, a noticeably increased risk of the composite end point (odds ratio [OR], 2.62; 95% confidence interval [CI], 1.19-5.79; P = .017), amputation (OR, 3.14; 95% CI, 1.35-7.31; P = .008), and hemodynamic failure (OR, 2.19; 95% CI, 1.19-4.04; P = .012) was observed. Conclusions CLI patients on dialysis represent a challenging cohort prone to in-hospital death, amputation, and hemodynamic failure. Two-thirds of these high-risk patients are treated with EVT. Present data suggest that this modality is generally considered as the most favorable treatment option in this patient subgroup.

Critical limb ischemia (CLI) is growing in global prevalence and is associated with high rates of limb loss and mortality. However, a relevant gap of evidence about the most optimal treatment strategy still exists. The aim of this study of the prospective, multicenter First-Line Treatments in Patients With Critical Limb Ischemia (CRITISCH) registry was to assess the current practice of all first-line treatments strategies in CLI patients in German vascular centers.Between January 2013 and September 2014, five first-line treatment strategies-endovascular revascularization (ER), bypass surgery (BS), femoral/profundal artery patchplasty (FAP), conservative treatment, and primary amputation-were determined among CLI patients in 27 vascular tertiary centers. The main composite end point was major amputation or death, or both, during the hospital stay. Secondary outcomes were hemodynamic failure, major adverse cardiovascular and cerebral events, and reintervention. Univariate logistic models were additionally built to preselect possible risk factors for either event, which were then used as candidates for a multivariate logistic model.The study included 1200 consecutive patients. First-line treatment of choice was ER in 642 patients (53.4%), BS in 284 (23.7%), FAP in 126 (10.5%), conservative treatment in 118 (9.8%), and primary amputation in 30 (2.5%). The composite end point was met in 24 patients (4%) after ER, in 17 (6%) after BS, in 8 (6%) after FAP, and in 9 (8%) after conservative treatment (P = .172). The highest rate of in-hospital death was observed after primary amputation (10%) and of hemodynamic failure after conservative treatment (91%). Major adverse cardiovascular and cerebral events developed in 4% of patients after ER, in 5% after BS, in 6% after FAP, in 5% after conservative treatment, and in 13% after primary amputation. The reintervention rate was 8%, 14%, 6%, 5%, and 3% in each group, respectively. In the multivariate regression model, coronary artery disease (odds ratio [OR], 2.96; 95% confidence interval [CI], 1.42-6.17) and previous myocardial infarction (PMI)6 months (OR, 3.67, 95% CI, 1.51-8.88) were identified as risk factors for the composite end point. Risk factors for amputation were dialysis (OR, 3.31, 95% CI, 1.44-7.58) and PMI (OR, 3.26, 95% CI, 1.23-8.36) and for death, BS compared with ER (OR, 3.32; 95% CI, 1.10-10.0), renal insufficiency without dialysis (OR, 6.34; 95% CI, 1.71-23.5), and PMI (OR, 7.41; 95% CI, 2.11-26.0).The CRITISCH registry revealed ER as the most common first-line approach in CLI patients. Coronary artery disease and PMI6 months were independent risk factors for the composite end point. Special attention should be also paid to CLI patients with renal insufficiency, with or without dialysis, and those undergoing BS.

Fragestellung Diese Datenerhebung wurde mit dem Ziel durchgefuhrt, beurteilen zu konnen, ob die In-situ-Rekonstruktion bei Gefasprotheseninfektion mit einem silberacetatbeschichten Dacrongraft ein sicheres und vertretbares Konzept ist. Wir etablierten ein fragebogenbasiertes prospektives, multizentrisches offenes Register. Die Teilnehmer waren zum Einsenden aller entsprechend therapierten Falle verpflichtet.

Mutation of tryptophan-101 in Clostridium difficile toxin A, a 308-kDa glucosyltransferase, resulted in a 50-fold-reduced cytopathic activity in cell culture experiments. The mutant toxin A was characterized and applied to distinguish between glucosyltransferase-dependent and -independent effects with respect to RhoB up-regulation as a cellular stress response.