RESUMEN
Smart hydrogels with versatile properties, including a tunable gelation time, nonswelling attributes, and biocompatibility, are in great need in the biomedical field. To meet this urgent demand, we explored novel biomaterials with the desired properties from sessile marine organisms. To this end, a novel protein, Sbp9, derived from scallop byssus was extensively investigated, which features typical epidermal growth factor-like (EGFL) multiple repetitive motifs. Our current work demonstrated that the key fragment of Sbp9 (calcium-binding domain (CBD) and 4 EGFL repeats (CE4)) was able to form a smart hydrogel driven by noncovalent interactions and facilitated by disulfide bonds. More importantly, this smart hydrogel demonstrates several desirable and beneficial features, which could offset the drawbacks of typical protein-based hydrogels, including (1) a redox-responsive gelation time (from <1 to 60 min); (2) tunable mechanical properties, nonswelling abilities, and an appropriate microstructure; and (3) good biocompatibility and degradability. Furthermore, proof-of-concept demonstrations showed that the newly discovered hydrogel could be used for anticancer drug delivery and cell encapsulation. Taken together, a smart hydrogel inspired by marine sessile organisms with desirable properties was generated and characterized and demonstrated to have extensive applicability potential in biomedical applications, including tissue engineering and drug release.
Asunto(s)
Proteínas de Unión al Calcio/química , Encapsulación Celular/métodos , Portadores de Fármacos/química , Hidrogeles/química , Pectinidae/química , Materiales Inteligentes/química , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Antineoplásicos/química , Proteínas de Unión al Calcio/toxicidad , Línea Celular Tumoral , Doxorrubicina/química , Portadores de Fármacos/toxicidad , Liberación de Fármacos , Humanos , Hidrogeles/toxicidad , Peróxido de Hidrógeno/química , Masculino , Células Madre Mesenquimatosas/efectos de los fármacos , Oxidación-Reducción , Porosidad , Dominios Proteicos , Ratas Sprague-Dawley , Materiales Inteligentes/toxicidadRESUMEN
Necrotic enteritis (NE) is a multifactorial disease in broiler that is caused by colonization of Clostridium perfringens in their gastrointestinal tract. Recently several immunogenic proteins from virulent C. perfringens have been considered as vaccines to provide protection against NE. In this study, a novel trivalent fusion protein including immunogenic epitopes of three virulence factors of, NetB, alpha toxin and a metallopeptidase protein (NAM) was designed using in silico studies. Circular dichroism spectra was applied for determination of secondary structure and folding properties of the purified recombinant NAM (rNAM) expressed in E. coli. The antigenicity of rNAM was confirmed by induction of immune response in rabbit and neutralization experiments of the toxins in cell culture studies. To this end, anti-rNAM antisera neutralized the crude toxins produced by a wild type virulent C. perfringens strain using chicken hepatocellular carcinoma (LMH) cell lines. The cells were exposed to a mixture of anti-rNAM antisera and 2 × LD50 doses of the toxins. The result showed 94% viability of the cells against the crude toxins, in the presence of anti-rNAM antisera. Our study suggests that combination of metallopeptidase protein along with alpha toxin and NetB toxins is a potent immunogen which is able to neutralize the toxicity of crude extracellular toxins. The recombinant chimeric NAM could be a suitable and effective subunit vaccine candidate to prevent NE disease caused by C. perfringens.
Asunto(s)
Vacunas Bacterianas/inmunología , Clostridium perfringens/inmunología , Simulación por Computador , Proteínas Recombinantes de Fusión/inmunología , Factores de Virulencia/inmunología , Animales , Anticuerpos Antibacterianos/inmunología , Toxinas Bacterianas/toxicidad , Proteínas de Unión al Calcio/toxicidad , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Pollos , Epítopos de Linfocito B/inmunología , Metaloproteasas/metabolismo , Pruebas de Neutralización , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , ARN Mensajero/química , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/aislamiento & purificación , Reproducibilidad de los Resultados , Fosfolipasas de Tipo C/toxicidadRESUMEN
Clostridium perfringens type A causes gas gangrene characterized by myonecrosis and development of an effective therapy for treating affected patients is of clinical importance. It was recently reported that the expression of granulocyte colony-stimulating factor (G-CSF) is greatly up-regulated by C. perfringens infection. However, the role of G-CSF in C. perfringens-mediated myonecrosis is still unclear. Here, we assessed the destructive changes in C. perfringens-infected skeletal muscles and tested whether inhibition of G-CSF receptor (G-CSFR) signaling or administration of recombinant G-CSF affects the tissue injury. Severe edema, contraction of muscle fiber diameter, and increased plasma creatine kinase activity were observed in mice intramuscularly injected with C. perfringens type A, and the destructive changes were α-toxin-dependent, indicating that infection induces the destruction of skeletal muscle in an α-toxin-dependent manner. G-CSF plays important roles in the protection of tissue against damage and in the regeneration of injured tissue. However, administration of a neutralizing antibody against G-CSFR had no profound impact on the destructive changes to skeletal muscle. Moreover, administration of recombinant human G-CSF, filgrastim, imparted no inhibitory effect against the destructive changes caused by C. perfringens. Together, these results indicate that G-CSF is not beneficial for treating C. perfringens α-toxin-mediated myonecrosis, but highlight the importance of revealing the mechanism by which C. perfringens negates the protective effects of G-CSF in skeletal muscle.
Asunto(s)
Toxinas Bacterianas/toxicidad , Proteínas de Unión al Calcio/toxicidad , Filgrastim/farmacología , Gangrena Gaseosa/etiología , Músculo Esquelético/efectos de los fármacos , Fosfolipasas de Tipo C/toxicidad , Animales , Ratones Endogámicos C57BL , Músculo Esquelético/patología , Necrosis , Receptores de Factor Estimulante de Colonias de Granulocito/antagonistas & inhibidores , Proteínas Recombinantes/farmacologíaRESUMEN
Around the world, Clostridium perfringens type A is known to be a common foodborne pathogen. Therefore, the control and treatment of food poisoning caused by this pathogen are important. This study investigated, in vitro, the effects of Bacillus coagulans and its culture extracts on alpha toxin gene expression, growth inhibition, cytotoxicity, and apoptosis induced by C. perfringens spore, germinated spore and its enterotoxin. Flow cytometry was used to evaluate the apoptosis rate, and MTT test was used to evaluate cytotoxicity. Minimum inhibitory concentration was also used to measure the percentage of inhibition in the broth medium. Finally, RT-qPCR was used to evaluate alpha toxin gene expression. The results showed that the B. coagulans culture extract was able to inhibit the growth of the germinated spore of C. perfringens. Moreover, treating the extract with pepsin can reduce growth in the broth medium. MTT and flow cytometry showed that both B. coagulans and its extract can significantly reduce the cytotoxicity and apoptosis rate induced by C. perfringens type A. In addition, it was shown that the co-culture of B. coagulans and C. perfringens decreases alpha toxin gene expression. The findings of this study indicate that B. coagulans, with growth inhibition and reduced expression of alpha toxin in C. perfringens, can reduce the cytotoxicity and apoptosis rate induced on HT-29â¯cells.
Asunto(s)
Antibiosis , Bacillus coagulans/crecimiento & desarrollo , Toxinas Bacterianas/biosíntesis , Toxinas Bacterianas/toxicidad , Proteínas de Unión al Calcio/biosíntesis , Proteínas de Unión al Calcio/toxicidad , Clostridium perfringens/crecimiento & desarrollo , Clostridium perfringens/metabolismo , Probióticos , Fosfolipasas de Tipo C/biosíntesis , Fosfolipasas de Tipo C/toxicidad , Apoptosis , Supervivencia Celular/efectos de los fármacos , Perfilación de la Expresión Génica , Humanos , Pruebas de Sensibilidad Microbiana , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
During bacterial infection, granulocyte colony-stimulating factor (G-CSF) is produced and accelerates neutrophil production from their progenitors. This process, termed granulopoiesis, strengthens host defense, but Clostridium perfringens α-toxin impairs granulopoiesis via an unknown mechanism. Here, we tested whether G-CSF accounts for the α-toxin-mediated impairment of granulopoiesis. We find that α-toxin dramatically accelerates G-CSF production from endothelial cells in response to Toll-like receptor 2 (TLR2) agonists through activation of the c-Jun N-terminal kinase (JNK) signaling pathway. Meanwhile, α-toxin inhibits G-CSF-mediated cell proliferation of Ly-6G+ neutrophils by inducing degradation of G-CSF receptor (G-CSFR). During sepsis, administration of α-toxin promotes lethality and tissue injury accompanied by accelerated production of inflammatory cytokines in a TLR4-dependent manner. Together, our results illustrate that α-toxin disturbs G-CSF-mediated granulopoiesis by reducing the expression of G-CSFR on neutrophils while augmenting septic shock due to excess inflammatory cytokine release, which provides a new mechanism to explain how pathogenic bacteria modulate the host immune system.
Asunto(s)
Toxinas Bacterianas/toxicidad , Proteínas de Unión al Calcio/toxicidad , Clostridium perfringens/patogenicidad , Gangrena Gaseosa/genética , Factor Estimulante de Colonias de Granulocitos/genética , Lipopolisacáridos/toxicidad , Receptores de Factor Estimulante de Colonias de Granulocito/genética , Choque Séptico/genética , Fosfolipasas de Tipo C/toxicidad , Animales , Clostridium perfringens/genética , Clostridium perfringens/inmunología , Citocinas/genética , Citocinas/inmunología , Modelos Animales de Enfermedad , Femenino , Gangrena Gaseosa/inmunología , Gangrena Gaseosa/microbiología , Gangrena Gaseosa/mortalidad , Regulación de la Expresión Génica , Factor Estimulante de Colonias de Granulocitos/inmunología , Hematopoyesis/efectos de los fármacos , Hematopoyesis/genética , Hematopoyesis/inmunología , Interacciones Huésped-Patógeno/genética , Interacciones Huésped-Patógeno/inmunología , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos/genética , Proteínas Quinasas JNK Activadas por Mitógenos/inmunología , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Neutrófilos/efectos de los fármacos , Neutrófilos/inmunología , Neutrófilos/microbiología , Receptores de Factor Estimulante de Colonias de Granulocito/inmunología , Choque Séptico/inmunología , Choque Séptico/microbiología , Choque Séptico/mortalidad , Transducción de Señal , Análisis de Supervivencia , Receptor Toll-Like 2/genética , Receptor Toll-Like 2/inmunología , Receptor Toll-Like 4/genética , Receptor Toll-Like 4/inmunologíaRESUMEN
ãWhereas granulopoiesis during Gram-negative bacterial infection is accelerated through activation of toll-like receptor 4 (TLR4), it has not been elucidated whether Gram-positive bacterial infection can stimulate granulopoiesis. Using the well-known TLR2 agonist peptidoglycan (PGN), it was shown that neutrophils in bone marrow and spleen and plasma granulocyte colony-stimulating factor were increased in mice that had received intraperitoneal administration of PGN. Incorporation of bromodeoxyuridine into bone marrow neutrophils increased in mice administered PGN, demonstrating that PGN promotes granulopoiesis. These results illustrate that bacterial recognition by TLR2 facilitates granulopoiesis during Gram-positive bacterial infection. Thus, granulopoiesis is accelerated to suppress bacterial infection, but some bacteria can still cause severe infections. Clostridium perfringens is a Gram-positive, anaerobic pathogenic bacterium and causes life-threatening gas gangrene in humans. Of the many toxins produced by C. perfringens, α-toxin is known to be a major virulence factor during infection. Recently, it has been revealed that C. perfringens α-toxin impairs the innate immune system by inhibiting neutrophil differentiation, which is crucial for the pathogenesis of C. perfringens. Moreover, the toxin also attenuates erythropoiesis, which would cause severe anemia in clinical settings. The findings provide new insight to understand how hosts strengthen innate immunity to fight pathogenic bacteria and how they evade the hosts' immune systems.
Asunto(s)
Toxinas Bacterianas/toxicidad , Infecciones por Bacterias Gramnegativas/inmunología , Infecciones por Bacterias Grampositivas/inmunología , Interacciones Huésped-Patógeno/inmunología , Inmunidad Innata/efectos de los fármacos , Inmunidad Innata/inmunología , Neutropenia/inmunología , Peptidoglicano/farmacología , Animales , Bromodesoxiuridina/metabolismo , Proteínas de Unión al Calcio/toxicidad , Diferenciación Celular/efectos de los fármacos , Clostridium perfringens/patogenicidad , Gangrena/microbiología , Factor Estimulante de Colonias de Granulocitos/metabolismo , Humanos , Ratones , Neutropenia/etiología , Neutropenia/metabolismo , Neutrófilos/efectos de los fármacos , Neutrófilos/fisiología , Peptidoglicano/administración & dosificación , Receptor Toll-Like 2/agonistas , Receptor Toll-Like 4/inmunología , Fosfolipasas de Tipo C/toxicidadRESUMEN
Clostridium perfringens α-toxin induces hemolysis of erythrocytes from various species, but it has not been elucidated whether the toxin affects erythropoiesis. In this study, we treated bone marrow cells (BMCs) from mice with purified α-toxin and found that TER119+ erythroblasts were greatly decreased by the treatment. A variant α-toxin defective in enzymatic activities, phospholipase C and sphingomyelinase, had no effect on the population of erythroblasts, demonstrating that the decrease in erythroblasts was dependent of its enzymatic activities. α-Toxin reduced the CD71+TER119+ and CD71-TER119+ cell populations but not the CD71+TER119- cell population. In addition, α-toxin decreased the number of colony-forming unit erythroid colonies but not burst-forming unit erythroid colonies, indicating that α-toxin preferentially reduced mature erythroid cells compared with immature cells. α-Toxin slightly increased annexinV+ cells in TER119+ cells. Additionally, simultaneous treatment of BMCs with α-toxin and erythropoietin greatly attenuated the reduction of TER119+ erythroblasts by α-toxin. Furthermore, hemin-induced differentiation of human K562 erythroleukemia cells was impaired by α-toxin, whereas the treatment exhibited no apparent cytotoxicity. These results suggested that α-toxin mainly inhibited erythroid differentiation. Together, our results provide new insights into the biological activities of α-toxin, which might be important to understand the pathogenesis of C. perfringens infection.
Asunto(s)
Toxinas Bacterianas/toxicidad , Proteínas de Unión al Calcio/toxicidad , Diferenciación Celular/efectos de los fármacos , Células Precursoras Eritroides/patología , Eritropoyesis/efectos de los fármacos , Fosfolipasas de Tipo C/toxicidad , Animales , Antígenos CD/metabolismo , Antígenos de Grupos Sanguíneos/metabolismo , Células Cultivadas , Células Precursoras Eritroides/efectos de los fármacos , Humanos , Células K562 , Ratones , Ratones Endogámicos C57BL , Receptores de Transferrina/metabolismoRESUMEN
Alpha-toxin produced by Clostridium perfringens is an important virulence factor, causing food poisoning and gas gangrene in humans. As such, it is considered a potential bioterrorism threat. To date, there is still no human effective therapeutic drug against alpha-toxin. In this study, a human single chain antibody against alpha-toxin was produced from synthetic (Tomlinson I + J) naive phage display libraries, and its preventive and therapeutic efficacy in mice was examined. To prove the neutralizing potential of the scFv, alpha-toxin was preincubated with scFv and subsequently tested for its lecithinase and hemolytic activity, as well as its lethal effect in mice following intravenous administration. The equilibrium association constant between scFv and CPA was 2.02 × 1010 (1/M), as analyzed by SPR. The scFv could inhibit lecithinase and hemolytic activity, and provided effective protection against alpha-toxin when mice were challenged 1-h post scFv injection. In addition, the survival rate reached 80% for mice treated with scFv within 30 min of being challenged with a 2 × LD50 dose of alpha-toxin. These results confirmed that we successfully prepared a human scFv against C. perfringens type A alpha-toxin, which can be used in the prevention and treatment of alpha-toxin-related illness.
Asunto(s)
Toxinas Bacterianas/inmunología , Proteínas de Unión al Calcio/inmunología , Anticuerpos de Cadena Única/uso terapéutico , Fosfolipasas de Tipo C/inmunología , Animales , Toxinas Bacterianas/toxicidad , Western Blotting , Proteínas de Unión al Calcio/toxicidad , Humanos , Dosificación Letal Mediana , Ratones , Biblioteca de Péptidos , Análisis de Secuencia de ADN , Anticuerpos de Cadena Única/química , Anticuerpos de Cadena Única/aislamiento & purificación , Fosfolipasas de Tipo C/toxicidadRESUMEN
Clostridium perfringens alpha-toxin (ATX) is considered as a prototype of cytotoxic bacterial phospholipases C, and is the major virulence factor in C. perfringens-induced gas gangrene. It is known that, depending on the dose, ATX causes membrane disruption and cytolysis or only limited hydrolysis of its substrates. In the latter case, toxin activity leads to the unregulated generation of bioactive lipids that can ultimately induce cell death. We have characterized apoptosis and necrosis in highly ATX-sensitive, ganglioside-deficient cells exposed to different concentrations of ATX and we have studied the lipidomic profile of cells treated with ATX as compared to native cells to detect the main changes in the lipidomic profile and the possible involvement of lipid signals in cell death. ATX causes both apoptosis and necrosis, depending on dose and time. ATX activates cell death, stimulating the release of cytochrome C from mitochondria and the consequent activation of caspases-3. Moreover GM95 cells treated with ATX showed important lipidomic alterations, among them we detected a general decrease in several phospholipid species and important changes in lipids involved in programmed cell death e.g. ceramide. The data suggest two different mechanisms of cell death caused by ATX, one leading to (mainly saturated) glycerophospholipid hydrolysis related to an increase in diacylglycerols and associated to membrane damage and necrosis, and a second mechanism involving chiefly sphingomyelin hydrolysis and generation of proapoptotic lipidic mediators such as ceramide, N-acylethanolamine and saturated non-esterified fatty acids.
Asunto(s)
Toxinas Bacterianas/toxicidad , Proteínas de Unión al Calcio/toxicidad , Lípidos/química , Fosfolipasas de Tipo C/toxicidad , Animales , Toxinas Bacterianas/química , Proteínas de Unión al Calcio/química , Muerte Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Ratones , Relación Estructura-Actividad , Células Tumorales Cultivadas , Fosfolipasas de Tipo C/químicaRESUMEN
BACKGROUND: Bovine necrohemorrhagic enteritis is caused by Clostridium perfringens type A. Due to the rapid progress and fatal outcome of the disease, vaccination would be of high value. In this study, C. perfringens toxins, either as native toxins or after formaldehyde inactivation, were evaluated as possible vaccine antigens. We determined whether antisera raised in calves against these toxins were able to protect against C. perfringens challenge in an intestinal loop model for bovine necrohemorrhagic enteritis. RESULTS: Alpha toxin and perfringolysin O were identified as the most immunogenic proteins in the vaccine preparations. All vaccines evoked a high antibody response against the causative toxins, alpha toxin and perfringolysin O, as detected by ELISA. All antibodies were able to inhibit the activity of alpha toxin and perfringolysin O in vitro. However, the antibodies raised against the native toxins were more inhibitory to the C. perfringens-induced cytotoxicity (as tested on bovine endothelial cells) and only these antibodies protected against C. perfringens challenge in the intestinal loop model. CONCLUSION: Although immunization of calves with both native and formaldehyde inactivated toxins resulted in high antibody titers against alpha toxin and perfringolysin O, only antibodies raised against native toxins protect against C. perfringens challenge in an intestinal loop model for bovine necrohemorrhagic enteritis.
Asunto(s)
Anticuerpos Neutralizantes/inmunología , Toxinas Bacterianas/administración & dosificación , Vacunas Bacterianas/administración & dosificación , Proteínas de Unión al Calcio/administración & dosificación , Enfermedades de los Bovinos/microbiología , Infecciones por Clostridium/veterinaria , Clostridium perfringens/inmunología , Enteritis/veterinaria , Proteínas Hemolisinas/administración & dosificación , Fosfolipasas de Tipo C/administración & dosificación , Animales , Toxinas Bacterianas/inmunología , Toxinas Bacterianas/toxicidad , Vacunas Bacterianas/inmunología , Vacunas Bacterianas/toxicidad , Proteínas de Unión al Calcio/inmunología , Proteínas de Unión al Calcio/toxicidad , Bovinos , Enfermedades de los Bovinos/inmunología , Enfermedades de los Bovinos/prevención & control , Infecciones por Clostridium/inmunología , Infecciones por Clostridium/patología , Infecciones por Clostridium/prevención & control , Modelos Animales de Enfermedad , Células Endoteliales/inmunología , Enteritis/inmunología , Enteritis/patología , Enteritis/prevención & control , Proteínas Hemolisinas/inmunología , Proteínas Hemolisinas/toxicidad , Yeyuno/inmunología , Masculino , Necrosis , Fosfolipasas de Tipo C/inmunología , Fosfolipasas de Tipo C/toxicidadRESUMEN
Although granulopoiesis is accelerated to suppress bacteria during infection, some bacteria can still cause life-threatening infections, but the mechanism behind this remains unclear. In this study, we found that mature neutrophils in bone marrow cells (BMCs) were decreased in C. perfringens-infected mice and also after injection of virulence factor α-toxin. C. perfringens infection interfered with the replenishment of mature neutrophils in the peripheral circulation and the accumulation of neutrophils at C. perfringens-infected sites in an α-toxin-dependent manner. Measurements of bacterial colony-forming units in C. perfringens-infected muscle revealed that α-toxin inhibited a reduction in the load of C. perfringens. In vitro treatment of isolated BMCs with α-toxin (phospholipase C) revealed that α-toxin directly decreased mature neutrophils. α-Toxin did not influence the viability of isolated mature neutrophils, while simultaneous treatment of BMCs with granulocyte colony-stimulating factor attenuated the reduction of mature neutrophils by α-toxin. Together, our results illustrate that impairment of the innate immune system by the inhibition of neutrophil differentiation is crucial for the pathogenesis of C. perfringens to promote disease to a life-threatening infection, which provides new insight to understand how pathogenic bacteria evade the host immune system.
Asunto(s)
Toxinas Bacterianas/toxicidad , Células de la Médula Ósea/efectos de los fármacos , Proteínas de Unión al Calcio/toxicidad , Clostridium perfringens/patogenicidad , Inmunidad Innata/inmunología , Neutrófilos/inmunología , Fosfolipasas de Tipo C/toxicidad , Factores de Virulencia/toxicidad , Animales , Bacillus subtilis/genética , Bacillus subtilis/patogenicidad , Toxinas Bacterianas/genética , Proteínas de Unión al Calcio/genética , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Infecciones por Clostridium/patología , Clostridium perfringens/genética , Factor Estimulante de Colonias de Granulocitos/farmacología , Inmunidad Innata/efectos de los fármacos , Recuento de Leucocitos , Ratones , Ratones Endogámicos C57BL , Músculo Esquelético/citología , Fosfolipasas de Tipo C/genética , Factores de Virulencia/genéticaRESUMEN
Clostridium perfringens alpha-toxin is a key mediator of gas gangrene, which is a life-threatening infection that manifests as fever, pain, edema, myonecrosis, and gas production. Alpha-toxin possesses phospholipase C and sphingomyelinase activities. The toxin is composed of an N-terminal domain (1-250 aa, N-domain), which is the catalytic site, and a C-terminal domain (251-370 aa, C-domain), which is the membrane-binding site. Immunization of mice with the C-domain of alpha-toxin prevents the gas gangrene caused by C. perfringens, whereas immunization with the N-domain has no effect. The central loop domain (55-93 aa), especially H .SW(84)Y(85) .G, plays an important role in the interaction with ganglioside GM1a. The toxin binds to lipid rafts in the presence of a GM1a/TrkA complex, and metabolites from phosphatidylcholine to diacylglycerol through the enzymatic activity of alpha-toxin itself. These membrane dynamics leads to the activation of endogenous PLCγ-1 via TrkA. In addition, treatment with alpha-toxin leads to the formation of diacylglycerol at membrane rafts in ganglioside-deficient DonQ cells; this in turn triggers endocytosis and cell death. This article summarizes the current the membrane-binding mechanism of alpha-toxin in detail.
Asunto(s)
Toxinas Bacterianas/toxicidad , Proteínas de Unión al Calcio/toxicidad , Membrana Celular/metabolismo , Fosfolipasas de Tipo C/toxicidad , Animales , Toxinas Bacterianas/química , Proteínas de Unión al Calcio/química , Endocitosis , Gangliósidos/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Fosfolipasas de Tipo C/químicaRESUMEN
The anorectic neuropeptide nesfatin-1 has recently been characterized as a potential mood regulator, but the accurate effect of nesfatin-1 on anxiety and learning and memory behavior and the possible mechanisms remains unknown. In the present study, to test the hypothesis that nesfatin-1 might affect the anxiety-like and learning and memory behaviors in rats via ERK/CREB/BDNF pathway, nesfatin-1 was administered intraperitoneally to rats with the doses (10, 20, 40µg/kg), and the behavioral performance was tested using the open field task, the Morris water maze (MWM), and the Y maze. Moreover, the protein expression of brain-derived neurotrophic factor (BDNF), total and phosphorylated-ERK in the hippocampus and the prefrontal cortex (PFC) were evaluated. The results showed that chronic administration of nesfatin-1 could decrease the moving distance, the duration in the center, and the frequencies of rearing and grooming in the open field task, decrease the moving distance, frequency, and the preference index of new arm in the Y maze, although there was no significant difference of the performance in the MWM task among groups. Furthermore, 3 weeks' consecutive administration of nesfatin-1 resulted in the decrease of protein expression of BDNF and phosphorylated-ERK in the hippocampus and the PFC. These results provided evidence that exogenous nesfatin-1 could decrease exploration and induce anxiety-like behavior in rats, the mechanism of which might be related to the reduced protein expression of BDNF and phosphorylated-ERK in the hippocampus and the PFC.
Asunto(s)
Ansiedad/inducido químicamente , Depresores del Apetito/toxicidad , Proteínas de Unión al Calcio/toxicidad , Proteínas de Unión al ADN/toxicidad , Conducta Exploratoria/efectos de los fármacos , Aprendizaje por Laberinto/efectos de los fármacos , Trastornos de la Memoria/inducido químicamente , Proteínas del Tejido Nervioso/toxicidad , Animales , Ansiedad/psicología , Relación Dosis-Respuesta a Droga , Conducta Exploratoria/fisiología , Masculino , Aprendizaje por Laberinto/fisiología , Trastornos de la Memoria/psicología , Nucleobindinas , Ratas , Ratas Sprague-DawleyRESUMEN
The causative pathogen of necrotic enteritis is the Gram-positive bacterium Clostridium perfringens. Its main cell wall component, peptidoglycan (PGN), can be recognized by Toll-like receptor 2 and nucleotide-binding oligomerization domain (NOD). Consequently, the immune response is initiated via activation of nuclear factor kappa B (NF-κB) signalling pathway. An in vitro study was conducted to investigate chicken intestinal inflammatory responses to C. perfringens type A and one of its virulence factors, α-toxin. In primary intestinal epithelial cells, C. perfringens as well as commercially available PGN and α-toxin challenge upregulated mRNA expression of interleukin (IL)-6, IL-8 and inducible nitric oxide synthase (iNOS) with a dosage-dependent manner at 3 h post infection (p.i.; P ≤ 0.001). Time-course effects of three stimulators at high concentration were further examined. C. perfringens infection elevated IL-6, IL-8 and iNOS levels from 1 h to 9 h p.i., while PGN treatment increased IL-6 and IL-8 expression at 1 h and 3 h p.i. (P < 0.05). Bacterial and PGN treatments induced NOD1 expression at 6 h p.i. and only bacterial infection boosted NF-κB p65 expression at 6 h and 9 h p.i. (P < 0.05). α-Toxin treatment upregulated IL-6 and IL-8 expression throughout infection, as well as iNOS, TNF-α and NF-κB p65 expression at later hours p.i. (P < 0.05). In conclusion, both C. perfringens and α-toxin challenge induced intense cytokine expression associated with NF-κB activation in chicken intestinal epithelial cells. The receptors for the recognition of PGN component of C. perfringens need further investigation.
Asunto(s)
Toxinas Bacterianas/toxicidad , Proteínas de Unión al Calcio/toxicidad , Clostridium perfringens/inmunología , Células Epiteliales/efectos de los fármacos , Células Epiteliales/inmunología , Regulación de la Expresión Génica/inmunología , Mucosa Intestinal/citología , Transducción de Señal/inmunología , Fosfolipasas de Tipo C/toxicidad , Animales , Embrión de Pollo , Células Epiteliales/microbiología , Regulación de la Expresión Génica/efectos de los fármacos , Técnicas In Vitro , Interleucina-6/metabolismo , Interleucina-8/metabolismo , FN-kappa B/inmunología , Óxido Nítrico Sintasa de Tipo II/metabolismoRESUMEN
Calcium-binding proteins are ubiquitous modulators of cellular activity and function. Cells possess numerous calcium-binding proteins that regulate calcium concentration in the cytosol by buffering excess free calcium ion. Disturbances in intracellular calcium homeostasis are at the heart of many age-related conditions making these proteins targets for therapeutic intervention. A calcium-binding protein, apoaequorin, has shown potential utility in a broad spectrum of applications for human health and well-being. Large-scale recombinant production of the protein has been successful; enabling further research and development and commercialization efforts. Previous work reported a 90-day subchronic toxicity test that demonstrated this protein has no toxicity by oral exposure in Sprague-Dawley rodents. The current study assesses the allergenic potential of the purified protein using bioinformatic analysis and simulated gastric digestion. The results from the bioinformatics searches with the apoaequorin sequence show the protein is not a known allergen and not likely to cross-react with known allergens. Apoaequorin is easily digested by pepsin, a characteristic commonly exhibited by many non-allergenic dietary proteins. From these data, there is no added concern of safety due to unusual stability of the protein by ingestion.
Asunto(s)
Aequorina/genética , Aequorina/toxicidad , Apoproteínas/genética , Apoproteínas/toxicidad , Proteínas de Unión al Calcio/biosíntesis , Proteínas de Unión al Calcio/toxicidad , Escherichia coli/genética , Seguridad , Aequorina/administración & dosificación , Aequorina/biosíntesis , Aequorina/inmunología , Alérgenos/inmunología , Secuencia de Aminoácidos , Animales , Apoproteínas/administración & dosificación , Apoproteínas/biosíntesis , Apoproteínas/inmunología , Proteínas de Unión al Calcio/administración & dosificación , Proteínas de Unión al Calcio/inmunología , Biología Computacional , Escherichia coli/metabolismo , Mucosa Gástrica/metabolismo , Datos de Secuencia Molecular , Pepsina A/metabolismo , Estabilidad Proteica , Ratas , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/toxicidad , Medición de Riesgo , Pruebas de Toxicidad SubcrónicaRESUMEN
Clostridium perfringens phospholipase C (CpPLC), also called α-toxin, plays a key role in the pathogenesis of gas gangrene. CpPLC may lead to cell lysis at concentrations that cause extensive degradation of plasma membrane phospholipids. However, at sublytic concentrations it induces cytotoxicity without inducing evident membrane damage. The results of this work demonstrate that CpPLC becomes internalized in cells by a dynamin-dependent mechanism and in a time progressive process: first, CpPLC colocalizes with caveolin both at the plasma membrane and in vesicles, and later it colocalizes with early and late endosomes and lysosomes. Lysosomal damage in the target cells is evident 9 h after CpPLC exposure. Our previous work demonstrated that CpPLCinduces ERK1/2 activation, which is involved in its cytotoxic effect. In this work we found that cholesterol sequestration, dynamin inhibition, as well as inhibition of actin polymerization, prevent CpPLC internalization and ERK1/2 activation, involving endocytosis in the signalling events required for CpPLC cytotoxic effect at sublytic concentrations. These results provide new insights about the mode of action of this bacterial phospholipase C, previously considered to act only locally on cell membrane.
Asunto(s)
Toxinas Bacterianas/metabolismo , Toxinas Bacterianas/toxicidad , Proteínas de Unión al Calcio/metabolismo , Proteínas de Unión al Calcio/toxicidad , Endocitosis , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Sistema de Señalización de MAP Quinasas , Fosfolipasas de Tipo C/metabolismo , Fosfolipasas de Tipo C/toxicidad , Línea Celular , HumanosRESUMEN
Clostridium perfringens alpha toxin/phospholipase C (CP-PLC) is one of the most potent bacterial toxins known to cause soft tissue infections like gas gangrene in humans and animals. It is the first bacterial toxin demonstrated to be an enzyme with phospholipase, sphingomyelinase and lecithinase activities. The toxin is comprised of an enzymatic N-domain and a binding C-domain interconnected by a flexible linker. The N-domain alone is non-toxic to mammalian cells, but incubation with C-domain restores the toxicity, the mechanism of which is still not elucidated. The objectives of the current study were to investigate the formation of a stable N and C-domain complex, to determine possible interactions between the two domains in silico and to characterize the in vitro and in vivo correlates of the interaction. To establish the existence of a stable N and C-domain hybrid, in vitro pull down assay and dot-Far Western blotting assays were employed, where it was clearly revealed that the two domains bound to each other to form an intermediate. Using bioinformatics tools like MetaPPISP, PatchDock and FireDock, we predicted that the two domains may interact with each other through electrostatic interactions between at least six pairs of amino acids. This N and C-domains interacted with each other in 1:1 ratio and the hybrid lysed mouse erythrocytes in a slower kinetics when compared with wild type native Cp-PLC. BALB/c mice when challenged with N and C-domain hybrid demonstrated severe myonecrosis at the site of injection while no death was observed. Our results provide further insight into better understanding the mechanism for the toxicity of Cp-PLC N and C-domain mixture.
Asunto(s)
Toxinas Bacterianas/química , Proteínas de Unión al Calcio/química , Clostridium perfringens/química , Simulación de Dinámica Molecular , Fosfolipasas de Tipo C/química , Animales , Toxinas Bacterianas/genética , Toxinas Bacterianas/toxicidad , Sitios de Unión , Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/toxicidad , Clostridium perfringens/enzimología , Clostridium perfringens/genética , Eritrocitos/citología , Eritrocitos/efectos de los fármacos , Femenino , Expresión Génica , Hemólisis/efectos de los fármacos , Inyecciones Intramusculares , Ratones , Ratones Endogámicos BALB C , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/patología , Necrosis/inducido químicamente , Necrosis/patología , Unión Proteica , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/toxicidad , Electricidad Estática , Fosfolipasas de Tipo C/genética , Fosfolipasas de Tipo C/toxicidadRESUMEN
Bovine necrohemorrhagic enteritis is a major cause of mortality in veal calves. Clostridium perfringens is considered as the causative agent, but there has been controversy on the toxins responsible for the disease. Recently, it has been demonstrated that a variety of C. perfringens type A strains can induce necrohemorrhagic lesions in a calf intestinal loop assay. These results put forward alpha toxin and perfringolysin as potential causative toxins, since both are produced by all C. perfringens type A strains. The importance of perfringolysin in the pathogenesis of bovine necrohemorrhagic enteritis has not been studied before. Therefore, the objective of the current study was to evaluate the role of perfringolysin in the development of necrohemorrhagic enteritis lesions in calves and its synergism with alpha toxin. A perfringolysin-deficient mutant, an alpha toxin-deficient mutant and a perfringolysin alpha toxin double mutant were less able to induce necrosis in a calf intestinal loop assay as compared to the wild-type strain. Only complementation with both toxins could restore the activity to that of the wild-type. In addition, perfringolysin and alpha toxin had a synergistic cytotoxic effect on bovine endothelial cells. This endothelial cell damage potentially explains why capillary hemorrhages are an initial step in the development of bovine necrohemorrhagic enteritis. Taken together, our results show that perfringolysin acts synergistically with alpha toxin in the development of necrohemorrhagic enteritis in a calf intestinal loop model and we hypothesize that both toxins act by targeting the endothelial cells.
Asunto(s)
Toxinas Bacterianas/toxicidad , Proteínas de Unión al Calcio/toxicidad , Enfermedades de los Bovinos/microbiología , Clostridium perfringens/fisiología , Enteritis/veterinaria , Proteínas Hemolisinas/toxicidad , Fosfolipasas de Tipo C/toxicidad , Animales , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/metabolismo , Bovinos , Clostridium perfringens/genética , Células Endoteliales/microbiología , Células Endoteliales/patología , Enteritis/microbiología , Ensayo de Inmunoadsorción Enzimática/veterinaria , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/metabolismo , Intestinos/microbiología , Intestinos/patología , Mutación , Necrosis/microbiología , Necrosis/veterinaria , Fosfolipasas de Tipo C/genética , Fosfolipasas de Tipo C/metabolismoRESUMEN
Dystrophic neurites surrounding ß-amyloid (Aß) plaques precede neuronal death in Alzheimer disease. These neuritic alterations may be one of the initial stages for synaptic loss and dysfunction. However, intracellular pathways that cause local disruption of neuronal processes by Aß remain to be fully elucidated. The identification of Aß-induced genes that mediate neuritic pathology would provide considerable insight into the mechanisms of Alzheimer's disease. Previously, we reported that selective up-regulation of calsyntenin-3 (Cst-3) by Aß and accumulation of neurotoxic Cst-3 in dystrophic neurites surrounding Aß plaques may lead to local disruption of these neurites. Like amyloid precursor protein, Cst-3 undergoes two-step proteolytic processing: the primary cleavage with α-secretase generates an N-terminal ectodomain and a C-terminal fragment (CTF). The CTF is subsequently cleaved into p3 peptide and an intracellular domain via γ-secretase. It would be interesting to know whether accumulated Cst-3 in dystrophic neurites surrounding Aß plaques is the full-length version or a CTF. Herein, we show that the CTF but not full-length Cst-3 accumulated in dystrophic neurites surrounding Aß plaques in Tg2576 mouse and Alzheimer disease brains. In vitro experiments with Cst-3 fragments have revealed that only the CTF resulted in acceleration of neuronal death. These results indicate that accumulation of the neurotoxic CTF in neurites surrounding Aß plaques may lead to local disruption of neuronal processes and development of dystrophic neurites.
Asunto(s)
Enfermedad de Alzheimer/patología , Encéfalo/metabolismo , Proteínas de Unión al Calcio/toxicidad , Proteínas de la Membrana/toxicidad , Neuritas/patología , Placa Amiloide/metabolismo , Enfermedad de Alzheimer/metabolismo , Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Animales , Encéfalo/efectos de los fármacos , Encéfalo/patología , Proteínas de Unión al Calcio/química , Proteínas de Unión al Calcio/metabolismo , Muerte Celular/efectos de los fármacos , Medios de Cultivo Condicionados/química , Femenino , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Ratones , Ratones Transgénicos , Neuritas/efectos de los fármacos , Neuritas/metabolismo , Fármacos Neuroprotectores/farmacología , Placa Amiloide/patología , Complejo de la Endopetidasa Proteasomal/metabolismo , Inhibidores de Proteasoma/farmacología , RatasRESUMEN
Clostridium botulinum type C and D strains recently have been found to produce PLC on egg yolk agar plates. To characterize the gene, enzymatic and biological activities of C. botulinum PLCs (Cb-PLCs), the cb-plc genes from 8 strains were sequenced, and 1 representative gene was cloned and expressed as a recombinant protein. The enzymatic and hemolytic activities of the recombinant Cb-PLC were measured and compared with those of the Clostridium perfringens alpha-toxin. Each of the eight cb-plc genes encoded a 399 amino acid residue protein preceded by a 27 residue signal peptide. The protein consists of 2 domains, the N- and C-domains, and the overall amino acid sequence identity between Cb-PLC and alpha-toxin was greater than 50%, suggesting that Cb-PLC is homologous to the alpha-toxin. The key residues in the N-domain were conserved, whereas those in the C-domain which are important in membrane interaction were different than in the alpha-toxin. As expected, Cb-PLC could hydrolyze egg yolk phospholipid, p-nitrophenylphosphorylcholine, and sphingomyelin, and also exhibited hemolytic activity;however, its activities were about 4- to over 200-fold lower than those of alpha-toxin. Although Cb-PLC showed weak enzymatic and biological activities, it is speculated that Cb-PLC might play a role in the pathogenicity of botulism or for bacterial survival.