Plasma Membrane Of Target Cells Research Articles

Novel Cholesterol-Based Dihydropyrimidinone Derivatives with Potential Biological Activities: An In-silico Investigation

Steroids play essential physiological roles in the living systems, including their role as sex hormones, glucocorticoids, and mineralocorticoids. Usually, they engage in interactions with steroid receptors. Target cell nuclei, cytosol and plasma membranes all have steroid hormone receptors. Cytoplasmic or nuclear, intracellular receptors start the signal transduction process for steroid hormones and cause gene expression alterations over the course of several hours to days. Certain steroid hormones bind to cell surface receptors, including ion channels, G protein-coupled receptors and several nuclear receptors. Dihydropyrimidinones are also significant moieties with important physiological roles. The hybrid molecules can offer several potential biological activities. In this work, we have checked the in silico interaction of some novel cholesterol–dihydropyrimidinones interactions with the sex hormones, mineralocorticoid and glucocorticoid receptors to screen their potential use in the regulation of the sexual cycle or the carbohydrate metabolism or the mineral imbalance.

Near-infrared photoimmunotherapy and anti-cancer immunity.

The activation of the anti-cancer immune system is an important strategy to control cancer. A new form of cancer phototherapy, near-infrared photoimmunotherapy (NIR-PIT), was approved for clinical use in 2020 and uses IRDye® 700DX (IR700)-conjugated antibodies and NIR light. After irradiation with NIR light, the antibody-IR700 conjugate forms water-insoluble aggregations on the plasma membrane of target cells. This aggregation causes lethal damage to the plasma membrane, and effectively leads to immunogenic cell death (ICD). Subsequently, ICD activates anti-cancer immune cells such as dendritic cells and cytotoxic T cells. Combination therapy with immune-checkpoint blockade has synergistically improved the anti-cancer effects of NIR-PIT. Additionally, NIR-PIT can eliminate immunosuppressive immune cells in light-irradiated tumors by using specific antibodies against regulatory T cells and myeloid-derived suppressor cells. In addition to cancer-cell-targeted NIR-PIT, such immune-cell-targeted NIR-PIT has shown promising results by activating the anti-cancer immune system. Furthermore, NIR-PIT can be used to manipulate the tumor microenvironment by eliminating only targeted cells in the tumor, and thus it also can be used to gain insight into immunity in basic research.

Four Cholesterol-Recognition Motifs in the Pore-Forming and Translocation Domains of Adenylate Cyclase Toxin Are Essential for Invasion of Eukaryotic Cells and Lysis of Erythrocytes.

Adenylate Cyclase Toxin (ACT or CyaA) is one of the important virulence factors secreted by Bordetella pertussis, the bacterium causative of whooping cough. ACT debilitates host defenses by production of unregulated levels of cAMP into the cell cytosol upon delivery of its N-terminal domain with adenylate cyclase activity (AC domain) and by forming pores in the plasma membrane of macrophages. Binding of soluble toxin monomers to the plasma membrane of target cells and conversion into membrane-integrated proteins are the first and last step for these toxin activities; however, the molecular determinants in the protein or the target membrane that govern this conversion to an active toxin form are fully unknown. It was previously reported that cytotoxic and cytolytic activities of ACT depend on membrane cholesterol. Here we show that ACT specifically interacts with membrane cholesterol, and find in two membrane-interacting ACT domains, four cholesterol-binding motifs that are essential for AC domain translocation and lytic activities. We hypothesize that direct ACT interaction with membrane cholesterol through those four cholesterol-binding motifs drives insertion and stabilizes the transmembrane topology of several helical elements that ultimately build the ACT structure for AC delivery and pore-formation, thereby explaining the cholesterol-dependence of the ACT activities. The requirement for lipid-mediated stabilization of transmembrane helices appears to be a unifying mechanism to modulate toxicity in pore-forming toxins.

Inhibition of perforin enhances T cell and humoral responses after immunization

Abstract Cytotoxic lymphocytes such as natural killer (NK) cells release perforin to form pores on plasma membrane of target cells to allow delivery of granzymes that induce apoptosis. We previously showed that NK cells exert a perforin-dependent suppression of follicular helper CD4 T cell and germinal center B cell responses following infection and immunization. Consistent with this activity, genetic ablation of either perforin or NK cells non-additively enhanced the quality and magnitude of humoral immune responses. Therefore, we hypothesize that transient inhibition of perforin represents an innovative means to enhance vaccine efficacy to prevent viral infections such as HIV. Intraperitoneal injection of a selective inhibitor of perforin (35mg/kg) to mice reduced killing activity of NK cells by >50%. A single injection of this inhibitor to mice during alum adjuvanted NP-KLH immunization resulted in a greater than two-fold enhancement of follicular helper CD4 T cell, germinal center B cell, and antigen-specific antibody responses. In summary, transient inhibition of pore-forming protein perforin can block NK-cell immunosuppression to enhance vaccine-elicited T and B cell responses. National Institute of Heath (NIH) (R01-AI148080)

Granzyme F production by CD8 T cells in the tumor microenvironment

Abstract Granzymes are a class of cytotoxic proteases and are the primary mechanism utilized by T cells to directly eliminate cancer cells. Each granzyme acts upon a unique set of substrates in target cells to induce cytotoxicity through a range of different mechanisms. Granzymes are some of the most differentially regulated genes in CD8+ tumor infiltrating lymphocytes, relative to T cells outside of the tumor microenvironment (TME). We and others have determined by microarray and qPCR that granzyme F is highly upregulated in the TME. Granzyme F expression is restricted to a small subset of antigen-experienced and exhausted T cells, as determined by flow cytometry-based detection of granzyme F RNA transcripts and may represent a marker of a unique T cell cytotoxic function. Single cell RNA sequencing of CD8 TIL has revealed that granzyme F-high expressing cells are unique from both granzyme A and B expressing cells, and that it is therefore likely these TIL utilize a unique mechanism of cytotoxicity in their elimination of cancer cells. Recombinant granzyme F has previously been shown to induce a unique form of cell death, characterized as being caspase-independent and resulting in rupture of target cell plasma membrane. By over expressing granzyme F we are determining if this mechanism of cell death is leverageable to improve the cytotoxic capacity of TIL, and if induction of different forms of T cell-mediated cytotoxicity can modulate the immunogenicity of the TME. These experiments are designed to provide insight into how to improve adoptive T cell therapies by directly improving cytotoxicity, the terminal step of T cell interaction with tumor cells. This work was supported by the National Institutes of Health NIAID training grant (Training Program in Immunology; T32-AI07405) award to Zachary Hay

What Is IL-1 for? The Functions of Interleukin-1 Across Evolution.

Interleukin-1 is a cytokine with potent inflammatory and immune-amplifying effects, mainly produced by macrophages during defensive reactions. In mammals, IL-1 is a superfamily of eleven structurally similar proteins, all involved in inflammation or its control, which mainly act through binding to specific receptors on the plasma membrane of target cells. IL-1 receptors are also a family of ten structurally similar transmembrane proteins that assemble in heterocomplexes. In addition to their innate immune/inflammatory effects, the physiological role of IL-1 family cytokines seems to be linked to the development of adaptive immunity in vertebrates. We will discuss why IL-1 developed in vertebrates and what is its physiological role, as a basis for understanding when and how it can be involved in the initiation and establishment of pathologies.

Inflammation in Focus: The Beginning and the End.

The inflammation is an important biological response induced by various harmful stimuli, like viruses, bacterial infections, toxins, toxic compounds, tissue injury. During inflammation inflammatory cytokines and reactive oxygen species are produced. Inflammatory cytokines act on various receptors present on the plasma membrane of target cells. To initiate signaling cascade, and activate transcription factors, receptors should be internalized and enter the early endosomes, where the members of the signaling cascade can meet. The further cytoplasmic fate of the receptor plays crucial role in the progression and the course of inflammation. Usually acute inflammation removes injurious stimuli and helps to regain the normal healthy status of the organism. In contrast to this the uncontrolled chronic inflammation—stimulating other than immune cells, inducing transdifferentiation—can provide base of various serious diseases. This paper draws the attention of the long-lasting consequence of chronic inflammation, pointing out that one of the most important step in medication is to identify in time the factors initiating and maintaining inflammation.

Cell-surface glycans act as attachment factors for porcine hemagglutinating encephalomyelitis virus

Porcine hemagglutinating encephalomyelitis virus (PHEV) is a neurotropic coronavirus and highly pathogenic in veterinary clinic. Spike (S) protein of PHEV interplays with host components to cross the plasma membrane of target cells, but characterization of its functional receptors is limited. Here, we discovered that cell-surface glycans, i.e., sialic acid (SA) and heparan sulfate (HS), act as critical interacting factors of PHEV, involving in viral attachment. As shown in glycans depletion assay, removing SA or HS from N2a cells inhibits PHEV infection. Soluble sugar monomers were utilized for competitive binding tests, and we found that both SA and HS could specifically bind to PHEV and affect the viral infectivity. Furthermore, the expression of heparan sulfate proteoglycans (HSPGs), including syndecans and glypicans, and endoglycosidase heparinase which cleaves HS were regulated by PHEV RNA replication. Together, we newly identified specificity recognition of cellular glycans and PHEV during infection, providing novel cellular targets for antiviral therapies and better understanding of pathogenesis.

Abstract 14182: Diverse microRNA Cargo is Essential for the Cardioprotective Effects of Therapeutic Extracellular Vesicles

Background: Extracellular vesicles (EVs) deliver their protein and RNA cargo across the plasma membrane of target cells to exert a functional response. Injection of cardiosphere-derived cell (CDC) EVs have been shown to reduce cardiomyocyte (CM) cell death and improve cardiac function in large and small animal models of myocardial infarction (MI) and ischemia-reperfusion. While several EV miRNAs (miRs) have been identified to play a role in cardioprotection, it has not yet been established whether miR cargo is necessary for the therapeutic benefit of EVs. We hypothesized that miRNA cargo is essential in CDC-EV mediated cardioprotection post-MI. Methods: CDCs were transfected with siRNA against Drosha, the initial endonuclease in the miR biogenesis pathway. EVs were isolated from serum-free media and characterized by size, morphology, and protein/miR expression as previously described. We examined the role of EV miRNA on target cell apoptosis, proliferation, angiogenesis, and gene expression using a series of in vitro assays. To determine if miR cargo is essential for the therapeutic efficacy of CDC-EVs in vivo , we induced acute MI in adult C57BL/6 mice and randomized them to receive 1) EVs, 2) miR depleted EVs or 3) vehicle control. LVEF was assessed by echo at 1- and 28-days post-MI and tissue samples processed for assessment of histological endpoints. Results: Transfection of CDCs with siRNA against Drosha reduces miRNA in CDCs and their EVs by 81±5% and 65±4%, respectively (mean ± SEM, t-test, p<0.001). miR depleted EVs failed to recapitulate the reduction in cardiomyocyte apoptosis, decrease in fibroblast proliferation, and enhancement in angiogenesis seen with control EVs. Furthermore, while intramyocardial administered CDC-derived EVs improved LVEF compared with vehicle control treated animals 28 days post-MI (62 ± 4% vs 44 ± 4%), mice treated with miR-depleted EVs demonstrated no significant difference in LVEF (42 ± 6%) compared with vehicle control (n=8 mice/group, mean ± SEM, p<0.05, one-way ANOVA). Conclusions: miR deficient human CDC-EVs are unable to recapitulate the cardioprotective effects seen with control extracellular vesicles, providing important insight into the mechanism of action of therapeutic EVs.

Systemic mitochondrial disruption is a key event in the toxicity of bacterial pore-forming toxins to Caenorhabditis elegans.

Pore-forming toxins (PFTs) are important weapons of multiple bacterial pathogens to establish their infections. PFTs generally form pores in the plasma membrane of target cells; however, the intracellular pathogenic processes triggered after pore-formation remain poorly understood. Using Caenorhabditis elegans as a model and Bacillus thuringiensis nematicidal Cry PFTs, we show here that the localized PFT attack causes a systemic mitochondrial damage, important for the PFT toxicity. We find that PFTs punch pores only in gut cells of nematodes, but unexpectedly mitochondrial disruption is able to occur in distal unperforated regions, such as the head and muscle tissues. We demonstrate that PFTs affect the activity of the mitochondrial respiratory chain (MRC) complex I resulting in the loss of mitochondrial membrane potential (ΔΨm ), which causes further mitochondrial fragmentation and the reduction of total mitochondrial content. Worms with decreased ΔΨm or inhibited MRC activity show higher sensitivity to PFTs. The inhibition of mitochondrial fission or the increase of mitochondrial content markedly improves the survival of animals treated with PFTs. These findings suggest that mitochondrial changes underpin PFT-mediated toxicity against nematodes and that systemic mitochondrial disruption caused by localized pore-formation represents a conserved key intracellular event in the mode of action of PFTs.

Improved cytokine\u2013receptor interaction prediction by exploiting the negative sample space

BackgroundCytokines act by binding to specific receptors in the plasma membrane of target cells. Knowledge of cytokine–receptor interaction (CRI) is very important for understanding the pathogenesis of various human diseases—notably autoimmune, inflammatory and infectious diseases—and identifying potential therapeutic targets. Recently, machine learning algorithms have been used to predict CRIs. “Gold Standard” negative datasets are still lacking and strong biases in negative datasets can significantly affect the training of learning algorithms and their evaluation. To mitigate the unrepresentativeness and bias inherent in the negative sample selection (non-interacting proteins), we propose a clustering-based approach for representative negative sample selection.ResultsWe used deep autoencoders to investigate the effect of different sampling approaches for non-interacting pairs on the training and the performance of machine learning classifiers. By using the anomaly detection capabilities of deep autoencoders we deduced the effects of different categories of negative samples on the training of learning algorithms. Random sampling for selecting non-interacting pairs results in either over- or under-representation of hard or easy to classify instances. When K-means based sampling of negative datasets is applied to mitigate the inadequacies of random sampling, random forest (RF) together with the combined feature set of atomic composition, physicochemical-2grams and two different representations of evolutionary information performs best. Average model performances based on leave-one-out cross validation (loocv) over ten different negative sample sets that each model was trained with, show that RF models significantly outperform the previous best CRI predictor in terms of accuracy (+ 5.1%), specificity (+ 13%), mcc (+ 0.1) and g-means value (+ 5.1). Evaluations using tenfold cv and training/testing splits confirm the competitive performance.ConclusionsA comparative analysis was performed to assess the effect of three different sampling methods (random, K-means and uniform sampling) on the training of learning algorithms using different evaluation methods. Models trained on K-means sampled datasets generally show a significantly improved performance compared to those trained on random selections—with RF seemingly benefiting most in our particular setting. Our findings on the sampling are highly relevant and apply to many applications of supervised learning approaches in bioinformatics.

The Basement Membrane in a 3D Breast Acini Model Modulates Delivery and Anti-Proliferative Effects of Liposomal Anthracyclines

Breast cancer progression is marked by cancer cell invasion and infiltration, which can be closely linked to sites of tumor-connected basement membrane thinning, lesion, or infiltration. Bad treatment prognosis frequently accompanies lack of markers for targeted therapy, which brings traditional chemotherapy into play, despite its adverse effects like therapy-related toxicities. In the present work, we compared different liposomal formulations for the delivery of two anthracyclines, doxorubicin and aclacinomycin A, to a 2D cell culture and a 3D breast acini model. One formulation was the classical phospholipid liposome with a polyethylene glycol (PEG) layer serving as a stealth coating. The other formulation was fusogenic liposomes, a biocompatible, cationic, three-component system of liposomes able to fuse with the plasma membrane of target cells. For the lysosome entrapment-sensitive doxorubicin, membrane fusion enabled an increased anti-proliferative effect in 2D cell culture by circumventing the endocytic route. In the 3D breast acini model, this process was found to be limited to cells beneath a thinned or compromised basement membrane. In acini with compromised basement membrane, the encapsulation of doxorubicin in fusogenic liposomes increased the anti-proliferative effect of the drug in comparison to a formulation in PEGylated liposomes, while this effect was negligible in the presence of intact basement membranes.

Retargeting from the CR3 to the LFA-1 receptor uncovers the adenylyl cyclase enzyme–translocating segment of Bordetella adenylate cyclase toxin

The Bordetella adenylate cyclase toxin-hemolysin (CyaA) and the α-hemolysin (HlyA) of Escherichia coli belong to the family of cytolytic pore-forming Repeats in ToXin (RTX) cytotoxins. HlyA preferentially binds the αLβ2 integrin LFA-1 (CD11a/CD18) of leukocytes and can promiscuously bind and also permeabilize many other cells. CyaA bears an N-terminal adenylyl cyclase (AC) domain linked to a pore-forming RTX cytolysin (Hly) moiety, binds the complement receptor 3 (CR3, αMβ2, CD11b/CD18, or Mac-1) of myeloid phagocytes, penetrates their plasma membrane, and delivers the AC enzyme into the cytosol. We constructed a set of CyaA/HlyA chimeras and show that the CyaC-acylated segment and the CR3-binding RTX domain of CyaA can be functionally replaced by the HlyC-acylated segment and the much shorter RTX domain of HlyA. Instead of binding CR3, a CyaA1-710/HlyA411-1024 chimera bound the LFA-1 receptor and effectively delivered AC into Jurkat T cells. At high chimera concentrations (25 nm), the interaction with LFA-1 was not required for CyaA1-710/HlyA411-1024 binding to CHO cells. However, interaction with the LFA-1 receptor strongly enhanced the specific capacity of the bound CyaA1-710/HlyA411-1024 chimera to penetrate cells and deliver the AC enzyme into their cytosol. Hence, interaction of the acylated segment and/or the RTX domain of HlyA with LFA-1 promoted a productive membrane interaction of the chimera. These results help delimit residues 400-710 of CyaA as an "AC translocon" sufficient for translocation of the AC polypeptide across the plasma membrane of target cells.

Fluorescence Microscopy of the HIV-1 Envelope.

Human immunodeficiency virus (HIV) infection constitutes a major health and social issue worldwide. HIV infects cells by fusing its envelope with the target cell plasma membrane. This process is mediated by the viral Env glycoprotein and depends on the envelope lipid composition. Fluorescent microscopy has been employed to investigate the envelope properties, and the processes of viral assembly and fusion, but the application of this technique to the study of HIV is still limited by a number of factors, such as the small size of HIV virions or the difficulty to label the envelope components. Here, we review fluorescence imaging studies of the envelope lipids and proteins, focusing on labelling strategies and model systems.

Kill, but don't be killed

Cell Biology Cytotoxic T lymphocytes (CTLs) recognize and destroy infected or malignant cells. CTLs secrete the pore-forming protein perforin into the immune synapse, where perforin disrupts the target cell plasma membrane and initiates cell death pathways. Paradoxically, the secreted perforin only damages the target cell, leaving the producer lymphocytes unscathed and able to attack another target cell. What protects the lymphocyte is unclear. Using a variety of approaches, Rudd-Schmidt et al. found that two distinct but coordinated mechanisms were deployed by CTLs to protect themselves. Both mechanisms depended on the dynamic regulation of plasma membrane lipid composition and topology. High membrane lipid order made the CTL presynaptic membrane refractory to perforin binding. Furthermore, phosphatidylserine, exposed on the killer cell membrane, appeared to bind and inactivate perforin. Nat. Commun. 10 , 5396 (2019).

Collisional mechanism of ligand release by Bombyxmori JHBP, a member of the TULIP / Takeout family of lipid transporters

Juvenile hormones (JHs) regulate important processes in insects, such as postembryonic development and reproduction. In the hemolymph of Lepidoptera, these lipophilic sesquiterpenic hormones are transported from their site of synthesis to target tissues by high affinity carriers, the juvenile hormone binding proteins (JHBPs). Lepidopteran JHBPs belong to a recently uncovered, yet very ancient family of proteins sharing a common lipid fold (TULIP domain) and involved in shuttling various lipid ligands. One important, but poorly understood aspect of JHs action, is the mechanism of hormone transfer to or through the plasma membranes of target cells. Since many membrane-active peptides and proteins, such as the pore-forming bacterial toxins, are activated by low pH or interaction with phospholipid membranes, we have examined the effect of these factors on JH binding by JHBPs. The affinity of Bombyx mori and Manduca sexta JHBPs for JH III was determined by the DCC assay, equilibrium dialysis, and isothermal titration calorimetry, and found to be greatly reduced at low pH, in agreement with previous observations. Loss of binding was accompanied by changes in fluorescence and near-UV CD spectra, indicating significant changes in protein structure in the environment of aromatic residues. The apparent dissociation rate constant (koff) of the JHBP-JH III complex was greater at acidic pH, suggesting that low pH favors ligand release by opening of the binding pocket. The affinity of recombinant B. mori JHBP (rBmJHBP) was also decreased in the presence of anionic phospholipid vesicles. Measurements of steady-state fluorescence anisotropy with the lipophilic probe TMA-DPH demonstrated that rBmJHBP specifically interacts with anionic membranes. These results suggest the existence of a collisional mechanism for ligand release that may be important for delivery of JHs to the target cells, and could be relevant to the function of related members of this emerging family of lipid-transport proteins.

Bioengineering of Bordetella pertussis Adenylate Cyclase Toxin for Antigen-Delivery and Immunotherapy.

The adenylate cyclase toxin (CyaA) is one of the major virulence factors of Bordetella pertussis, the causative agent of whooping cough. CyaA is able to invade eukaryotic cells where, upon activation by endogenous calmodulin, it synthesizes massive amounts of cAMP that alters cellular physiology. The CyaA toxin is a 1706 residues-long bifunctional protein: the catalytic domain is located in the 400 amino-proximal residues, whereas the carboxy-terminal 1306 residues are implicated in toxin binding to the cellular receptor, the αMβ2 (CD11b/CD18) integrin, and subsequently in the translocation of the catalytic domain across the cytoplasmic membrane of the target cells. Indeed, this protein is endowed with the unique capability of delivering its N-terminal catalytic domain directly across the plasma membrane of eukaryotic target cells. These properties have been exploited to engineer the CyaA toxin as a potent non-replicating vector able to deliver antigens into antigen presenting cells and elicit specific cell-mediated immune responses. Antigens of interest can be inserted into the CyaA protein to yield recombinant molecules that are targeted in vivo to dendritic cells, where the antigens are processed and presented by the major class I and class II histocompatibility complexes (MHC-I and II). CyaA turned out to be a remarkably effective and versatile vaccine vector capable of inducing all the components of the immune response (T-CD4, T-CD8, and antibody). In this chapter, we summarize the basic knowledge on the adenylate cyclase toxin and then describe the application of CyaA in vaccinology, including some recent results of clinical trials of immunotherapy using a recombinant CyaA vaccine.

Toxoplasma Parasite Twisting Motion Mechanically Induces Host Cell Membrane Fission to Complete Invasion within a Protective Vacuole

To invade cells, the parasite Toxoplasma gondii injects a multi-unit nanodevice into the target cellplasma membrane (PM). The core nanodevice, which is composed of the RhOptry Neck (RON) protein complex, connects Toxoplasma and host cell through a circular tight junction (TJ). We now report that this RON nanodevice mechanically promotes membrane scission at the TJ-PM interface, directing a physical rotation driven by the parasite twisting motion that enables the budding parasitophorous vacuole (PV) to seal and separate from the host cell PM as a bona fide subcellular Toxoplasma-loaded PV. Mechanically impairing the process induces swelling of the budding PV and death of the parasite but not host cell. Moreover, this study reveals that the parasite nanodevice functions as a molecular trigger to promote PV membrane remodeling and rapid onset of T.gondii to intracellular lifestyle.

Mechanisms of Action and Cell Death Associated with Clostridium perfringens Toxins.

Clostridium perfringens uses its large arsenal of protein toxins to produce histotoxic, neurologic and intestinal infections in humans and animals. The major toxins involved in diseases are alpha (CPA), beta (CPB), epsilon (ETX), iota (ITX), enterotoxin (CPE), and necrotic B-like (NetB) toxins. CPA is the main virulence factor involved in gas gangrene in humans, whereas its role in animal diseases is limited and controversial. CPB is responsible for necrotizing enteritis and enterotoxemia, mostly in neonatal individuals of many animal species, including humans. ETX is the main toxin involved in enterotoxemia of sheep and goats. ITX has been implicated in cases of enteritis in rabbits and other animal species; however, its specific role in causing disease has not been proved. CPE is responsible for human food-poisoning and non-foodborne C. perfringens-mediated diarrhea. NetB is the cause of necrotic enteritis in chickens. In most cases, host–toxin interaction starts on the plasma membrane of target cells via specific receptors, resulting in the activation of intracellular pathways with a variety of effects, commonly including cell death. In general, the molecular mechanisms of cell death associated with C. perfringens toxins involve features of apoptosis, necrosis and/or necroptosis.

Binary Clostridium difficile toxin (CDT) - A virulence factor disturbing the cytoskeleton

Clostridium difficile infection causes antibiotics-associated diarrhea and pseudomembranous colitis. Major virulence factors of C. difficile are the Rho-glucosylating toxins TcdA and TcdB. In addition, many, so-called hypervirulent C. difficile strains produce the binary actin-ADP-ribosylating toxin CDT. CDT causes depolymerization of F-actin and rearrangement of the actin cytoskeleton. Thereby, many cellular functions, which depend on actin, are altered. CDT disturbs the dynamic balance between actin and microtubules in target cells. The toxin increases microtubule polymerization and induces the formation of microtubule-based protrusions at the plasma membrane of target cells. Moreover, CDT causes a redistribution of vesicles from the basolateral side to the apical side, where extracellular matrix proteins are released. These processes may increase the adherence of clostridia to target cells. Here, we review the effects of the action of CDT on the actin cytoskeleton and on the microtubule system.