RESUMO
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.
Assuntos
Claudinas/metabolismo , Enterotoxinas/metabolismo , Hepatócitos/metabolismo , Pele/metabolismo , Substituição de Aminoácidos , Linhagem Celular Tumoral , Claudinas/química , Enterotoxinas/química , Enterotoxinas/farmacologia , Epiderme/metabolismo , Hepacivirus/efeitos dos fármacos , Hepacivirus/fisiologia , Hepatite C/metabolismo , Hepatite C/virologia , Humanos , Modelos Moleculares , Conformação Molecular , Ligação Proteica , Pele/citologia , Internalização do Vírus/efeitos dos fármacos , Replicação ViralRESUMO
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 α, 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.
Assuntos
Claudinas/antagonistas & inibidores , Infecções por Clostridium/patologia , Clostridium perfringens/patogenicidade , Colo/patologia , Enterotoxinas/toxicidade , Doenças Transmitidas por Alimentos/patologia , Junções Íntimas/patologia , Linhagem Celular , Enterócitos/patologia , Humanos , Mucosa Intestinal/patologia , PermeabilidadeRESUMO
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. Ca2+ 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.
Assuntos
Clostridium perfringens/metabolismo , Enterotoxinas/metabolismo , Junções Íntimas/metabolismo , Junções Íntimas/microbiologia , Fatores de Virulência/metabolismo , Animais , Humanos , Proteínas de Junções Íntimas/metabolismoRESUMO
Clostridium perfringens enterotoxin (CPE) binds to distinct claudins (Clds), which regulate paracellular barrier functions in endo- and epithelia. The C-terminal domain (cCPE) has the potential for selective claudin modulation, since it only binds to a subset of claudins, e.g., Cld3 and Cld4 (cCPE receptors). Cld5 (non-CPE receptor) is a main constituent in tight junctions (TJ) of the blood-brain barrier. We aimed to reveal claudin recognition mechanisms of cCPE and to create a basis for a Cld5-binder. By utilizing structure-based interaction models, mutagenesis and assays of cCPE-binding to the TJ-free cell line HEK293, transfected with human Cld1 and murine Cld5, we showed how cCPE-binding to Cld1 and Cld5 is prevented by two residues in extracellular loop 2 of Cld1 (Asn(150) and Thr(153)) and Cld5 (Asp(149) and Thr(151)). Binding to Cld5 is especially attenuated by the lack of a bulky hydrophobic residue like leucine at position 151. By downsizing the binding pocket and compensating for the lack of this leucine residue, we created a novel cCPE-variant; cCPEY306W/S313H binds Cld5 with nanomolar affinity (K d 33 ± 10 nM). Finally, the effective binding to endogenously Cld5-expressing blood-brain barrier model cells (murine microvascular endothelial cEND cell line) suggests cCPEY306W/S313H as basis for Cld5-specific modulation to improve paracellular drug delivery, or to target claudin overexpressing tumors.
Assuntos
Claudina-1/metabolismo , Claudina-5/metabolismo , Enterotoxinas/metabolismo , Proteínas Mutantes/metabolismo , Sequência de Aminoácidos , Aminoácidos/química , Aminoácidos/genética , Aminoácidos/metabolismo , Animais , Sítios de Ligação/genética , Linhagem Celular , Claudina-1/química , Claudina-1/genética , Claudina-5/química , Claudina-5/genética , Clostridium perfringens/genética , Clostridium perfringens/metabolismo , Enterotoxinas/química , Enterotoxinas/genética , Células HEK293 , Humanos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Camundongos , Microscopia Confocal , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Proteínas Mutantes/química , Proteínas Mutantes/genética , Mutação , Ligação Proteica , Estrutura Terciária de Proteína , Homologia de Sequência de AminoácidosRESUMO
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.
Assuntos
Antineoplásicos/farmacologia , Claudinas/metabolismo , Clostridium perfringens/metabolismo , Enterotoxinas/farmacologia , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias da Glândula Tireoide/tratamento farmacológico , Animais , Antineoplásicos/uso terapêutico , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/terapia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Claudina-1/química , Claudina-1/genética , Claudina-1/metabolismo , Claudina-3/química , Claudina-3/genética , Claudina-3/metabolismo , Claudina-4/química , Claudina-4/genética , Claudina-4/metabolismo , Claudina-5/química , Claudina-5/genética , Claudina-5/metabolismo , Claudinas/química , Claudinas/genética , Enterotoxinas/química , Enterotoxinas/uso terapêutico , Feminino , Humanos , Imuno-Histoquímica , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/terapia , Camundongos , Mutagênese Sítio-Dirigida , Mutação , Necrose/induzido quimicamente , Ligação Proteica , Proteínas Recombinantes , Neoplasias da Glândula Tireoide/metabolismo , Neoplasias da Glândula Tireoide/terapia , Transfecção , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Early diagnosis of chronic hepatitis B virus (HBV) and hepatitis C virus (HCV) infections is pivotal for optimal disease management. Sensitivity and specificity of 19 rapid diagnostic test (RDT) kits by different manufacturers (ABON, CTK Biotech, Cypress Diagnostics, Green Gross, Human Diagnostic, Humasis, InTec, OraSure, SD Bioline, Wondfo) were assessed on serum samples of 270 Mongolians (90 seropositive for hepatitis B surface antigen (HBsAg), 90 seropositive for hepatitis C antibody (HCV-Ab), 90 healthy subjects). All tested RDTs for detection of HBsAg performed with average sensitivities and specificities of 100% and 99%, respectively. Albeit, overall sensitivity and specificity of RDTs for detection of HCV-Ab was somewhat lower compared to that of HBsAg RDTs (average sensitivity 98.9%, average specificity 96.7%). Specificity of RDTs for detection of HCV-Ab was dramatically lower among HBsAg positive individuals, who were 10.2 times more likely to show false positive test results. The results of our prospective study demonstrate that inexpensive, easy to handle RDTs are a promising tool in effective HBV- and HCV-screening especially in resource-limited settings.
Assuntos
Hepatite B/diagnóstico , Hepatite C/diagnóstico , Programas de Rastreamento/métodos , Kit de Reagentes para Diagnóstico/normas , Adolescente , Adulto , Idoso , Feminino , Antígenos de Superfície da Hepatite B/sangue , Anticorpos Anti-Hepatite C/sangue , Humanos , Masculino , Programas de Rastreamento/economia , Pessoa de Meia-Idade , Estudos Prospectivos , Sensibilidade e EspecificidadeRESUMO
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.
Assuntos
Barreira Hematoencefálica/metabolismo , Claudina-5/metabolismo , Clostridium perfringens/metabolismo , Enterotoxinas/metabolismo , Animais , Transporte Biológico , Barreira Hematoencefálica/ultraestrutura , Encéfalo/metabolismo , Encéfalo/ultraestrutura , Células Cultivadas , Células Endoteliais/metabolismo , Enterotoxinas/química , Técnica de Fratura por Congelamento , Células HEK293 , Humanos , Microscopia Eletrônica , Ligação Proteica , Suínos , Junções Íntimas/metabolismo , Junções Íntimas/ultraestruturaRESUMO
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.
Assuntos
Técnicas Biossensoriais/métodos , Claudina-4/metabolismo , Enterotoxinas/metabolismo , Imageamento por Ressonância Magnética/métodos , Xenônio/química , Avidina/química , Claudina-3/química , Claudina-3/genética , Claudina-3/metabolismo , Claudina-4/química , Claudina-4/genética , Enterotoxinas/química , Enterotoxinas/genética , Citometria de Fluxo , Fluoresceínas/química , Células HEK293 , Humanos , Microscopia Confocal , Modelos Moleculares , Compostos Policíclicos/química , Polietilenoglicóis/química , Ligação Proteica , Estrutura Terciária de Proteína , Reprodutibilidade dos TestesRESUMO
Zinc homoeostasis exerts protective effects in inflammatory intestinal diseases and zinc supplementation has been successfully used for treating infectious diarrhoea. This study aimed at a characterisation of zinc effects on focal leak induction by α-haemolysin (HlyA)-producing Escherichia coli (E. coli) as protective mechanism for colitis. We conducted in vivo experiments by oral challenge of gnotobiotic mice colonised with HlyA-expressing E. coli-536. Mice were either fed a defined normal or high zinc diet to analyse effects of zinc as a therapeutic regimen. HlyA-deficient E. coli-536 mutants were used as controls. Mice infected with HlyA-producing E. coli showed impaired barrier integrity when receiving normal zinc. High zinc supplementation in HlyA-producing E. coli-infected mice reduced epithelial dysfunction as indicated by ameliorated macromolecule permeability. Reduced size of focal leaks with diminished bacterial translocation was observed as inherent mechanisms of this zinc action. In human colon cell monolayers application of zinc rescued the HlyA-dependent decline in transepithelial electrical resistance via reduction of the calcium entry into HlyA-exposed cells. Calcium-dependent cell exfoliation was identified as mechanism for focal leak induction. In conclusion, zinc supplementation protects from HlyA-induced barrier dysfunction in vivo and in vitro, providing an explanation for the protective efficacy of zinc in intestinal disorders.
Assuntos
Colite , Infecções por Escherichia coli/complicações , Proteínas de Escherichia coli/metabolismo , Proteínas Hemolisinas/metabolismo , Zinco/administração & dosagem , Animais , Cálcio/metabolismo , Linhagem Celular Tumoral , Colite/metabolismo , Colite/microbiologia , Colite/prevenção & controle , Modelos Animais de Doenças , Escherichia coli/metabolismo , Escherichia coli/patogenicidade , Humanos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiologia , Intestinos/efeitos dos fármacos , CamundongosRESUMO
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 cCPE194-319 variant cCPEY306W/S313H was applied in this study to investigate its ability to modulate the permeability of zebrafish larval BBB. In vitro results showed that cCPEY306W/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 cCPEY306W/S313H. In an in vivo experiment, this study estimates the capability of cCPEY306W/S313H to modulate Cldn5 using a rhodamine B-Dextran dye diffusion assay in zebrafish larval BBB. The results show that cCPEY306W/S313H co-localized with Cldn5 in zebrafish cerebral vascular cells and modulated BBB permeability, resulting in dye leakage. Taken together, this study suggests that cCPEY306W/S313H has the capability - both in vitro and in vivo - to modulate BBB permeability temporarily by specific binding to Cldn5.