Clostridium Perfringens Enterotoxin Research Articles

Cryo-EM structures of Clostridium perfringens enterotoxin bound to its human receptor, claudin-4

Clostridium perfringens enterotoxin (CpE) causes prevalent and deadly gastrointestinal disorders. CpE binds to receptors called claudins on the apical surfaces of small intestinal epithelium. Claudins normally regulate paracellular transport but are hijacked from doing so by CpE and are instead led to form claudin/CpE complexes. Claudin/CpE complexes are the building blocks of oligomeric β-barrel pores that penetrate the plasma membrane and induce gut cytotoxicity. Here, we present the structures of CpE in complex with its native claudin receptor in humans, claudin-4, using cryogenic electron microscopy. The structures reveal the architecture of the claudin/CpE complex, the residues used in binding, the orientation of CpE relative to the membrane, and CpE-induced changes to claudin-4. Further, structures and modeling allude to the biophysical procession from claudin/CpE complexes to cytotoxic β-barrel pores during pathogenesis. In full, this work proposes a model of claudin/CpE assembly and provides strategies to obstruct its formation to treat CpE diseases.

Cryo-EM Structures of Clostridium perfringens Enterotoxin Bound to its Human Receptor, Claudin-4.

Pathogenic strains of Clostridium perfringens secrete an enterotoxin (CpE) that causes prevalent, severe, and sometimes deadly gastrointestinal disorders in humans and domesticated animals. CpE binds selectively to membrane protein receptors called claudins on the apical surfaces of small intestinal epithelium. Claudins normally construct tight junctions that regulate epithelial paracellular transport but are hijacked from doing so by CpE and are instead led to form claudin/CpE small complexes. Small complexes are building blocks for assembling oligomeric β-barrel pores that penetrate the plasma membrane and induce gut cytotoxicity. Here we present structures of CpE in complexes with its native claudin receptor in humans, claudin-4, at 4.0 and 2.8 Å using cryogenic electron microscopy. The structures reveal the overall architecture of the small complex, that the small complex can be kinetically trapped, and resolve its key features; like the residues used in claudin/CpE complex binding, the orientation of CpE relative to the membrane, and CpE-induced structural changes to claudin-4. Further, the structures allude to the biophysical procession from small complex to cytotoxic β-barrel pore used by CpE during pathogenesis and the role of trypsin in this process. In full, this work elucidates the structure and mechanism of claudin-bound CpE pore assembly and provides strategies to obstruct its formation to treat CpE-induced gastrointestinal diseases.

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.

Overexpressing the cpr1953 Orphan Histidine Kinase Gene in the Absence of cpr1954 Orphan Histidine Kinase Gene Expression, or Vice Versa, Is Sufficient to Obtain Significant Sporulation and Strong Production of Clostridium perfringens Enterotoxin or Spo0A by Clostridium perfringens Type F Strain SM101.

The CPR1953 and CPR1954 orphan histidine kinases profoundly affect sporulation initiation and Clostridium perfringens enterotoxin (CPE) production by C. perfringens type F strain SM101, whether cultured in vitro (modified Duncan-Strong sporulation medium (MDS)) or ex vivo (mouse small intestinal contents (MIC)). To help distinguish whether CPR1953 and CPR1954 act independently or in a stepwise manner to initiate sporulation and CPE production, cpr1953 and cpr1954 null mutants of SM101 were transformed with plasmids carrying the cpr1954 or cpr1953 genes, respectively, causing overexpression of cpr1954 in the absence of cpr1953 expression and vice versa. RT-PCR confirmed that, compared to SM101, the cpr1953 mutant transformed with a plasmid encoding cpr1954 expressed cpr1954 at higher levels while the cpr1954 mutant transformed with a plasmid encoding cpr1953 expressed higher levels of cpr1953. Both overexpressing strains showed near wild-type levels of sporulation, CPE toxin production, and Spo0A production in MDS or MIC. These findings suggest that CPR1953 and CPR1954 do not function together in a step-wise manner, e.g., as a novel phosphorelay. Instead, it appears that, at natural expression levels, the independent kinase activities of both CPR1953 and CPR1954 are necessary for obtaining sufficient Spo0A production and phosphorylation to initiate sporulation and CPE production.

Abstract 4153: PET/CT imaging of pancreatic ductal adenocarcinoma with a claudin-4 binding peptide in preclinical mouse model

Abstract There is a critical need to develop new imaging and therapeutic strategies for pancreatic cancer. Current positron emission tomography (PET) imaging assessments of pancreatic ductal adenocarcinoma (PDAC) rely on 18F-FDG as a radiopharmaceutical. To increase the sensitivity of PDAC detection, we identified Claudin-4 (CLDN4), a tight junction transmembrane protein, as a PDAC biomarker through spatial transcriptomic and proteomic analyses. CLDN4 expression has previously been reported to be enhanced in 99% of primary and 100% of metastatic pancreatic cancer tissue samples and in 10 of 11 precancerous pancreatic intraepithelial neoplasia (PanIN) specimens. A small fragment of naturally occurring Clostridium perfringens enterotoxin (CPE) was previously reported to bind to the extracellular loop domain of CLDN4 protein (PDB:7kp4) with nanomolar (nM) affinity (Kd). A CLDN4 binding peptide (C4BP) was developed as a radioligand by modifying the n-terminus of C4BP which does not impact receptor binding. Here, we designed and synthesized a DOTA-PEG1-C4BP ligand on a rink amide resin using a microwave-assisted peptide synthesizer. Radiolabeling of DOTA-PEG1-C4BP (2 nmol) with 64CuCl2 (70 MBq) conferred 64Cu-C4BP (28 MBq/nmol) with 80% non-decay corrected yield and >99% radiochemical purity. PET/CT imaging of 64Cu-C4BP (~0.1 μg/mouse) performed in an inducible PDAC (KrasLSL-G12D/+;Rosa26LSL-tdTomato/LSL-tdTomato(KT);Ptf1aCreER;Trp53fl/fl) model at 84 day after tamoxifen injection resulted in ~25% injected activity per cubic centimeter (IA/cc) accumulation in metastases and at ~18% IA/cc retention in pancreatic tumors within 30 minutes of tail vein injection with minimal background accumulation. Ex vivo PET imaging confirmed the localized accumulation of 64Cu-C4BP in transgenic mice within the colon, pancreas, and liver. Blocking of 64Cu-C4BP accumulation with the cold compound in a xenograft model (NOD/SCID) bearing Capan-1 PDAC tumors demonstrated the specificity of 64Cu-C4BP binding. Here we report that CLDN4 is a promising target and 64Cu-C4BP is a promising ligand for the detection of pancreatic cancer. Citation Format: Jai Woong Seo, James Wang, Aris J. Kare, Spencer K. Tumbale, Bo Wu, Marina N. Raie, Mallesh Pandrala, Katherine W. Ferrara. PET/CT imaging of pancreatic ductal adenocarcinoma with a claudin-4 binding peptide in preclinical mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4153.

Structural basis of Clostridium perfringens enterotoxin activation and oligomerization by trypsin

Structural basis of Clostridium perfringens enterotoxin activation and oligomerization by trypsin

Effect of Chitosan (Chi)-C Terminal 30 Amino Acids of Clostridium Perfringens Enterotoxin (CPE30)-pVP1 Nanoparticles on Rats with Viral Myocarditis

Coxsackie B3 virus (CVB3) is the most common pathogen of viral myocarditis (VMC), and it is necessary to study an efficient vaccine to prevent the VMC. In this research, chitosan (chi)-C-terminal 30 amino acid (CPE30) was prepared by chemical coupling, and then chi-CPE30-pcDNA3.1-VP1 plasmid (pVP) complex particles were formed by co-aggregation method. The biological characteristics of the chi-CPE30-pVP1 complex particles were analyzed. It was immunized into SD rats intranasally at different time points as a vaccine together with other by-products (such as chi-pVP1, chi-CPE30-pcDNA3.1, and chi-pcDNA3.1). 100 μg of plasmid was inoculated each time, with 4 times in total, and the specific antibody level and cellular immune response of all rats were detected. It was revealed that based on the coupling effect of ethylcarbodiimide hydrochloride and N-hydroxysuccinimide (EDC/NHS) chemical coupling reagent, nearly 70% of CPE30 was coupled to chi, and the efficiency of chi-CPE30 to wrap DNA was close to 100%. After a certain concentration of pVP1 solution was added, the chi-CPE30-pVP1 composite particles were obtained, and the surface of the chi-CPE30-pVP1 composite was scanned as spherical particles. When used as a vaccine, the composite particles can induce high serum immunoglobulin G (IgG) and mucosal IgA antibody levels in rats. Meantime, the specific lymphocyte proliferation test confirmed that chi-CPE30-pVP1 effectively induced the proliferative response of CVB specific lymphocytes in the spleen and mesenteric lymph nodes (MLN). After the rats were infected with 3LD50CVB3, it was found that the weight of rats changed slightly under the action of chi-CPE30-pVP1 vaccine (P < 0.05). The creatine kinase and creatine kinase-myoglobin binding (CK-MB) levels of rats in this group were lower than those of chi-pVP1 rats and control group (P < 0.05). Applying the prepared chi-CPE30-pVP1 vaccine to immunize rats in this research could provide a new immune method for the molecular design of new vaccines and the prevention and treatment of CVB3 infection.

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

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.

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.

Chimeric Claudins Provide a Novel Method to Research Neural Tube Defects

Tight junctions (TJ) play a major role in the formation of various embryonic structures, including the neural tube. Disruptions of claudins (CLDN), a family of proteins critical to the TJ function, has been shown to induce neural tube defects (NTD) in chicken embryos. Clostridium perfringens enterotoxin (CPE) induces NTDs through this mechanism as it inhibits CLDNs 3, 4, 6, 7, 8, 9, 14, and 19, creating a pore in the TJ and causing cell death. CPE broadly targets these CLDNs in a nonspecific manner. Our research utilizes chimeric-claudin (chCLDN) proteins to target individual CLDN interactions, increasing understanding of their specific contributions to NTDs. Using chicken embryos, we have evaluated the role of chCLDN3 when compared to CPE and solvent. Gallus gallus chCLDN-3 (GG3) induced NTDs in chicken embryos at a significantly higher rate than negative controls and statistically similar rate to CPE, our positive control. Additionally, GG3 exhibited different NTD patterns from CPE, allowing us to investigate the unique contributions of CLDN3 in NTD formation and establish the value of this research method.

Claudin-4: A New Molecular Target for Epithelial Cancer Therapy.

Claudin-4 (CLDN4) is a key component of tight junctions (TJs) in epithelial cells. CLDN4 is overexpressed in many epithelial malignancies and correlates with cancer progression. Changes in CLDN4 expression have been associated with epigenetic factors (such as hypomethylation of promoter DNA), inflammation associated with infection and cytokines, and growth factor signaling. CLDN4 helps to maintain the tumor microenvironment by forming TJs and acts as a barrier to the entry of anticancer drugs into tumors. Decreased expression of CLDN4 is a potential marker of epithelial-mesenchymal transition (EMT), and decreased epithelial differentiation due to reduced CLDN4 activity is involved in EMT induction. Non-TJ CLDN4 also activates integrin beta 1 and YAP to promote proliferation, EMT, and stemness. These roles in cancer have led to investigations of molecular therapies targeting CLDN4 using anti-CLDN4 extracellular domain antibodies, gene knockdown, clostridium perfringens enterotoxin (CPE), and C-terminus domain of CPE (C-CPE), which have demonstrated the experimental efficacy of this approach. CLDN4 is strongly involved in promoting malignant phenotypes in many epithelial cancers and is regarded as a promising molecular therapeutic target.

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

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. 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). 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 200µg/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 50µg/kg. The combination of the product ion spectra and relative ion ratio supported CPE identification at the LOQ level. 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.

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.

Construction of a Lactobacillus plantarum-based claudin-3 targeting delivery system for the development of vaccines against Eimeria tenella.

Avian coccidiosis causes huge economic losses to the poultry industry worldwide and currently lacks effective live vector vaccines. Achieving efficient antigen delivery to mucosa-associated lymphoid tissue (MALT) is critical for improving the effectiveness of vaccines. Here, chicken claudin-3 (CLDN3), a tight junction protein expressed in MALT, was identified as a target, and the C-terminal region of Clostridium perfringens enterotoxin (C-CPE) was proven to bind to chicken CLDN3. Then, a CLDN3-targeting Lactobacillus plantarum NC8-expressing C-CPE surface display system (NC8/GFP-C-CPE) was constructed to successfully express the heterologous protein on the surface of L. plantarum. The colonization level of NC8/GFP-C-CPE was significantly increased compared to the non-targeting strain and could persist in the intestine for at least 72h. An oral vaccine strain expressing five EGF domains of Eimeria tenella microneme protein 8 (EtMIC8-EGF) (NC8/EtMIC8-EGF-C-CPE) was constructed to evaluate the protective efficacy against E. tenella infection. The results revealed that CLDN3-targeting L. plantarum induced stronger mucosal immunity in gut-associated lymphoid tissues (GALT) as well as humoral responses and conferred better protection in terms of parasite replication and pathology than the non-targeting strain. Overall, we successfully constructed a CLDN3-targeting L. plantarum NC8 surface display system characterized by MALT-targeting, which is an efficient antigen delivery system to confer enhanced protective efficacy in chickens against E. tenella infection.

Clostridium perfringens associated with dairy farm systems show diverse genotypes

Clostridium perfringens is a bacterial species of importance to both public and animal health. Frequently found in food system environments, it presents a risk to food animal health such as dairy herds, and may cross contaminate associated ingredients or food products, with potential to cause sporadic and outbreaks of disease in human populations, including gastroenteric illness. In this study, we characterized C. perfringens isolated from bovine, caprine, and ovine dairy farm systems (n = 8, 11 and 4, respectively). Isolates were phenotypically screened for antimicrobial sensitivity profiling, and subjected to whole genome sequencing to elucidate related genetic markers, as well as examine virulence gene markers, mobile genetic elements, and other features. Both toxin type A and type D isolates were identified (78 % and 22 % of isolates, respectively), including 20 novel sequence types. Resistance to clindamycin was most prevalent among antibiotics screened (30 %), followed by erythromycin (13 %), then penicillin and tetracycline (4 %), although an additional 3 isolates were non-susceptible to tetracycline. Most isolates harboured plasmids, which mobilised virulence markers such as etx, cpb2, and resistance markers tetA(P), tetB(P), and erm(Q), on conjugative plasmids. The presence of type D isolates on caprine farms emphasizes the need for control efforts to prevent infection and potential enterotoxemia. Clostridium perfringens enterotoxin (cpe) was not identified, suggesting lower risk of gastrointestinal illness from contaminated foods, the presence of other virulence and antimicrobial resistance markers suggests farm hygiene remains an important consideration to help ensure food safety of associated dairy foods produced.

Characterizing the Contributions of Various Clostridium perfringens Enterotoxin Properties to In Vivo and In Vitro Permeability Effects.

ABSTRACTClostridium perfringens enterotoxin (CPE) is thought to cause lethal enterotoxemia when absorbed from the intestinal lumen into the circulation. CPE action sequentially involves receptor-binding, oligomerization into a prepore, and pore formation. To explore the mechanistic basis by which CPE alters permeability, this study tested the permeability effects of several recombinant CPE (rCPE) species: rCPE and rCPEC186A (which form pores), rC-CPE and rCPED48A (which bind to receptors but cannot oligomerize), rCPEC186A/F91C (which binds and oligomerizes without pore formation), and rCPEY306A/L315A (which has poor receptor-binding ability). On Caco-2 cells, i) only rCPE and rCPEC186A were cytotoxic; ii) rCPE and rCPEC186A affected transepithelial resistance (TEER) and 4 kDa fluorescent dextran (FD4) transit more quickly than binding-capable, but noncytotoxic, rCPE variants; whereas iii) rCPEY306A/L315A did not affect TEER or FD4 transit. Using mouse intestinal loops, rCPE (but not noncytotoxic rC-CPE, rCPED48A or rCPEY306A/L315A) was lethal and caused intestinal histologic damage within 4 h. After 2 h of treatment, rCPE was more strongly absorbed into the serum than those noncytotoxic rCPE species but by 4 h rC-CPE and rCPED48A became absorbed similarly as rCPE, while rCPEY306A/L315A absorption remained low. This increased rC-CPE and rCPED48A absorption from 2 to 4 h did not involve a general intestinal permeability increase because Evans Blue absorption from the intestines did not increase between 2 and 4 h of treatment with rC-CPE or rCPED48A. Collectively, these results indicate that CPE receptor binding is sufficient to slowly affect permeability, but CPE-induced cytotoxicity is necessary for rapid permeability changes and lethality.IMPORTANCE Clostridium perfringens enterotoxin (CPE) causes lethal enterotoxemia when absorbed from the intestines into the bloodstream. Testing recombinant CPE (rCPE) or rCPE variants impaired for various specific steps in CPE action showed that full CPE-induced cytotoxicity causes rapid Caco-2 monolayer permeability alterations, as well as enterotoxemic lethality and rapid CPE absorption in mouse small intestinal loops. However, receptor binding-capable, but noncytotoxic, rCPE variants did cause slow-developing in vitro and in vivo permeability effects. Absorption of binding-capable, noncytotoxic rCPE variants from the intestines did not correlate with general intestinal permeability alterations, suggesting that CPE binding can induce its own uptake. These findings highlight the importance of binding and, especially, cytotoxicity for CPE absorption during enterotoxemia and may assist development of permeability-altering rCPE variants for translational purposes.

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

Simple SummaryBrain metastasis occurs in primary cancers, such as breast cancer, and is correlated with mortality. There are limited options available for treatment, but Clostridium perfringens Enterotoxin (CPE) and its interaction with Claudin-4, a possible diagnostic biomarker for breast cancer, can provide a molecular pathway basis for the development of treatment options for metastatic brain cancer. Analysis of the literature reveals that Claudin-4 plays an important role as a receptor for CPE, allowing for the disruption of cell membrane permeability, an influx of calcium ions, and subsequent cell death. The negligible presence of Claudin-4 in normal brain cancer cells and the high abundance of Claudin-4 in breast cancer cells metastasized to the brain, allow for the targeted binding of CPE to tumor cells in the brain. We show that the C-terminal of CPE conjugated to nanoparticles that cross the blood–brain barrier could serve as a drug delivery tool to treat metastatic cells in the brain.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 targeting as an effective tool for directed barrier modulation of the viable epidermis.

Tight junction (TJ) formation is vital for epidermal barrier function. We aimed to specifically manipulate TJ barriers in the reconstructed human epidermis (RHE) by claudin-1 and -4 knockdown (KD) and by claudin-binding fusion proteins of glutathione S-transferase and modified C-terminal fragments of Clostridium perfringens enterotoxin (GST-cCPE). Impedance spectroscopy and tracer permeability imaging were employed for functional barrier assessment and investigation of claudin contribution. KD of claudin-1, but not claudin-4, impaired the paracellular barrier in vitro. Similarly, claudin-binding GST-cCPE variants weakened the paracellular but not the stratum corneum barrier. Combining both TJ targeting methods, we found that claudin-1 targeting by GST-cCPE after claudin-4 KD led to a marked decrease in paracellular barrier properties. Conversely, after claudin-1 KD, GST-cCPE did not further impair the barrier. Comparison of GST-cCPE variants with different claudin-1/claudin-4 affinities, NHS-fluorescein tracer detection, and immunostaining of RHE paraffin sections showed that GST-cCPE variants bind to extrajunctional claudin-1 and -4, which are differentially distributed along the stratum basale-stratum granulosum axis. GST-cCPE binding blocks these claudins, thereby specifically opening the paracellular barrier of RHE. The data indicate a critical role for claudin-1 in regulating paracellular permeability for ions and small molecules in the viable epidermis. Claudin targeting is presented as a proof-of-concept for precise barrier modulation.

Development, structure, and mechanism of synthetic antibodies that target claudin and Clostridium perfringens enterotoxin complexes

Strains of Clostridium perfringens produce a two-domain enterotoxin (CpE) that afflicts humans and domesticated animals, causing prevalent gastrointestinal illnesses. CpE’s C-terminal domain (cCpE) binds cell surface receptors, followed by a restructuring of its N-terminal domain to form a membrane-penetrating β-barrel pore, which is toxic to epithelial cells of the gut. The claudin family of membrane proteins are known receptors for CpE and also control the architecture and function of cell-cell contacts (tight junctions) that create barriers to intercellular molecular transport. CpE binding and assembly disables claudin barrier function and induces cytotoxicity via β-pore formation, disrupting gut homeostasis; however, a structural basis of this process and strategies to inhibit the claudin–CpE interactions that trigger it are both lacking. Here, we used a synthetic antigen-binding fragment (sFab) library to discover two sFabs that bind claudin-4 and cCpE complexes. We established these sFabs’ mode of molecular recognition and binding properties and determined structures of each sFab bound to claudin-4–cCpE complexes using cryo-EM. The structures reveal that the sFabs bind a shared epitope, but conform distinctly, which explains their unique binding equilibria. Mutagenesis of antigen/sFab interfaces observed therein result in binding changes, validating the structures, and uncovering the sFab’s targeting mechanism. From these insights, we generated a model for CpE’s claudin-bound β-pore that predicted sFabs would not prevent cytotoxicity, which we then verified in vivo. Taken together, this work demonstrates the development and mechanism of claudin/cCpE-binding sFabs that provide a framework and strategy for obstructing claudin/CpE assembly to treat CpE-linked gastrointestinal diseases.

Tight junction membrane protein's role in neural tube defects

More than 300,000 infants worldwide are born with neural tube defects (NTD), making it the second most common birth defect in the world. Many genetic and environmental factors have been identified as drivers of NTDs, some of which have been tied to damage of tight junctions (TJ), which are critical for the success of neural tube folding. Claudins (CLDN), occludin (OCLN) and junctional adhesion molecules (JAMs) are essential components of the TJ and are key players in its function. In the chicken embryo, clostridium perfringens enterotoxin (CPE) induces NTDs, by interacting broadly with CLDN3, ‐4, and ‐8. These interactions increase TJ permeability to an influx of calcium ions, causing cell death. There is no information for the role of OCLN or JAMs in NTDs. Our laboratory has created Synthetic Biology approaches to produce soluble CLDNs, OCLN and JAMs. These are detergent independent proteins that result in molecules that retain structure and adhesive properties of the native membrane protein. We present evidence that specific interactions, rather than the broad specificity of CPE, can be used to identify specific mechanisms leading to NTD formation. Our approach expands to the role of OCLN and JAMs in the formation of NTDs. These new tools may be used in developmental research as an alternative for genetic manipulation with equal value.