In 1965, he received his doctoral degree in Tübingen with a thesis on substance P in the brain under the supervision of Prof. Fred Lembeck.The habilitation was awarded in Essen, Germany, where Klaus Starke opened a field of research that he subsequently strongly influenced-presynaptic receptors.He investigated how angiotensin acts on sympathetic nerve fibers, thereby increasing the release of the neurotransmitter noradrenaline.Presynaptic receptors have been the focus of Klaus Starke's scientific work since his habilitation.His research significantly contributed to clarifying the question of how antagonists at α 2 -adrenoceptors increase the release of noradrenaline from sympathetic nerves.Klaus Starke showed that these antagonists block presynaptic α 2adrenoceptors, which normally inhibit transmitter release.Presynaptic α 2 -adrenoceptors are part of a feedback loop that controls sympathetic noradrenaline release.This regulatory principle has been identified for many other transmitters and is one of the basic principles of neurotransmitter release in the central and peripheral nervous system.In 1977,

Research on the human gut pathogen Clostridioides (C.) difficile and its toxins continues to attract much attention as a consequence of the threat to human health posed by hypervirulent strains. Toxin A (TcdA) and Toxin B (TcdB) are the two major virulence determinants of C. difficile. Both are single-chain proteins with a similar multidomain architecture. Certain hypervirulent C. difficile strains also produce a third toxin, namely binary toxin CDT (C. difficile transferase). C. difficile toxins are the causative agents of C. difficile-associated diseases (CDADs), such as antibiotics-associated diarrhea and pseudomembranous colitis. For that reason, considerable efforts have been expended to unravel their molecular mode-of-action and the cellular mechanisms responsible for their uptake. Many of these studies have been conducted in European laboratories. Here, we provide an update on our previous review (Papatheodorou et al. Adv Exp Med Biol, 2018) on important advances in C. difficile toxins research.

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

Infection by the bacterium Clostridioides difficile can be fatal in a clinical setting. Insights into the molecular mechanisms underlying such infection might offer targets for the search to develop new treatments.

In the last decade, it was discovered that protein mucin-type O-glycosylation and O-GlcNAcylation modify Tyr residues besides the well explored Thr and Ser amino acids. Several glycoproteomic studies have identified α-GalNAc-O-Tyr modifications, and studies propose that β-GlcNAc-O-Tyr also exists as a new group of posttranslational modifications (PTMs). Specific bacterial toxins have further been identified to modify host GTPases with α-GlcNAc-O-Tyr to promote bacterial virulence. Despite being identified on numerous proteins, the biological roles, biosynthesis and expression of GalNAc- and GlcNAc-O-Tyr modifications are poorly understood. A major obstacle is the lack of tools to specifically detect and identify proteins containing these modifications. With this in mind, we prepared vaccine constructs and raised antibodies to enable selective detection of proteins carrying these new PTMs. The obtained polyclonal antibody sera were evaluated using ELISA and glycopeptide microarrays and were found to be highly selective for GlcNAc- and GalNAc-O-Tyr glycopeptides over the corresponding Ser- and Thr-modifications. For microarray analysis, synthetic GlcNAc- and GalNAc-O-Tyr Fmoc-amino acids were prepared and applied in Fmoc-SPPS to obtain an extensive O-glycopeptide library. After affinity purification, the antibodies were applied in western blot analysis and showed specific detection of α-GlcNAc-O-Tyr modified RhoA GTPase.

Microbial communities are found throughout the biosphere, from human guts to glaciers, from soil to activated sludge. Understanding the statistical properties of such diverse communities can pave the way to elucidate the common mechanisms ...Multiple ecological forces act together to shape the composition of microbial communities. Phyloecology approaches—which combine phylogenetic relationships between species with community ecology—have the potential to disentangle such forces but are often ...

Clostridioides difficile infection (CDI) represents a significant burden on the health care system, one that is exacerbated by the emergence of binary toxin (CDT)-producing hypervirulent C. difficile strains. Previous work from our laboratory has shown that Toll-like receptor 2 (TLR2) recognizes CDT to induce inflammation. Here we explore the interactions of CDT with TLR2 and the impact on host immunity during CDI. We found that the TLR2/6 heterodimer, not TLR2/1, is responsible for CDT recognition, and that gene pathways including nuclear factor-κB and MAPK downstream of TLR2/6 are upregulated in mice with intact TLR2/6 signaling during CDI.

The intestinal pathogen Clostridioides (C.) difficile is a major cause of diarrhea both in hospitals and outpatient in industrialized countries. This bacterium produces two large exotoxins, toxin A (TcdA) and toxin B (TcdB), which are directly responsible for the onset of clinical symptoms of C. difficile-associated diseases (CDADs), such as antibiotics-associated diarrhea and the severe, life-threatening pseudomembranous colitis. Both toxins are multidomain proteins and taken up into host eukaryotic cells via receptor-mediated endocytosis. Within the cell, TcdA and TcdB inactivate Rho and/or Ras protein family members by glucosylation, which eventually results in cell death. The cytotoxic mode of action of the toxins is the main reason for the disease. Thus, compounds capable of inhibiting the cellular uptake and/or mode-of-action of both toxins are of high therapeutic interest. Recently, we found that the sterol regulatory element-binding protein 2 (SREBP-2) pathway, which regulates cholesterol content in membranes, is crucial for the intoxication of cells by TcdA and TcdB. Furthermore, it has been shown that membrane cholesterol is required for TcdA- as well as TcdB-mediated pore formation in endosomal membranes, which is a key step during the translocation of the glucosyltransferase domain of both toxins from endocytic vesicles into the cytosol of host cells. In the current study, we demonstrate that intoxication by TcdA and TcdB is diminished in cultured cells preincubated with the compound U18666A, an established inhibitor of cholesterol biosynthesis and/or intracellular transport. U18666A-pretreated cells were also less sensitive against TcdA and TcdB variants from the epidemic NAP1/027 C. difficile strain. Our study corroborates the crucial role of membrane cholesterol for cell entry of TcdA and TcdB, thus providing a valuable basis for the development of novel antitoxin strategies in the context of CDADs.

Clostridioides bacteria are responsible for life threatening infections. Here, we show that in addition to actin, the binary toxins CDT, C2I, and Iota from Clostridioides difficile, botulinum, and perfrigens, respectively, ADP-ribosylate the actin-related protein Arp2 of Arp2/3 complex and its additional components ArpC1, ArpC2, and ArpC4/5. The Arp2/3 complex is composed of seven subunits and stimulates the formation of branched actin filament networks. This activity is inhibited after ADP-ribosylation of Arp2. Translocation of the ADP-ribosyltransferase component of CDT toxin into human colon carcinoma Caco2 cells led to ADP-ribosylation of cellular Arp2 and actin followed by a collapse of the lamellipodial extensions and F-actin network. Exposure of isolated mouse colon pieces to CDT toxin induced the dissolution of the enterocytes leading to luminal aggregation of cellular debris and the collapse of the mucosal organization. Thus, we identify the Arp2/3 complex as hitherto unknown target of clostridial ADP-ribosyltransferases.

Nicotinamide adenine dinucleotide (NAD+) is an important biomolecule with essential roles at the intersection of energy metabolism, epigenetic regulation and cell signalling. Synthetic analogues of NAD+ are therefore of great interest as chemical tools for medicinal chemistry, chemical biology and drug discovery. Herein, we report the chemical synthesis and full analytical characterisation of three stereoisomers of 2″-amino NAD+, and their biochemical evaluation against two classes of NAD+-consuming enzymes: the human sirtuins 1-3, and the bacterial toxin TccC3. To rationalise the observed activities, molecular docking experiments were carried out with SIRT1 and SIRT2, which identified the correct orientation of the pyrophosphate linkage as a major determinant for activity in this series. These results, together with results from stability tests and a conformational analysis, allow, for the first time, a side-by-side comparison of the chemical and biochemical features, and analytical properties, of different 2″-amino NAD+ stereoisomers. Our findings provide insight into the recognition of co-substrate analogues by sirtuins, and will greatly facilitate the application of these important NAD+ analogues as chemical tool compounds for mechanistic studies with these as well as other NAD+-dependent enyzmes.

Clostridioides difficile (formerly named Clostridium difficile) is the dominant causative agent of a spectrum of illnesses, which frequently occur as the consequence of antibiotic treatment of patients. C. difficile infections (CDIs) cause mild to severe diarrhea (so-called antibiotic-associated diarrhea) but also pseudomembranous enterocolitis with complications like toxic megacolon, bowel perforation, and death. Three protein toxins are involved in the pathology of CDIs, toxin A (TcdA) and toxin B (TcdB), which are the prototypes of large clostridial glucosylating toxins, and C. difficile ADP-ribosyltransferase CDT. TcdB is most likely the driving toxin responsible for the major pathology of the infection. Recent studies showed that C. difficile exhibits considerable genome diversity, requiring the classification into different C. difficile clades (at least 5 major clades), which may have different impact on infections. Beside others, C. difficile clade 2 is of special interest because it contains strains like ribotype 027 (NAP1), which caused major clinical outbreaks with high morbidity and mortality. The enormous diversity in C. difficile strains leads to the question of diversity in toxins. Indeed, numerous subtypes of TcdB were described, which appear to differ in cell targeting and toxicity. At least 8 toxin subtypes (TcdB1–8) with >200 different members have been reported for TcdBs. All toxins (including TcdBs and all other “large clostridial glucosylating toxins”) share a very similar overall structure and consist of 4 major domains: a glucosyltransferase domain (GTD) at the N terminus, a cysteine protease domain (CPD), a delivery/receptor-binding domain (DRBD), and a so-called combined repetitive oligopeptides (CROPs) domain at the C terminus (Figure 1). The GTDs of all toxins modify small GTPases of the Rho/Ras family, mainly resulting in inhibition of Rho-dependent signaling.1Aktories K. Schwan C. Jank T. Clostridium difficile toxin biology.Annu. Rev. Microbiol. 2017; 71: 281-307Crossref PubMed Scopus (132) Google Scholar However, it is now clear that the biological activities of the various toxins are not identical, a fact that is most important for therapy of CDIs. Up to now, several receptors have been described for TcdB toxins, including chondroitin sulfate proteoglycan 4 (CSPG4) and the heptahelical Frizzled receptors (FZD 1, 2, and 7).2Chen P. Tao L. Wang T. et al.Structural basis for recognition of frizzled proteins by Clostridium difficile toxin B.Science. 2018; 360: 664-669Crossref PubMed Scopus (62) Google Scholar For both receptor types, which bind independently from each other, the pathophysiological relevance is evident. In addition, nectin 3 (also called PVRL3) and low-density lipoprotein receptor-related protein 1 (LRP1) have been described as potential receptors, but their roles in toxin functions are not clear. Recently, in a mouse model, it was shown that various clade 2 C. difficile strains including the hypervirulent ribotype 027 strains cause severe colonic damage with massive stem cell injury by subtype toxins, which do not bind to FZD receptors.3Mileto S.J. Jarde T. Childress K.O. et al.Clostridioides difficile infection damages colonic stem cells via TcdB, impairing epithelial repair and recovery from disease.Proc. Natl. Acad. Sci. U S A. 2020; 117: 8064-8073Crossref PubMed Scopus (23) Google Scholar So far, FZD binding and inhibition of FZD-dependent Wnt signaling has been attributed to stem cell damage. By contrast, CSPG4, the other well-known toxin receptor, is not expressed in colon epithelium but rather in subepithelial myofibroblasts and is probably not involved in stem cell damage, which might be of major impact for the pathology of CDIs. What, then, is the relevant toxin receptor? Now, using CRISPR-Cas9-dependent genome-wide screening, Liang Tao, Yanyan Li, and co-workers show that tissue factor pathway inhibitor (TFPI) is a colonic crypt receptor for TcdBs from hypervirulent clade 2 C. difficile strains.4Luo J. Yang Q. Zhang X. et al.TFPI is a colonic crypt receptor for TcdB from hypervirulent clade 2 C. difficile.Cell. 2022; 185: 980-994.e15Abstract Full Text Full Text PDF PubMed Scopus (3) Google Scholar Especially TcdB4, another clade 2 toxin, which is ∼85% identical with the prototype TcdB1, depends totally on the TFPI receptor. The essential role of TFPI in binding and action of clade 2 toxins was convincingly verified by various knockout cell and animal models. TFPI is well known for its role as an anticoagulant protein produced primarily by endothelium and megakaryocytes. It inhibits coagulation factor Xa, the function of the TF-FVIIa complex and the initial prothrombinase complex.5Broze Jr., G.J. Girard T.J. Tissue factor pathway inhibitor: structure-function.Front. Biosci. 2012; 17: 262-280Crossref PubMed Scopus (116) Google Scholar TFPI occurs in two alternatively spliced isoforms, TFPIα and TFPIβ. TFPIα is secreted and found in plasma and on cell membranes. It consists of three multivalent Kunitz domains (K1–3) and a basic C terminus and is most likely responsible for membrane attachment. TFPIβ has only K1,2 domains but possesses a GPI anchor for membrane insertion. The K1 domain of TFPI binds FVIIa, and the K2 domain (TFPIK2) binds and inhibits FXa. The researchers found that TcdB binds both TFPI isoforms at their K2 domain (and blocks interaction with FXa). TFPIβ might be particularly important as a receptor for TcdB because the CRISPR-Cas9-dependent genome-wide screenings found, in addition to TFPI, numerous enzymes involved in GPI-anchor formation. Using cryoelectron microscopy (cryo-EM), the interaction of full-length TcdB4 with TFPI at high resolution (3.1–3.7 Å) was analyzed, revealing that the TFPIK2 binds to a convex region of the delivery domain of TcdB4, forming a receptor-binding interface (RBI) that covers residues 1,431–1,606 of TcdB4. This part is identical with the region for binding of the FZD receptor by toxin subtype TcdB1. They identified amino acids that favor binding to TFPI and block binding to FZD, and vice versa. Moreover, phylogenetic analysis of various TcdBs revealed 2 major toxin classes, with class I RBIs common in TcdB1, TcdB3, and TcdB5 and class II interfaces for binding of TcdB2, TcdB4, TcdB6, and TcdB7, which mainly represent clade 2 C. difficile toxins. Knockout of TFPI resulted in increased resistance of the cells toward TcdB2, TcdB4, TcdB6, and TcdB7. Worth mentioning is that an equivalent, almost identical region of the related TcsL toxin from Paeniclostridium sordellii (formerly Clostridium sordellii), sharing ∼76% identity with TcdB1, is involved in binding to Semaphorin A and B, the receptors of TcsL. In human intestine, TFPI is highly expressed in endothelial and colon crypt cells. In wild-type mice, TcdB4 caused acute kidney damage and death after intraperitoneal (i.p.) injection. By contrast, in Tfpiβ-knockout (KO) mice, TcdB4 was much less toxic and showed normal kidneys. Further studies revealed the important role of TFPI in TcdB2 intoxication. While co-injection of TcdB4 with the construct TFPIK2-Fc in ligated colon segments efficiently prevented colon crypts damage, the decoy-based inhibition of TcdB2 was suboptimal, suggesting that TcdB2 could damage gut tissue via its CSPG4 receptor. Therefore, a fusion construct consisting of domain K2 of TFPI and the first repeat of CSPG4 (residues 410–551), which is involved in toxin binding, was employed and showed enhanced protection against colon damage. Thus, it is speculated that such constructs might be of therapeutic value in clade 2 CDIs. Taken together, the recent findings are a significant step forward in the understanding of the pathology of CDIs and give answers to important questions about the diversity and evolutionary development of C. difficile toxins and the variability of toxin effects. The new results may open novel perspectives for treatment of CDI. Moreover, CRISPR-Cas9-dependent screenings and cryo-EM analyses of toxin-receptor complexes may hopefully lead to further “surprising” results on receptor binding of other subtypes of TcdBs and related large clostridial glycosylating toxins. The author declares no competing interests.

Selected findings about Clostridioides difficile (formerly Clostridium difficile ) toxins are presented in a narrative review. Starting with a personal view on research about G proteins, adenylyl cyclase, and ADP-ribosylating toxins in the laboratory of Günter Schultz in Heidelberg, milestones of C. difficile toxin research are presented with the focus on toxin B (TcdB), covering toxin structure, receptor binding, toxin up-take and refolding, the intracellular actions of TcdB, and the treatment of C. difficile infection.

Photorhabdus luminescens Tc toxins are large tripartite ABC-type toxin complexes, composed of TcA, TcB and TcC proteins. Tc toxins are widespread and have shown a tropism for a variety of targets including insect, mammalian and human cells. However, their receptors and the specific mechanisms of uptake into target cells remain unknown. Here, we show that the TcA protein TcdA1 interacts with N-glycans, particularly Lewis X/Y antigens. This is confirmed using N-acetylglucosamine transferase I (Mgat1 gene product)-deficient Chinese hamster ovary (CHO) Lec1 cells, which are highly resistant to intoxication by the Tc toxin complex most likely due to the absence of complex N-glycans. Restoring Mgat1 gene activity, and hence complex N-glycan biosynthesis, recapitulated the sensitivity of these cells to the toxin. Exogenous addition of Lewis X trisaccharide partially inhibits intoxication in wild-type cells. Additionally, sialic acid also largely reduced binding of the Tc toxin. Moreover, proteolytic activation of TcdA1 alters glycan-binding and uptake into target cells. The data suggest that TcdA1-binding is most likely multivalent, and carbohydrates probably work cooperatively to facilitate binding and intoxication.

The human pathogenic bacterium Clostridioides difficile produces two exotoxins TcdA and TcdB, which inactivate Rho GTPases thereby causing C. difficile-associated diseases (CDAD) including life-threatening pseudomembranous colitis. Hypervirulent strains produce additionally the binary actin ADP-ribosylating toxin CDT. These strains are hallmarked by more severe forms of CDAD and increased frequency and severity. Once in the cytosol, the toxins act as enzymes resulting in the typical clinical symptoms. Therefore, targeting and inactivation of the released toxins are of peculiar interest. Prompted by earlier findings that human α-defensin-1 neutralizes TcdB, we investigated the effects of the defensin on all three C. difficile toxins. Inhibition of TcdA, TcdB, and CDT was demonstrated by analyzing toxin-induced changes in cell morphology, substrate modification, and decrease in transepithelial electrical resistance. Application of α-defensin-1 protected cells and human intestinal organoids from the cytotoxic effects of TcdA, TcdB, CDT, and their combination which is attributed to a direct interaction between the toxins and α-defensin-1. In mice, the application of α-defensin-1 reduced the TcdA-induced damage of intestinal loops in vivo. In conclusion, human α-defensin-1 is a specific and potent inhibitor of the C. difficile toxins and a promising agent to develop novel therapeutic options against C. difficile infections.

Anthrax toxin is the major virulence factor secreted by Bacillus anthracis, causing high mortality in humans and other mammals. It consists of a membrane translocase, known as protective antigen (PA), that catalyzes the unfolding of its cytotoxic substrates lethal factor (LF) and edema factor (EF), followed by translocation into the host cell. Substrate recruitment to the heptameric PA pre-pore and subsequent translocation, however, are not well understood. Here, we report three high-resolution cryo-EM structures of the fully-loaded anthrax lethal toxin in its heptameric pre-pore state, which differ in the position and conformation of LFs. The structures reveal that three LFs interact with the heptameric PA and upon binding change their conformation to form a continuous chain of head-to-tail interactions. As a result of the underlying symmetry mismatch, one LF binding site in PA remains unoccupied. Whereas one LF directly interacts with a part of PA called α-clamp, the others do not interact with this region, indicating an intermediate state between toxin assembly and translocation. Interestingly, the interaction of the N-terminal domain with the α-clamp correlates with a higher flexibility in the C-terminal domain of the protein. Based on our data, we propose a model for toxin assembly, in which the relative position of the N-terminal α-helices in the three LFs determines which factor is translocated first.

Open access funding provided by Projekt DEAL. Lee, H. et al. Recognition of semaphorin proteins by P. sordellii lethal toxin reveals principles of receptor specificity in clostridial toxins. Cell 182, 345–356.e16 (2020). CAS Article Google Scholar Aktories, K., Schwan, C. & Jank, T. Clostridium difficile toxin biology. Annu. Rev. Microbiol. 71, 281–307 (2017). CAS Article Google Scholar Geny, B. et al. Clostridium sordellii lethal toxin kills mice by inducing a major increase in lung vascular permeability. Am. J. Pathol. 170, 1003–1017 (2007). CAS Article Google Scholar Tian, S. et al. Genome-wide CRISPR screen identifies semaphorin 6A and 6B as receptors for Paeniclostridium sordellii toxin TcsL. Cell Host Microbe 27, 782–792.e787 (2020). CAS Article Google Scholar Chen, P. et al. Structural basis for recognition of frizzled proteins by Clostridium difficile toxin B. Science 360, 664–669 (2018). CAS Article Google Scholar Download references Institute of Experimental and Clinical Pharmacology and Toxicology, Medical Faculty, University of Freiburg, Albertstr. 25, 79104, Freiburg, Germany Klaus Aktories You can also search for this author in PubMed Google Scholar Correspondence to Klaus Aktories. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Reprints and Permissions Aktories, K. Semaphorins or Frizzled –it is the receptor that direct the action of clostridial glucosylating toxins. Sig Transduct Target Ther 5, 206 (2020). https://doi.org/10.1038/s41392-020-00307-3 Download citation Received: 27 July 2020 Revised: 05 August 2020 Accepted: 19 August 2020 Published: 19 September 2020 DOI: https://doi.org/10.1038/s41392-020-00307-3

Infections with the pathogenic bacterium Clostridioides (C.) difficile are coming more into focus, in particular in hospitalized patients after antibiotic treatment. C. difficile produces the exotoxins TcdA and TcdB. Since some years, hypervirulent strains are described, which produce in addition the binary actin ADP-ribosylating toxin CDT. These strains are associated with more severe clinical presentations and increased morbidity and frequency. Once in the cytosol of their target cells, the catalytic domains of TcdA and TcdB glucosylate and thereby inactivate small Rho-GTPases whereas the enzyme subunit of CDT ADP-ribosylates G-actin. Thus, enzymatic activity of the toxins leads to destruction of the cytoskeleton and breakdown of the epidermal gut barrier integrity. This causes clinical symptoms ranging from mild diarrhea to life-threatening pseudomembranous colitis. Therefore, pharmacological inhibition of the secreted toxins is of peculiar medical interest. Here, we investigated the neutralizing effect of the human antimicrobial peptide α-defensin-5 toward TcdA, TcdB, and CDT in human cells. The toxin-neutralizing effects of α-defensin-5 toward TcdA, TcdB, and CDT as well as their medically relevant combination were demonstrated by analyzing toxins-induced changes in cell morphology, intracellular substrate modification, and decrease of trans-epithelial electrical resistance. For TcdA, the underlying mode of inhibition is most likely based on the formation of inactive toxin-defensin-aggregates whereas for CDT, the binding- and transport-component might be influenced. The application of α-defensin-5 delayed intoxication of cells in a time- and concentration-dependent manner. Due to its effect on the toxins, α-defensin-5 should be considered as a candidate to treat severe C. difficile-associated diseases.