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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. [ABSTRACT FROM AUTHOR]

Clostridium perfringens enterotoxin (CpE) is a β-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 β-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 β-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 β-pore formation. Its findings can incite new approaches to inhibit CpE-based cytotoxicity with oligomer-disrupting therapeutics. [ABSTRACT FROM AUTHOR]

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.

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 Adomain 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. [ABSTRACT FROM AUTHOR]

Clostridium perfringens enterotoxin (CpE) is a β-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 β-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 β-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 β-pore formation. Its findings can incite new approaches to inhibit CpE-based cytotoxicity with oligomer-disrupting therapeutics.

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.

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. [ABSTRACT FROM AUTHOR]

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.

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 β-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.

ABSTRACT 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 vivo. IMPORTANCE C. 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.

Background: Detection of Clostridium perfringens enterotoxin (CPE) is critical for disease surveillance; however, commercial testing kits produce contrasting results. Methods: We examined the cause of the differing results from a reversed passive latex agglutination (RPLA) assay (PET-RPLA Toxin Detection Kit) and an enzyme-linked immunosorbent assay (C. perfringens Enterotoxin ELISA Kit) using 73 human norovirus-positive fecal samples from gastroenteritis patients across 22 episodes in Japan. Results: CPE was detected in 39/73 samples using the RPLA method; however, ELISA-based examination of 10 RPLA-positive samples produced negative results. Moreover, cpe was not detected in any of the RPLA-positive (n = 32) or -negative (n = 5) samples, and C. perfringens was only isolated from one RPLA-positive sample. Conclusions: An ELISA-based testing approach may be more reliable than RPLA assays for CPE detection from human fecal samples. These findings may also be applicable to the detection of other foodborne diseases. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Clostridium perfringens is both an ubiquitous environmental bacterium and the fourth most common causative agent of foodborne outbreaks (FBOs) in France and Europe. These outbreaks are known to be caused by C. perfringens enterotoxin (CPE) encoded by the cpe gene. However, additional information on the toxin/virulence gene content of C. perfringens has become available in the last few years. Therefore, to understand the enteropathogenicity of this bacterium, we need to describe the toxin and virulence genes content of strains involved in FBOs. In this study, we used a new real-time PCR typing technique based on a comprehensive set of 17 genes encoding virulence factors. The analysis was performed on a collection of 141 strains involved in 42 FBOs in the Paris region. It was combined with whole genome sequence (WGS) phylogenomic reconstruction, based on the coregenome single nucleotide polymorphisms (SNPs) of 58 isolates, representatives of the identified virulence gene profiles. Two or three different virulence gene profiles were detected in 10 FBOs, demonstrating that C. perfringens FBOs may be associated with heterogeneous strains. cpe-positive strains were isolated in 23 outbreaks, confirming the prominent role of CPE in pathogenicity. However, while C. perfringens was the sole pathogen isolated from the incriminated food, the cpe gene was not detected in strains related to 13 outbreaks. This result indicates either that the standard method was not able to isolate cpe+ strains or that the cpe gene may not be the only determinant of the enterotoxigenic potential of C. perfringens strains. Using phylogenomic reconstruction, we identified two clades distinguishing chromosomal cpe-positive from cpe-negative and plasmid-borne cpe. Important epidemiological information was also garnered from this phylogenomic reconstruction that revealed unexpected links between different outbreaks associated with closely related strains (seven SNP differences) and having common virulence gene profiles. This study provides new insight into the characterization of foodborne C. perfringens and highlights the potential of WGS for the investigation of FBOs.

Clostridium perfringens is both an ubiquitous environmental bacterium and the fourth most common causative agent of foodborne outbreaks (FBOs) in France and Europe. These outbreaks are known to be caused by C. perfringens enterotoxin (CPE) encoded by the cpe gene. However, additional information on the toxin/virulence gene content of C. perfringens has become available in the last few years. Therefore, to understand the enteropathogenicity of this bacterium, we need to describe the toxin and virulence genes content of strains involved in FBOs. In this study, we used a new real-time PCR typing technique based on a comprehensive set of 17 genes encoding virulence factors. The analysis was performed on a collection of 141 strains involved in 42 FBOs in the Paris region. It was combined with whole genome sequence (WGS) phylogenomic reconstruction, based on the coregenome single nucleotide polymorphisms (SNPs) of 58 isolates, representatives of the identified virulence gene profiles. Two or three different virulence gene profiles were detected in 10 FBOs, demonstrating that C. perfringens FBOs may be associated with heterogeneous strains. cpe- positive strains were isolated in 23 outbreaks, confirming the prominent role of CPE in pathogenicity. However, while C. perfringens was the sole pathogen isolated from the incriminated food, the cpe gene was not detected in strains related to 13 outbreaks. This result indicates either that the standard method was not able to isolate cpe+ strains or that the cpe gene may not be the only determinant of the enterotoxigenic potential of C. perfringens strains. Using phylogenomic reconstruction, we identified two clades distinguishing chromosomal cpe -positive from cpe -negative and plasmid-borne cpe. Important epidemiological information was also garnered from this phylogenomic reconstruction that revealed unexpected links between different outbreaks associated with closely related strains (seven SNP differences) and having common virulence gene profiles. This study provides new insight into the characterization of foodborne C. perfringens and highlights the potential of WGS for the investigation of FBOs. [ABSTRACT FROM AUTHOR]

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 (CPE) is a common cause of food poisoning and hyperkalemia-associated death. Previously, we reported that fusion of pneumococcal surface protein A (PspA) to C-terminal fragment of CPE (C-CPE) efficiently bound mucosal epithelium so that PspA-specific immune responses could be provoked. In this study, we found that fusion of C-CPE with PspA augmented the antigenicity of C-CPE itself. These findings allowed us to hypothesize that fusion of C-CPE and another food poisoning vaccine act as a bivalent food poisoning vaccine. Therefore, we constructed an adjuvant-free bivalent vaccine against CPE and cholera toxin (CT), which is a major food poisoning in developing country, by genetically fusing CT B subunit to C-CPE. Because of the low antigenicity of C-CPE, immunization of mice with C-CPE alone did not induce C-CPE-specific immune responses. However, immunization with our vaccine induced both C-CPE- and CT-specific neutralizing antibody. The underlying mechanism of the augmented antigenicity of C-CPE included the activation of T cells by CTB. Moreover, neutralizing antibodies lasted for at least 48 weeks and the quality of the antibody was dependent on the binding activity of CTB–C-CPE to its receptors. These findings suggest that our fusion protein is a potential platform for the development of an adjuvant-free bivalent vaccine against CPE and CT.

Saccharomyces boulardii, a subspecies of Saccharomyces cerevisiae, is a well-known eukaryotic probiotic with many benefits for human health. In the present study, a recombinant strain of S. boulardii was prepared to use as a potential oral vaccine delivery vehicle. In this sense, a ura3 auxotroph strain of S. boulardii CNCM I-745 (known as S. cerevisiae HANSEN CBS 5926, Yomogi®) was generated using CRISPR/Cas9 methodology. Then a gene construct encoding a highly immunogenic protein, ovalbumin (OVA), was prepared and transformed into the ura3- S. boulardii. To facilitate the transport of the recombinant immunogen across the intestinal barrier, a claudin-targeting sequence from Clostridium perfringens enterotoxin (CPE) was added to the C-terminus of the expression cassette. The recombinant S. boulardii strain expressing the OVA-CPE fusion protein was then administered orally to a group of mice, and serum IgG and fecal IgA levels were evaluated by ELISA. Our results demonstrated that anti-OVA IgG in serum significantly increased in test group (P < 0.001) compared to control groups (receiving wild type S. boulardii or PBS), and the fecal IgA titer was significantly higher in test group (P < 0.05) than control groups. In parallel, a recombinant S. boulardii strain expressing the similar construct lacking C-terminal CPE was also administered orally. The result showed an increased level of serum IgG in group receiving yeasts expressing the CPE negative construct compared to control groups; however, the fecal IgA levels did not increase significantly. In conclusion, our findings indicated that the yeast S. boulardii, as a delivery vehicle with possible immunomodulatory effects, and c-CPE, as a targeting tag, synergistically assist to stimulate systemic and local immunity. This proposed recombinant S. boulardii system might be useful in the expression of other antigenic peptides, making it as a promising tool for oral delivery of vaccines or therapeutic proteins.

Clostridium perfringens enterotoxin (CPE) is a three-domain polypeptide, which binds to Claudin-3 and Claudin-4 with high affinity. Because these receptors are highly differentially expressed in many human tumors, claudin-3 and claudin-4 may provide an efficient molecular tool to specifically identify and target biologically aggressive human cancer cells for CPE-specific binding and cytolysis. In this review we will discuss these surface proteins as targets for the detection and treatment of chemotherapy-resistant gynecologic malignancies overexpressing claudin-3 and -4 using CPE-based theranostic agents. We will also discuss the use of fluorescent c-CPE peptide in the operative setting for real time detection of micro-metastatic tumors during surgery and review the potential role of CPE in other medical applications.

Clostridium perfringens enterotoxin (CPE) is a common cause of food poisoning and hyperkalemia-associated death. Previously, we reported that fusion of pneumococcal surface protein A (PspA) to C-terminal fragment of CPE (C-CPE) efficiently bound mucosal epithelium so that PspA-specific immune responses could be provoked. In this study, we found that fusion of C-CPE with PspA augmented the antigenicity of C-CPE itself. These findings allowed us to hypothesize that fusion of C-CPE and another food poisoning vaccine act as a bivalent food poisoning vaccine. Therefore, we constructed an adjuvant-free bivalent vaccine against CPE and cholera toxin (CT), which is a major food poisoning in developing country, by genetically fusing CT B subunit to C-CPE. Because of the low antigenicity of C-CPE, immunization of mice with C-CPE alone did not induce C-CPE-specific immune responses. However, immunization with our vaccine induced both C-CPE- and CT-specific neutralizing antibody. The underlying mechanism of the augmented antigenicity of C-CPE included the activation of T cells by CTB. Moreover, neutralizing antibodies lasted for at least 48 weeks and the quality of the antibody was dependent on the binding activity of CTB–C-CPE to its receptors. These findings suggest that our fusion protein is a potential platform for the development of an adjuvant-free bivalent vaccine against CPE and CT. [ABSTRACT FROM AUTHOR]

According to numerous studies, failures in treatment of ovarian cancer, i.e., a relapse and metastases, result from a small population of cancer stem cells (CSCs). They may also be responsible for tumor initiation. Cancer stem cells are resistant to chemo- and radiotherapy. Eradication of CSCs may involve the application of salinomycin, metformin and Clostridium perfringens; the effect of anti-angiogenic factors remains controversial. Salinomycin is an antibiotic isolated from Streptomyces albus bacteria. Its CSC-eradicating effect has been demonstrated both in ovarian cancer cell lines and in women with breast cancer. Clostridium perfringens enterotoxin (CPE) has been demonstrated to destroy CSCs in ovarian cancer both in vivo and in vitro. Metformin, apart from its hypoglycemic effect, reduces the CSC population and inhibits the proliferation of neoplastic cells and angiogenesis. Cancer stem cells with expression of VEGFR1+ have been described as affecting circulating cancer cells and influencing the formation of metastases. Both positive and negative effects of anti-angiogenic therapy on the CSC population have been documented. [ABSTRACT FROM AUTHOR]

The blood-brain barrier (BBB) prevents entry of neurotoxic substances but also that of drugs into the brain. Here, the paracellular barrier is formed by tight junctions (TJs) with claudin-5 (Cldn5) being the main sealing constituent. Transient BBB opening by targeting Cldn5 could improve paracellular drug delivery. The non-toxic C-terminal domain of Clostridium perfringens enterotoxin (cCPE) binds to a subset of claudins, e.g., Cldn3, -4. Structure-based mutagenesis was used to generate Cldn5-binding variants (cCPE-Y306W/S313H and cCPE-N218Q/Y306W/S313H). These cCPE-variants were tested for transient TJ opening using multiple in vitro BBB models: Primary porcine brain endothelial cells, coculture of primary rat brain endothelial cells with astrocytes and mouse cerebEND cells. cCPE-Y306W/S313H and cCPE-N218Q/Y306W/S313H but neither cCPE-wt nor cCPE-Y306A/L315A (not binding to claudins) decreased transendothelial electrical resistance in a concentration-dependent and reversible manner. Furthermore, permeability of carboxyfluorescein (with size of CNS drugs) was increased. cCPE-Y306W/S313H but neither cCPE-wt nor cCPE-Y306A/L315A bound to Cldn5-expressing brain endothelial cells. However, freeze-fracture EM showed that cCPE-Y306W/S313H did not cause drastic TJ breakdown. In sum, Cldn5-binding cCPE-variants enabled mild and transient opening of brain endothelial TJs. Using reliable in vitro BBB models, the results demonstrate that cCPE-based biologics designed to bind Cldn5 improve paracellular drug delivery across the BBB. [ABSTRACT FROM AUTHOR]

Saccharomyces boulardii, a subspecies of Saccharomyces cerevisiae, is a well-known eukaryotic probiotic with many benefits for human health. In the present study, a recombinant strain of S. boulardii was prepared to use as a potential oral vaccine delivery vehicle. In this sense, a ura3 auxotroph strain of S. boulardii CNCM I-745 (known as S. cerevisiae HANSEN CBS 5926, Yomogi®) was generated using CRISPR/Cas9 methodology. Then a gene construct encoding a highly immunogenic protein, ovalbumin (OVA), was prepared and transformed into the ura3- S. boulardii. To facilitate the transport of the recombinant immunogen across the intestinal barrier, a claudintargeting sequence from Clostridium perfringens enterotoxin (CPE) was added to the C-terminus of the expression cassette. The recombinant S. boulardii strain expressing the OVA-CPE fusion protein was then administered orally to a group of mice, and serum IgG and fecal IgA levels were evaluated by ELISA. Our results demonstrated that anti-OVA IgG in serum significantly increased in test group (P < 0.001) compared to control groups (receiving wild type S. boulardii or PBS), and the fecal IgA titer was significantly higher in test group (P < 0.05) than control groups. In parallel, a recombinant S. boulardii strain expressing the similar construct lacking C-terminal CPE was also administered orally. The result showed an increased level of serum IgG in group receiving yeasts expressing the CPE negative construct compared to control groups; however, the fecal IgA levels did not increase significantly. In conclusion, our findings indicated that the yeast S. boulardii, as a delivery vehicle with possible immunomodulatory effects, and c-CPE, as a targeting tag, synergistically assist to stimulate systemic and local immunity. This proposed recombinant S. boulardii system might be useful in the expression of other antigenic peptides, making it as a promising tool for oral delivery of vaccines or therapeutic proteins. [ABSTRACT FROM AUTHOR]

Clostridium perfringens enterotoxin (CPE) causes food poisoning and antibiotic-associated diarrhea. It uses some claudin tight junction proteins (eg, claudin-4) as receptors to form Ca2+-permeable pores in the membrane, damaging epithelial cells in small intestine and colon. We demonstrate that only a subpopulation of colonic enterocytes which are characterized by apical dislocation of claudins are CPE-susceptible. CPE-mediated damage was enhanced if paracellular barrier was impaired by Ca2+ depletion, proinflammatory cytokine tumor necrosis factor a, or dedifferentiation. Microscopy, Ca2+ monitoring, and electrophysiological data showed that CPE-mediated cytotoxicity and barrier disruption was limited by extent of CPE-binding. The latter was restricted by accessibility of non-junctional claudin molecules such as claudin-4 at apical membranes. Focal-leaks detected in HT-29/B6 colonic monolayers were verified for native tissue using colon biopsies. These mechanistic findings indicate how CPE-mediated effects may turn from self-limiting diarrhea into severe clinical manifestation such as colonic necrosis--if intestinal barrier dysfunction, eg, during inflammation facilitates claudin accessibility. [ABSTRACT FROM AUTHOR]

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.

The tight junction (TJ) is an intercellular sealing component found in epithelial and endothelial tissues that regulates the passage of solutes across the paracellular space. Research examining the biology of TJs has revealed that they are complex biochemical structures constructed from a range of proteins including claudins, occludin, tricellulin, angulins and junctional adhesion molecules. The transient disruption of the barrier function of TJs to open the paracellular space is one means of enhancing mucosal and transdermal drug absorption and to deliver drugs across the blood−brain barrier. However, the disruption of TJs can also open the paracellular space to harmful xenobiotics and pathogens. To address this issue, the strategies targeting TJ proteins have been developed to loosen TJs in a size- or tissue-dependent manner rather than to disrupt them. As several TJ proteins are overexpressed in malignant tumors and in the inflamed intestinal tract, and are present in cells and epithelia conjoined with the mucosa-associated lymphoid immune tissue, these TJ-protein-targeted strategies may also provide platforms for the development of novel therapies and vaccines. Here, this paper reviews two TJ-protein-targeted technologies, claudin binders and an angulin binder, and their applications in drug development.

The majority of malignant tumors originate from epithelial cells, and many of them are characterized by an overexpression of claudins (Cldns) and their mislocalization out of tight junctions. We utilized the C-terminal claudin-binding domain of Clostridium perfringens enterotoxin (cCPE), with its high affinity to specific members of the claudin family, as the targeting unit for a claudin-sensitive cancer biosensor. To overcome the poor sensitivity of conventional relaxivity-based magnetic resonance imaging (MRI) contrast agents, we utilized the superior sensitivity of xenon Hyper-CEST biosensors. We labeled cCPE for both xenon MRI and fluorescence detection. As one readout module, we employed a cryptophane (CrA) monoacid and, as the second, a fluorescein molecule. Both were conjugated separately to a biotin molecule via a polyethyleneglycol chemical spacer and later via avidin linked to GST-cCPE. Nontransfected HEK293 cells and HEK293 cells stably expressing Cldn4-FLAG were incubated with the cCPE-based biosensor. Fluorescence-based flow cytometry and xenon MRI demonstrated binding of the biosensor specifically to Cldn4-expressing cells. This study provides proof of concept for the use of cCPE as a carrier for diagnostic contrast agents, a novel approach for potential detection of Cldn3/-4-overexpressing tumors for noninvasive early cancer detection. [ABSTRACT FROM AUTHOR]

Given that most malignant tumors are derived from epithelium, developing a strategy for treatment of epithelium-derived cancers (i.e., carcinomas) is a pivotal issue in cancer therapy. Carcinomas, including ovarian, breast, prostate, and pancreatic cancers, are known to overexpress various claudins (CLDNs); in particular, CLDN-3 and -4 are frequently overexpressed in malignant case. The generation of CLDN binders is a key for expanding CLDN-targeted cancer therapy but has been delayed due to the small size of CLDN extracellular domains (approximately 50 amino acids for the first domain and 15 amino acids for the second) and their high homology among species. Interestingly, however, the receptors for Clostridium perfringens enterotoxin (CPE), a foodborne toxin in humans, happen to be identical to CLDN-3 and -4. Thus, the first CLDN binder, CPE, has provided us CLDN-targeted cancer therapy from a concept into a potential reality. In this review, we describe roles of CPE technology in cancer therapy and discuss future directions in the CLDN-targeting concept-to-therapy process. [ABSTRACT FROM AUTHOR]

The integrity of tight junctions, which regulate paracellular permeability, is challenged by many bacterial pathogens. This is caused by inflammatory responses triggered by pathogens and direct interaction of bacteria or their toxins with host epithelial cells. In some cases, tight junction proteins represent receptors for cell surface proteins or toxins of the pathogen, such as Clostridium perfringens enterotoxin (CPE). CPE causes diarrhea and cramps-the symptoms of a common foodborne illness, caused by C. perfringens type A. It uses a subgroup of the claudin family of tight junction proteins as receptors and forms pores in the membrane of intestinal epithelial cells. Ca influx through these pores finally triggers cell damage. In this review, we summarize tight junction targeting and alteration by a multitude of different microorganisms such as C. perfringens, Escherichia coli, Helicobacter pylori, Salmonella typhimurium, Shigella flexneri, Vibrio cholerae, Yersinia enterocolitica, protozoan parasites, and their proteins. A focus is drawn towards CPE, the interaction with its receptors, cellular, and pathophysiological consequences for the intestinal epithelium. In addition, we portend to the use of CPE-based claudin modulators for drug delivery as well as diagnosis and therapy of cancer. [ABSTRACT FROM AUTHOR]

Claudins are a family of tight junction proteins regulating paracellular permeability and cell polarity with different patterns of expression in benign and malignant human tissues. There are approximately 27 members of the claudin family identified to date with varying cell and tissue-specific expression. Claudins-3, -4 and -7 represent the most highly differentially expressed claudins in ovarian cancer. While their exact role in ovarian tumors is still being elucidated, these proteins are thought to be critical for ovarian cancer cell invasion/dissemination and resistance to chemotherapy. Claudin-3 and claudin-4 are the natural receptors for the Clostridium perfringens enterotoxin (CPE), a potent cytolytic toxin. These surface proteins may therefore represent attractive targets for the detection and treatment of chemotherapy-resistant ovarian cancer and other aggressive solid tumors overexpressing claudin-3 and -4 using CPE-based theranostic agents.

Clostridium perfringens causes one of the most common foodborne illnesses, which is largely mediated by the Clostridium perfringens enterotoxin (CPE). The toxin consists of two functional domains. The N-terminal region mediates the cytotoxic effect through pore formation in the plasma membrane of the mammalian host cell. The C-terminal region (cCPE) binds to the second extracellular loop of a subset of claudins. Claudin-3 and claudin-4 have been shown to be receptors for CPE with very high affinity. The toxin binds with weak affinity to claudin-1 and -2 but contribution of these weak binding claudins to CPE-mediated disease is questionable. cCPE is not cytotoxic, however, it is a potent modulator of tight junctions. This review describes recent progress in the molecular characterization of the cCPE-claudin interaction using mutagenesis, in vitro binding assays and permeation studies. The results promote the development of recombinant cCPE-proteins and CPE-based peptidomimetics to modulate tight junctions for improved drug delivery or to treat tumors overexpressing claudins.

The current intravesical treatment of bladder cancer (BC) is limited to a few chemotherapeutics that show imperfect effectiveness and are associated with some serious complications. Thus, there is an urgent need for alternative therapies, especially for patients with high-risk non-muscle invasive (NMIBC). Clostridium perfringens enterotoxin (CPE), cytolytic protein binds to its receptors: claudin 3 and 4 that are expressed in epithelial cells. This binding is followed by rapid cell death. Claudin 4 is present in several epithelial tissue including bladder urothelium and its expression is elevated in some forms of BC. In addition to directly targeting BC cells, binding of CPE to claudins increases urothelium permeability that creates conditions for better accession of the tumor. Therefore, we evaluated CPE as a candidate for intravesical treatment of BC using a cellular model. We examined cytotoxicity of CPE against BC cells lines and 3D cultures of cells derived from surgical samples. To better elucidate cellular mechanisms, activated by CPE and to consider the use of CPE non-toxic fragment (C-CPE) for combination treatment with other drugs we synthesized C-CPE, compared its cytotoxic activity with CPE and examined claudin 4 expression and intracellular localization after C-CPE treatment. CPE induced cell death after 1 h in low aggressive RT4 cells, in moderately aggressive 5637 cells and in the primary 3D cultures of BC cells derived from NMIBC. Conversely, non-transformed urothelial cells and cells derived from highly aggressive tumor (T24) survived this treatment. The reason for this resistance to CPE might be the lower expression of CLDNs or their inaccessibility for CPE in these cells. C-CPE treatment for 48 h did not affect cell viability in tested cells, but declined expression of CLDN4 in RT4 cells. C-CPE increased sensitivity of RT4 cells to Mitommycin C and Dasatinib. To better understand mechanisms of this effect we examined expression and phosphorylation status of EphA2 and Src after C-CPE treatment and found changes in expression and phosphorylated status of these regulatory molecules. These observations show that after additional preclinical studies CPE and C-CPE in combinations with other drugs can be considered as a potential modalities for intravesical treatment of BC because of its ability to effectively destroy BC cells expressing claudin 4 and low toxicity against normal urothelium. [ABSTRACT FROM AUTHOR]

The vertebrate blood–brain barrier (BBB) creates an obstacle for central nervous system-related drug delivery. Claudin-5 (Cldn5), expressed in large quantities in BBB, plays a vital role in restricting BBB permeability. The C-terminal domain of Clostridium perfringens enterotoxin (cCPE) has been verified as binding to a subset of claudins (Cldns). The Cldn5-binding cCPE 194 – 319 variant cCPE Y306W/S313H was applied in this study to investigate its ability to modulate the permeability of zebrafish larval BBB. In vitro results showed that cCPE Y306W/S313H is able to bind specifically to Cldn5 in murine brain vascular endothelial (bEnd.3) cells, and is transported along with Cldn5 from the cell membrane to the cytoplasm, which in turn results in a reduction in transendothelial electrical resistance (TEER). Conversely, this effect can be reversed by removal of cCPE Y306W/S313H . In an in vivo experiment, this study estimates the capability of cCPE Y306W/S313H to modulate Cldn5 using a rhodamine B-Dextran dye diffusion assay in zebrafish larval BBB. The results show that cCPE Y306W/S313H co-localized with Cldn5 in zebrafish cerebral vascular cells and modulated BBB permeability, resulting in dye leakage. Taken together, this study suggests that cCPE Y306W/S313H has the capability – both in vitro and in vivo – to modulate BBB permeability temporarily by specific binding to Cldn5. [ABSTRACT FROM AUTHOR]

Identification of micrometastatic disease at the time of surgery remains extremely challenging in ovarian cancer patients. We used fluorescence microscopy, an in vivo imaging system and a fluorescence stereo microscope to evaluate fluorescence distribution in Claudin-3- and -4-overexpressing ovarian tumors, floating tumor clumps isolated from ascites and healthy organs. To do so, mice harboring chemotherapy-naïve and chemotherapy-resistant human ovarian cancer xenografts or patient-derived xenografts (PDXs) were treated with the carboxyl-terminal binding domain of the Clostridium perfringens enterotoxin (c-CPE) conjugated to FITC (FITC-c-CPE) or the near-infrared (NIR) fluorescent tag IRDye CW800 (CW800-c-CPE) either intraperitoneally (IP) or intravenously (IV). We found tumor fluorescence to plateau at 30 min after IP injection of both the FITC-c-CPE and the CW800-c-CPE peptides and to be significantly higher than in healthy organs ( p < 0.01). After IV injection of CW800-c-CPE, tumor fluorescence plateaued at 6 hr while the most favorable tumor-to-background fluorescence ratio (TBR) was found at 48 hr in both mouse models. Importantly, fluorescent c-CPE was highly sensitive for the in vivo visualization of peritoneal micrometastatic tumor implants and the identification of ovarian tumor spheroids floating in malignant ascites that were otherwise not detectable by conventional visual observation. The use of the fluorescent c-CPE peptide may represent a novel and effective optical approach at the time of primary debulking surgery for the real-time detection of micrometastatic ovarian disease overexpressing the Claudin-3 and -4 receptors or the identification of residual disease at the time of interval debulking surgery after neoadjuvant chemotherapy treatment. [ABSTRACT FROM AUTHOR]