The Effect of Caco-2 Cells on Sporulation and Enterotoxin Expression by Foodborne Clostridium perfringens.

Clostridium perfringens enterotoxin (Cpe)-producing strains cause gastrointestinal infections in humans and account for the second-largest number of all foodborne outbreaks caused by bacterial toxins. The Cpe toxin is only produced during sporulation; this process might be affected when C. perfringens comes into contact with host cells. The current study determined how the cpe expression levels and spore formation changed over time during co-culture with Caco-2 cells (as a model of intestinal epithelial cells). In co-culture with Caco-2 cells, total C. perfringens cell counts first decreased and then remained more or less stable, whereas spore counts were stable over the whole incubation period. The cpe mRNA level in the co-culture with Caco-2 cells increased more rapidly than in the absence of Caco-2 cells (3.9-fold higher levels in coculture than in the absence of Caco-2 cells after 8 h of incubation). Finally, we found that cpe expression is inhibited by a cue released by Caco-2 cells (8.3-fold lower levels in the presence of supernatants of Caco-2 cells than in the absence of the supernatants after 10 h of incubation); as a consequence, the increased expression in co-culture with Caco-2 cells must be caused by a factor associated with the Caco-2 cells.

Structural Basis of Clostridium perfringens Enterotoxin Activation and Oligomerization by Trypsin.

Clostridium perfringens enterotoxin (CpE) is a ß-pore forming toxin that disrupts gastrointestinal homeostasis in mammals by binding membrane protein receptors called claudins. Although structures of CpE fragments bound to claudins have been determined, the mechanisms that trigger CpE activation and oligomerization that lead to the formation of cytotoxic ß-pores remain undetermined. Proteolysis of CpE in the gut by trypsin has been shown to play a role in this and subsequent cytotoxicity processes. Here, we report solution structures of full-length and trypsinized CpE using small-angle X-ray scattering (SAXS) and crystal structures of trypsinized CpE and its C-terminal claudin-binding domain (cCpE) using X-ray crystallography. Mass spectrometry and SAXS uncover that removal of the CpE N-terminus by trypsin alters the CpE structure to expose areas that are normally unexposed. Crystal structures of trypsinized CpE and cCpE reveal unique dimer interfaces that could serve as oligomerization sites. Moreover, comparisons of these structures to existing ones predict the functional implications of oligomerization in the contexts of cell receptor binding and ß-pore formation. This study sheds light on trypsin's role in altering CpE structure to activate its function via inducing oligomerization on its path toward cytotoxic ß-pore formation. Its findings can incite new approaches to inhibit CpE-based cytotoxicity with oligomer-disrupting therapeutics.

cCPE Fusion Proteins as Molecular Probes to Detect Claudins and Tight Junction Dysregulation in Gastrointestinal Cell Lines, Tissue Explants and Patient-Derived Organoids.

Claudins regulate paracellular permeability, contribute to epithelial polarization and are dysregulated during inflammation and carcinogenesis. Variants of the claudin-binding domain of Clostridium perfringens enterotoxin (cCPE) are highly sensitive protein ligands for generic detection of a broad spectrum of claudins. Here, we investigated the preferential binding of YFP- or GST-cCPE fusion proteins to non-junctional claudin molecules. Plate reader assays, flow cytometry and microscopy were used to assess the binding of YFP- or GST-cCPE to non-junctional claudins in multiple in vitro and ex vivo models of human and rat gastrointestinal epithelia and to monitor formation of a tight junction barrier. Furthermore, YFP-cCPE was used to probe expression, polar localization and dysregulation of claudins in patient-derived organoids generated from gastric dysplasia and gastric cancer. Live-cell imaging and immunocytochemistry revealed cell polarity and presence of tight junctions in glandular organoids (originating from intestinal-type gastric cancer and gastric dysplasia) and, in contrast, a disrupted diffusion barrier for granular organoids (originating from discohesive tumor areas). In sum, we report the use of cCPE fusion proteins as molecular probes to specifically and efficiently detect claudin expression, localization and tight junction dysregulation in cell lines, tissue explants and patient-derived organoids of the gastrointestinal tract.

Computational design of a chimeric toxin against Claudin-4-expressing cancer cells: molecular modeling, docking and molecular dynamics simulation analysis.

Cancer is one of the main reasons of mortality all over the world. Over the time, the major ways for cancer-therapy were based on radiotherapy, chemotherapy and surgery. These methods are not specific enough for that purpose, therefore, new ideas for design of new drugs with higher specificity are considered. Chimeric protein toxins are hybrid proteins consisting of a targeting portion and a toxic one which specifically bind and kill the target cancer cells. The main purpose of this study was designing a recombinant chimeric toxin with biding capability to one of the most key receptors namely claudin-4 which is over-expressed in almost all cancer cells. To design it, we utilized the last 30 C-terminal amino acids of Clostridium perfringens enterotoxin (CPE) as a binding module for claudin-4 and the toxic module which is the A-domain of Shiga toxin from Shigella dysenteriae. Using molecular modeling and docking methods, appropriate binding affinity of the recombinant chimeric toxin to its specific receptor was demonstrated. In the next step, the stability of this interaction was investigated by molecular dynamics simulation. Although partial instability was detected at some time points, however, sufficient stable situation of hydrogens bonds and high binding affinity between the chimeric toxin and receptor were observed in the in silico studies which in turn suggested that this complex could be formed successfully.

Proteomic identification and quantification of Clostridium perfringens enterotoxin using a stable isotope-labelled peptide via liquid chromatography-tandem mass spectrometry.

PURPOSE: Detection of Clostridium perfringens enterotoxin (CPE) in human stool is critical evidence of food poisoning. However, processing patient-derived samples is difficult and very few methods exist to confirm the presence of CPE. In this study, a technique was developed using proteomic analysis to identify and quantify CPE in artificial gut fluid as an alternative. METHODS: The standard CPE was spiked into artificial gut fluids, and effective methods were developed by employing both a stable isotope-labelled internal standard peptide and liquid chromatography-tandem mass spectrometry (LC-MS/MS). RESULTS: Proteotypic peptide EILDLAAATER formed by tryptic digestion was selected for quantitation of CPE. The peptide was identified using product ion spectra. Although the nontoxic peptides originating from CPE showed very low detectability in extraction and tryptic digestion, they could be detected with sufficient sensitivity using the method we developed. Based on a spiked recovery test at two concentrations (50 and 200 µg/kg), the recovery values were 85 and 78%, respectively. The relative standard deviations of repeatability and within-laboratory reproducibility were less than 8 and 11%, respectively. These standard deviations satisfied the criteria of the Japanese validation guidelines for residues (MHLW 2010, Director Notice, Syoku-An No. 1224-1). The limit of quantification (LOQ) was estimated to be 50 µg/kg. The combination of the product ion spectra and relative ion ratio supported CPE identification at the LOQ level. CONCLUSIONS: To the best of our knowledge, this is the first report of proteomic analysis of CPE using LC-MS/MS. The method would greatly help in assessing CPE reliably.

Multiscale modelling of claudin-based assemblies: A magnifying glass for novel structures of biological interfaces.

Claudins (Cldns) define a family of transmembrane proteins that are the major determinants of the tight junction integrity and tissue selectivity. They promote the formation of either barriers or ion-selective channels at the interface between two facing cells, across the paracellular space. Multiple Cldn subunits form complexes that include cis- (intracellular) interactions along the membrane of a single cell and trans- (intercellular) interactions across adjacent cells. The first description of Cldn assemblies was provided by electron microscopy, while electrophysiology, mutagenesis and cell biology experiments addressed the functional role of different Cldn homologs. However, the investigation of the molecular details of Cldn subunits and complexes are hampered by the lack of experimental native structures, currently limited to Cldn15. The recent implementation of computer-based techniques greatly contributed to the elucidation of Cldn properties. Molecular dynamics simulations and docking calculations were extensively used to refine the first Cldn multimeric model postulated from the crystal structure of Cldn15, and contributed to the introduction of a novel, alternative, arrangement. While both these multimeric assemblies were found to account for the physiological properties of some family members, they gave conflicting results for others. In this review, we illustrate the major findings on Cldn-based systems that were achieved by using state-of-the-art computational methodologies. The information provided by these results could be useful to improve the characterization of the Cldn properties and help the design of new efficient strategies to control the paracellular transport of drugs or other molecules.

Application of C-Terminal Clostridium Perfringens Enterotoxin in Treatment of Brain Metastasis from Breast Cancer.

Claudin-4 is part of the Claudin family of transmembrane tight junction (TJ) proteins found in almost all tissues and, together with adherens junctions and desmosomes, forms epithelial and endothelial junctional complexes. Although the distribution of Claudin-4 occurs in many cell types, the level of expression is cell-specific. Claudin proteins regulate cell proliferation and differentiation by binding cell-signaling ligands, and its expression is upregulated in several cancers. As a result, alterations in Claudin expression patterns or distribution are vital in the pathology of cancer. Profiling the genetic expression of Claudin-4 showed that Claudin-4 is also a receptor for the clostridium perfringens enterotoxin (CPE) and that Claudin-4 has a high sequence similarity with CPE's high-affinity receptor. CPE is cytolytic due to its ability to form pores in cellular membranes, and CPE treatment in breast cancer cells have shown promising results due to the high expression of Claudin-4. The C-terminal fragment of CPE (c-CPE) provides a less toxic alternative for drug delivery into breast cancer cells, particularly metastatic tumors in the brain, especially as Claudin-4 expression in the central nervous system (CNS) is low. Therefore, c-CPE provides a unique avenue for the treatment of breast-brain metastatic tumors.

Claudin-Targeted Suicide Gene Therapy for Claudin-Overexpressing Tumor Cells by Using Modified Clostridium perfringens Enterotoxin (CPE).

Bacterial toxins gain growing attention as potential cancer treatment due to their potent cytotoxic effects. Among the very different toxins with diverse modes of action, the Clostridium perfringens enterotoxin (CPE) is in focus to treat solid cancers. This toxin targets the tight junction proteins claudin-3 and -4 (Cldn-3/4), which are frequently overexpressed in solid cancers. Binding to these claudins induces pore formation in the host cell plasma membrane leading to rapid oncoleaking cell death of tumor cells. Based on this, extending the targeting of CPE beyond Cldn-3/4 is of interest, since other claudins, such as claudin-1 or -5 are often overexpressed in various cancer entities such as non-small-cell lung cancer (NSCLC) or papillary thyroid carcinoma. In this chapter we describe the modification of a CPE-encoding vector by structure-directed mutagenesis to either preferentially target Cldn-1 and -5 or to expand targeting to Cldn1-9 for improved broadened cytotoxic targeting of claudin-overexpressing tumors such as but not limited to lung cancer via CPE gene transfer.

Disruption of Claudin-Made Tight Junction Barriers by Clostridium perfringens Enterotoxin: Insights from Structural Biology.

Claudins are a family of integral membrane proteins that enable epithelial cell/cell interactions by localizing to and driving the formation of tight junctions. Via claudin self-assembly within the membranes of adjoining cells, their extracellular domains interact, forming barriers to the paracellular transport of small molecules and ions. The bacterium Clostridium perfringens causes prevalent gastrointestinal disorders in mammals by employing an enterotoxin (CpE) that targets claudins. CpE binds to claudins at or near tight junctions in the gut and disrupts their barrier function, potentially by disabling their assembly or via cell signaling means-the mechanism(s) remain unclear. CpE ultimately destroys claudin-expressing cells through the formation of a cytotoxic membrane-penetrating ß-barrel pore. Structures obtained by X-ray crystallography of CpE, claudins, and claudins in complex with CpE fragments have provided the structural bases of claudin and CpE functions, revealing potential mechanisms for the CpE-mediated disruption of claudin-made tight junctions. This review highlights current progress in this space-what has been discovered and what remains unknown-toward efforts to elucidate the molecular mechanism of CpE disruption of tight junction barriers. It further underscores the key insights obtained through structure that are being applied to develop CpE-based therapeutics that combat claudin-overexpressing cancers or modulate tight junction barriers.

Autophagic degradation of claudin-5 mediated by its binding to a Clostridium perfringens enterotoxin fragment modulates endothelial barrier permeability.

The blood-brain barrier (BBB) protects the central nervous system (CNS) from harmful elements, while it also restricts efficient drug delivery into the CNS. Previously, we generated a mutated fragment of Clostridium perfringens enterotoxin (cCPEYWSH ) which specifically binds to the endothelial tight junction protein claudin-5. Here, we explore the mechanisms regulating the dynamics of membranous claudin-5 and BBB permeability. Following cCPEYWSH binding to claudin-5, caveolin-1 mediates the redistribution of claudin-5 to the cytosol. This abnormal cytosolic aggregation triggers the autophagic degradation of claudin-5, leading to an increase in BBB permeability. Enhancement or inhibition of autophagy accelerates or inhibits the degradation of cytosolic claudin-5, respectively. Our findings may pave the way for improving BBB permeability for drug delivery.

Effective Oncoleaking Treatment of Pancreatic Cancer by Claudin-Targeted Suicide Gene Therapy with Clostridium perfringens Enterotoxin (CPE).

Pancreatic cancer (PC) is one of the most lethal cancers worldwide, associated with poor prognosis and restricted therapeutic options. Clostridium perfringens enterotoxin (CPE), is a pore-forming (oncoleaking) toxin, which binds to claudin-3 and -4 (Cldn3/4) causing selective cytotoxicity. Cldn3/4 are highly upregulated in PC and represent an effective target for oncoleaking therapy. We utilized a translation-optimized CPE vector (optCPE) for new suicide approach of PC in vitro and in cell lines (CDX) and patient-derived pancreatic cancer xenografts (PDX) in vivo. The study demonstrates selective toxicity in Cldn3/4 overexpressing PC cells by optCPE gene transfer, mediated by pore formation, activation of apoptotic/necrotic signaling in vitro, induction of necrosis and of bystander tumor cell killing in vivo. The optCPE non-viral intratumoral in vivo jet-injection gene therapy shows targeted antitumoral efficacy in different CDX and PDX PC models, leading to reduced tumor viability and induction of tumor necrosis, which is further enhanced if combined with chemotherapy. This selective oncoleaking suicide gene therapy improves therapeutic efficacy in pancreas carcinoma and will be of value for better local control, particularly of unresectable or therapy refractory PC.

Targeting of claudin-4 by Clostridium perfringens enterotoxin-conjugated polysialic acid nanoparticles for pancreatic cancer therapy.

Despite recent advances in chemotherapy, pancreatic cancer remains a leading cause of cancer-related deaths. Moreover, although targeted therapy has shown promising therapeutic efficacy in many types of cancers, no such effective targeting strategy for treatment of pancreatic cancer, which is in desperate need for new treatment approaches. Here, we developed claudin-4-targeting Clostridium perfringens enterotoxin (CPE) peptide-conjugated polysialic acid nanoparticles (C-SNPs) for pancreatic cancer-targeted therapy. Doxorubicin-loaded C-SNPs (DOX-C-SNPs) higly accumulated in the targeted pancreatic cancer via enhanced peameability and retention (EPR) effect, targeting claudin-4 in pancreatic cancer that becomes superficially exposed owing to the disruption of tight junctions. Notably, DOX-C-SNP accumulation in the non-targeted, normal pancreas was significantly reduced because of hindered access to claudin-4 in tight junctions. As a result, DOX-C-SNPs substantially suppressed tumor growth in an orthotopic pancreatic cancer model while exerting minimal toxicity against non-targeted, normal tissues. Collectively, these findings indicate that claudin-4-targeting DOX-C-SNPs may have promise in treating pancreatic cancers through targeting of exposed claudin-4.

Tight Junction Modulating Bioprobes for Drug Delivery System to the Brain: A Review.

The blood-brain barrier (BBB), which is composed of endothelial cells, pericytes, astrocytes, and neurons, separates the brain extracellular fluid from the circulating blood, and maintains the homeostasis of the central nervous system (CNS). The BBB endothelial cells have well-developed tight junctions (TJs) and express specific polarized transport systems to tightly control the paracellular movements of solutes, ions, and water. There are two types of TJs: bicellular TJs (bTJs), which is a structure at the contact of two cells, and tricellular TJs (tTJs), which is a structure at the contact of three cells. Claudin-5 and angulin-1 are important components of bTJs and tTJs in the brain, respectively. Here, we review TJ-modulating bioprobes that enable drug delivery to the brain across the BBB, focusing on claudin-5 and angulin-1.

Anti-tumor effect of a recombinant Bifidobacterium strain secreting a claudin-targeting molecule in a mouse breast cancer model.

Bifidobacterium is a nonpathogenic strain of anaerobic bacteria that selectively localizes and proliferates in tumors. It has emerged as a specific carrier of anticancer proteins against malignant tumors. Claudins are tetraspanin transmembrane proteins that form tight junctions. Claudin-4 is overexpressed in certain epithelial malignant cancers. The C-terminal fragment of the Clostridium perfringens enterotoxin (C-CPE), an exotoxin without the cytotoxic domain, strongly binds to claudin-4. The C-CPE fusion toxin (C-CPE-PE23), which targets claudin-4, strongly suppresses tumor growth; however, C-CPE fusion toxins exhibit hepatic toxicity. In this study, we successfully generated a strain of Bifidobacterium longum that secreted C-CPE-PE23 (B. longum-C-CPE-PE23) and was specific to and cross reactive with human and mouse claudin-4. We evaluated the therapeutic potential of this strain against triple-negative breast cancer using a mouse model. C-CPE-PE23 decreased cell viability in a dose-dependent manner in human and mouse breast cancer cell lines. After intravenous injection, Bifidobacterium was specifically distributed in the tumors of mice bearing breast cancer tumors. Moreover, B. longum-C-CPE-PE23 significantly suppressed tumor growth in mice with breast cancer without serious side effects, such as weight loss or hepatic and renal damage. We suggest that B. longum-C-CPE-PE23 is a good candidate for breast cancer treatment. Bifidobacterium could also be used as a drug delivery system for hepatotoxic agents.

Role of Clostridium perfringens Enterotoxin on YAP Activation in Colonic Sessile Serrated Adenoma/ Polyps with Dysplasia.

Sessile serrated adenoma/polyp with dysplasia (SSA/P-D) is an SSA/P with cellular dysplasia and has a higher risk of progressing to colon carcinogenesis. Previously, we reported that tight junction impairment by Clostridium perfringens enterotoxin (CPE) leads to activation of the transcriptional co-activator yes-associated protein (YAP) in oral squamous cell carcinoma. Here, we investigated whether CPE activates YAP to promote the malignant progression of SSA/P. E-cadherin expression was lower in the 12 cases with SSA/P-D examined than that in normal mucosa, SSA/P, or tubular adenoma (TA). Furthermore, intracellular translocation of claudin-4 (CLDN4) and nuclear translocation of YAP were observed. The CPE gene was detected in DNA extracted from SSA/P-D lesions, but not in SSA/P or TA. Treatment of the rat intestinal epithelial cell line IEC6 with low-dose CPE resulted in intracellular translocation of CLDN4 to the cytoplasmic membrane. Cytoplasmic CLDN4 showed co-precipitation with transcriptional co-activator with PDZ-binding motif, zonula occludens (ZO)-1, large tumor suppressor, and mammalian Ste20-like. Additionally, YAP co-precipitated with ZO-2 under CPE treatment led to decreased YAP phosphorylation and nuclear translocation. YAP activation promoted increase in nuclear TEA domain family member level, expression of cyclin D1, snail, vimentin, CD44, NS and decrease in E-cadherin levels, thereby inducing stemness and epithelial-mesenchymal-transition (EMT). The Hippo complex with the incorporation of CLDN4 increased stability. Upon low-dose CPE treatment, HT29 cells with BRAFV600E gene mutation showed increased growth, enhanced invasive potential, stemness, and induced EMT phenotype, whereas HCT116 cells, which carry KRASG13D gene mutation, did not show such changes. In an examination of 10 colorectal cancers, an increase in EMT and stemness was observed in CPE (+) and BRAF mutation (+) cases. These findings suggest that C. perfringens might enhance the malignant transformation of SSA/P-D via YAP activation. Our findings further highlight the importance of controlling intestinal flora using probiotics or antibiotics.

Clostridium perfringens enterotoxin induces claudin-4 to activate YAP in oral squamous cell carcinomas.

Claudin (CLDN)-4 expression has been associated with malignancy in various cancers. When CLDN4 expression was examined in oral squamous cell carcinoma (OSCC), 22 out of 57 (39%) cases showed immunoreactivity in the nucleus. Nuclear CLDN4-positive cases showed a stronger correlation with cancer progression than the negative cases. Intratumoral anaerobic bacterial DNA examination revealed nuclear CLDN4 expression in 81% of Clostridium perfringens-positive cases. Treatment of human oral squamous cell carcinoma cell lines HSC3 and HSC4 with Clostridium perfringens enterotoxin (CPE), induced CLDN4 nuclear translocation to enhance epithelial-mesenchymal transition (EMT), stemness, cell proliferation and invasive ability. In addition, CPE treatment suppressed phosphorylation of yes-associated protein-1 (YAP1) and promoted YAP1 nuclear translocation, resulting in increased expression of YAP1 target genes; cyclin D1 and connective tissue growth factor. Moreover, it was revealed that the complex of YAP1, CLDN4 and zona occludens-2 (ZO-2) was formed by CPE treatment, further suppressing YAP1 phosphorylation by LATS1 and activating it. Thus YAP activation in OSCC was regarded important in promoting malignant phenotypes. Our research suggested that the control of oral anaerobic bacteria may suppress YAP activation and in turn tumor progression.

Targeting claudin-overexpressing thyroid and lung cancer by modified Clostridium perfringens enterotoxin.

Clostridium perfringens enterotoxin (CPE) can be used to eliminate carcinoma cells that overexpress on their cell surface CPE receptors - a subset of claudins (e.g., Cldn3 and Cldn4). However, CPE cannot target tumors expressing solely CPE-insensitive claudins (such as Cldn1 and Cldn5). To overcome this limitation, structure-guided modifications were used to generate CPE variants that can strongly bind to Cldn1, Cldn2 and/or Cldn5, while maintaining the ability to bind Cldn3 and Cldn4. This enabled (a) targeting of the most frequent endocrine malignancy, namely, Cldn1-overexpressing thyroid cancer, and (b) improved targeting of the most common cancer type worldwide, non-small-cell lung cancer (NSCLC), which is characterized by high expression of several claudins, including Cldn1 and Cldn5. Different CPE variants, including the novel mutant CPE-Mut3 (S231R/S313H), were applied on thyroid cancer (K1 cells) and NSCLC (PC-9 cells) models. In vitro, CPE-Mut3, but not CPEwt, showed Cldn1-dependent binding and cytotoxicity toward K1 cells. For PC-9 cells, CPE-Mut3 improved claudin-dependent cytotoxic targeting, when compared to CPEwt. In vivo, intratumoral injection of CPE-Mut3 in xenograft models bearing K1 or PC-9 tumors induced necrosis and reduced the growth of both tumor types. Thus, directed modification of CPE enables eradication of tumor entities that cannot be targeted by CPEwt, for instance, Cldn1-overexpressing thyroid cancer by using the novel CPE-Mut3.

Impacts of the prostate stem cell antigen (PSCA) and Clostridium perfringens enterotoxin (CPE) on the apoptosis and cell cycle regulatory genes in PC3.

Clostridium perfringens enterotoxin (CPE) has anti-prostate cancer effects and the prostate stem cell antigen (PSCA) has been used as a plasmid-based vaccine. So we expressed both of them in the PC3 cells to evaluate their effects on cell cycling and apoptosis. The PC3 cells were transfected either by the pBudCE4.1-CPE-PSCA or empty plasmid. The expression of the cpe and PSCA genes in transfected PC3 was evaluated. The apoptosis genes (Fas, P53, Bak, and Bax) as well as cell cycling genes (cyclin D1 and E) expression was evaluated by qPCR. Successful expression of cpe and PSCA in PC3 cells was confirmed. The flow cytometry results showed the cellular death rates of 62.6% and 21.8% for PC3 cells transformed with recombinant and empty plasmids respectively. Bak, Fas, Bax and P53 genes were significantly upregulated in PC3 cells transformed with pBudCE4.1-CPE-PSCA, while cyclin D1 and E were downregulated when compared with the pBudCE4.1-transfected PC3 and normal cells (p < .05). The results showed the lethal consequences of cpe and PSCA genes expression on PC3 transfected cells. Expression of the cpe and PSCA genes affects the PC3 cell death so it could be a suitable candidate for further researches in prostate cancer vaccine development.

RIP1, RIP3, and MLKL Contribute to Cell Death Caused by Clostridium perfringens Enterotoxin.

Clostridium perfringens type F strains cause gastrointestinal disease when they produce a pore-forming toxin named C. perfringens enterotoxin (CPE). In human enterocyte-like Caco-2 cells, low CPE concentrations cause caspase-3-dependent apoptosis, while high CPE concentrations cause necrosis. Since necrosis or apoptosis sometimes involves receptor-interacting serine/threonine-protein kinase-1 or 3 (RIP1 or RIP3), this study examined whether those kinases are important for CPE-induced apoptosis or necrosis. Highly specific RIP1 or RIP3 inhibitors reduced both CPE-induced apoptosis and necrosis in Caco-2 cells. Those findings suggested that the form of necrosis induced by treating Caco-2 cells with high CPE concentrations involves necroptosis, which was confirmed when high, but not low, CPE concentrations were shown to induce oligomerization of mixed-lineage kinase domain-like pseudokinase (MLKL), a key late step in necroptosis. Furthermore, an MLKL oligomerization inhibitor reduced cell death caused by high, but not low, CPE concentrations. Supporting RIP1 and RIP3 involvement in CPE-induced necroptosis, inhibitors of those kinases also reduced MLKL oligomerization during treatment with high CPE concentrations. Calpain inhibitors similarly blocked MLKL oligomerization induced by high CPE concentrations, implicating calpain activation as a key intermediate in initiating CPE-induced necroptosis. In two other CPE-sensitive cell lines, i.e., Vero cells and human enterocyte-like T84 cells, low CPE concentrations also caused primarily apoptosis/late apoptosis, while high CPE concentrations mainly induced necroptosis. Collectively, these results establish that high, but not low, CPE concentrations cause necroptosis and suggest that RIP1, RIP3, MLKL, or calpain inhibitors can be explored as potential therapeutics against CPE effects in vivoIMPORTANCEC. perfringens type F strains are a common cause of food poisoning and antibiotic-associated diarrhea. Type F strain virulence requires production of C. perfringens enterotoxin (CPE). In Caco-2 cells, high CPE concentrations cause necrosis while low enterotoxin concentrations induce apoptosis. The current study determined that receptor-interacting serine/threonine-protein kinases 1 and 3 are involved in both CPE-induced apoptosis and necrosis in Caco-2 cells, while mixed-lineage kinase domain-like pseudokinase (MLKL) oligomerization is involved in CPE-induced necrosis, thereby indicating that this form of CPE-induced cell death involves necroptosis. High CPE concentrations also caused necroptosis in T84 and Vero cells. Calpain activation was identified as a key intermediate for CPE-induced necroptosis. These results suggest inhibitors of RIP1, RIP3, MLKL oligomerization, or calpain are useful therapeutics against CPE-mediated diseases.

Use of Modified Clostridium perfringens Enterotoxin Fragments for Claudin Targeting in Liver and Skin Cells.

Claudins regulate paracellular permeability in different tissues. The claudin-binding domain of Clostridium perfringens enterotoxin (cCPE) is a known modulator of a claudin subset. However, it does not efficiently bind to claudin-1 (Cldn1). Cldn1 is a pharmacological target since it is (i) an essential co-receptor for hepatitis C virus (HCV) infections and (ii) a key element of the epidermal barrier limiting drug delivery. In this study, we investigated the potential of a Cldn1-binding cCPE mutant (i) to inhibit HCV entry into hepatocytes and (ii) to open the epidermal barrier. Inhibition of HCV infection by blocking of Cldn1 with cCPE variants was analyzed in the Huh7.5 hepatoma cell line. A model of reconstructed human epidermis was used to investigate modulation of the epidermal barrier by cCPE variants. In contrast to cCPEwt, the Cldn1-binding cCPE-S305P/S307R/S313H inhibited infection of Huh7.5 cells with HCV in a dose-dependent manner. In addition, TJ modulation by cCPE variant-mediated targeting of Cldn1 and Cldn4 opened the epidermal barrier in reconstructed human epidermis. cCPE variants are potent claudin modulators. They can be applied for mechanistic in vitro studies and might also be used as biologics for therapeutic claudin targeting including HCV treatment (host-targeting antivirals) and improvement of drug delivery.