RESUMO
Infection by Proteus mirabilis causes urinary stones and catheter incrustation due to ammonia formed by urease (PMU), one of its virulence factors. Non-enzymatic properties, such as pro-inflammatory and neurotoxic activities, were previously reported for distinct ureases, including that of the gastric pathogen Helicobacter pylori. Here, PMU was assayed on isolated cells to evaluate its non-enzymatic properties. Purified PMU (nanomolar range) was tested in human (platelets, HEK293 and SH-SY5Y) cells, and in murine microglia (BV-2). PMU promoted platelet aggregation. It did not affect cellular viability and no ammonia was detected in the cultures' supernatants. PMU-treated HEK293 cells acquired a pro-inflammatory phenotype, producing reactive oxygen species (ROS) and cytokines IL-1ß and TNF-α. SH-SY5Y cells stimulated with PMU showed high levels of intracellular Ca2+ and ROS production, but unlike BV-2 cells, SH-SY5Y did not synthesize TNF-α and IL-1ß. Texas Red-labeled PMU was found in the cytoplasm and in the nucleus of all cell types. Bioinformatic analysis revealed two bipartite nuclear localization sequences in PMU. We have shown that PMU, besides urinary stone formation, can potentially contribute in other ways to pathogenesis. Our data suggest that PMU triggers pro-inflammatory effects and may affect cells beyond the renal system, indicating a possible role in extra-urinary diseases.
Assuntos
Proteus mirabilis/enzimologia , Proteus mirabilis/patogenicidade , Urease/metabolismo , Urease/toxicidade , Sequência de Aminoácidos , Animais , Cálcio/metabolismo , Linhagem Celular , Núcleo Celular/metabolismo , Células HEK293 , Humanos , Técnicas In Vitro , Camundongos , Microglia/efeitos dos fármacos , Microglia/metabolismo , Microglia/microbiologia , Modelos Moleculares , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/microbiologia , Neurotoxinas/química , Neurotoxinas/metabolismo , Neurotoxinas/toxicidade , Sinais de Localização Nuclear , Agregação Plaquetária/efeitos dos fármacos , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/toxicidade , Urease/química , Virulência/fisiologiaRESUMO
Ureases are microbial virulence factors either because of the enzymatic release of ammonia or due to many other non-enzymatic effects. Here we studied two neurotoxic urease isoforms, Canatoxin (CNTX) and Jack Bean Urease (JBU), produced by the plant Canavalia ensiformis, whose mechanisms of action remain elusive. The neurotoxins provoke convulsions in rodents (LD50 â¼2 mg/kg) and stimulate exocytosis in cell models, affecting intracellular calcium levels. Here, electrophysiological and brain imaging techniques were applied to elucidate their mode of action. While systemic administration of the toxins causes tonic-clonic seizures in rodents, JBU injected into rat hippocampus induced spike-wave discharges similar to absence-like seizures. JBU reduced the amplitude of compound action potential from mouse sciatic nerve in a tetrodotoxin-insensitive manner. Hippocampal slices from CNTX-injected animals or slices treated in vitro with JBU failed to induce long term potentiation upon tetanic stimulation. Rat cortical synaptosomes treated with JBU released L-glutamate. JBU increased the intracellular calcium levels and spontaneous firing rate in rat hippocampus neurons. MicroPET scans of CNTX-injected rats revealed increased [18]Fluoro-deoxyglucose uptake in epileptogenesis-related areas like hippocampus and thalamus. Curiously, CNTX did not affect voltage-gated sodium, calcium or potassium channels currents, neither did it interfere on cholinergic receptors, suggesting an indirect mode of action that could be related to the ureases' membrane-disturbing properties. Understanding the neurotoxic mode of action of C. ensiformis ureases could help to unveil the so far underappreciated relevance of these toxins in diseases caused by urease-producing microorganisms, in which the human central nervous system is affected.
Assuntos
Canavalia/química , Síndromes Neurotóxicas/etiologia , Proteínas de Plantas/toxicidade , Toxinas Biológicas/toxicidade , Urease/toxicidade , Animais , Convulsivantes/isolamento & purificação , Convulsivantes/toxicidade , Feminino , Masculino , Camundongos , Sistema Nervoso/efeitos dos fármacos , Sistema Nervoso/patologia , Síndromes Neurotóxicas/fisiopatologia , Proteínas de Plantas/isolamento & purificação , Ratos , Ratos Wistar , Toxinas Biológicas/isolamento & purificação , Urease/isolamento & purificação , Xenopus laevisRESUMO
Jaburetox is a recombinant peptide derived from one of the Canavalia ensiformis urease isoforms. This peptide induces several toxic effects on insects of different orders, including interference on muscle contractility in cockroaches, modulation of UDP-N-acetylglucosamine pyrophosphorylase (UAP) and nitric oxide synthase (NOS) activities in the central nervous system of triatomines, as well as activation of the immune system in Rhodnius prolixus. When injected, the peptide is lethal for R. prolixus and Triatoma infestans. Here, we evaluated Jaburetox toxicity to Nauphoeta cinerea cockroaches, exploring the effects on the central nervous system through the activities of UAP, NOS, acid phosphatases (ACP), and acetylcholinesterase (AChE). The results indicated that N. cinerea is not susceptible to the lethal effect of the peptide. Moreover, both in vivo and in vitro treatments with Jaburetox inhibited NOS activity, without modifying the protein levels. No alterations on ACP activity were observed. In addition, the enzyme activity of UAP only had its activity affected at 18 hr after injection. The peptide increased the AChE activity, suggesting a mechanism involved in overcoming the toxic effects. In conclusion, our findings indicate that Jaburetox affects the nitrinergic signaling as well as the AChE and UAP activities and establishes N. cinerea as a Jaburetox-resistant model for future comparative studies.
Assuntos
Baratas/efeitos dos fármacos , Baratas/enzimologia , Proteínas de Plantas/toxicidade , Urease/toxicidade , Acetilcolinesterase/efeitos dos fármacos , Fosfatase Ácida/efeitos dos fármacos , Animais , Sistema Nervoso Central/efeitos dos fármacos , Feminino , Masculino , Óxido Nítrico Sintase/efeitos dos fármacos , Nucleotidiltransferases/efeitos dos fármacos , Proteínas Recombinantes/toxicidadeRESUMO
Jaburetox (JBTX) is an insecticidal and antifungal peptide derived from jack bean (Canavalia ensiformis) urease that has been considered a candidate for developing genetically modified crops. This study aimed to perform the risk assessment of the peptide JBTX following the general recommendations of the two-tiered, weight-of-evidence approach proposed by International Life Sciences Institute. The urease of C. ensiformis (JBU) and its isoform JBURE IIb (the JBTX parental protein) were assessed. The history of safe use revealed no hazard reports for the studied proteins. The available information shows that JBTX possesses selective activity against insects and fungi. JBTX and JBU primary amino acids sequences showed no relevant similarity to toxic, antinutritional or allergenic proteins. Additionally, JBTX and JBU were susceptible to in vitro digestibility, and JBU was also susceptible to heat treatment. The results did not identify potential risks of adverse effects and reactions associated to JBTX. However, further allergen (e.g. serum IgE binding test) and toxicity (e.g. rodent toxicity tests) experimentation can be done to gather additional safety information on JBTX, and to meet regulatory inquiries for commercial approval of transgenic cultivars expressing this peptide.
Assuntos
Antifúngicos/toxicidade , Inseticidas/toxicidade , Proteínas de Plantas/toxicidade , Medição de Risco , Urease/toxicidade , Animais , Antifúngicos/química , Canavalia/enzimologia , Biologia Computacional , Fungos/efeitos dos fármacos , Insetos/efeitos dos fármacos , Inseticidas/química , Proteínas de Plantas/química , Isoformas de Proteínas/química , Isoformas de Proteínas/toxicidade , Proteólise , Urease/químicaRESUMO
Jaburetox is a recombinant peptide derived from a Canavalia ensiformis urease that presents toxic effects upon several species of insects, phytopathogenic fungi and yeasts of medical importance. So far, no toxicity of Jaburetox to mammals has been shown. Previous reports have identified biochemical targets of this toxic peptide in insect models, although its mechanism of action is not completely understood. In this work, we aimed to characterize the effects of Jaburetox in hemolymphatic insect cells. For this purpose, the model insect and Chagas' disease vector Rhodnius prolixus was used. In vivo and in vitro experiments indicated that Jaburetox interacts with a subset of hemocytes and it can be found in various subcellular compartments. In insects injected with Jaburetox there was an increase in the gene expression of the enzymes UDP-N-acetylglucosamine pyrophosphorylase (UAP), chitin synthase and nitric oxide synthase (NOS). Nevertheless, the expression of NOS protein, the enzyme activities of UAP and acid phosphatase (a possible link between UAP and NOS) as well as the phosphorylation state of proteins remained unchanged upon the in vivo Jaburetox treatment. Nitric oxide (NO) imaging using fluorescent probes showed that Jaburetox augmented NO production in the hemocyte aggregates when compared to controls. Even though Jaburetox activated the hemocytes, as demonstrated by wheat germ agglutinin binding assays, the peptide did not lead to an increase of their phagocytic behavior. Taken together, these findings contribute to our understanding of toxic effects of Jaburetox, a peptide with biotechnological applications and a prospective tool for rational insect control.
Assuntos
Hemócitos/efeitos dos fármacos , Praguicidas/toxicidade , Rhodnius , Urease/toxicidade , Animais , Células Cultivadas , Ninfa/efeitos dos fármacos , Proteínas de Plantas , Proteínas Recombinantes/toxicidadeRESUMO
Ammonia is putatively the major toxin associated with hepatic encephalopathy (HE), a neuropsychiatric manifestation that results in cognitive impairment, poor concentration and psychomotor alterations. The hippocampus, a brain region involved in cognitive impairment and depressive behavior, has been studied less than neocortical regions. Herein, we investigated hippocampal astrocyte parameters in a hyperammonemic model without hepatic lesion and in acute hippocampal slices exposed to ammonia. We also measured hippocampal BDNF, a neurotrophin commonly related to synaptic plasticity and cognitive deficit, and peripheral S100B protein, used as a marker for brain damage. Hyperammonemia directly impaired astrocyte function, inducing a decrease in glutamate uptake and in the activity of glutamine synthetase, in turn altering the glutamine-glutamate cycle, glutamatergic neurotransmission and ammonia detoxification itself. Hippocampal BDNF was reduced in hyperammonemic rats via a mechanism that may involve astrocyte production, since the same effect was observed in astrocyte cultures exposed to ammonia. Ammonia induced a significant increase in S100B secretion in cultured astrocytes; however, no significant changes were observed in the serum or in cerebrospinal fluid. Data demonstrating hippocampal vulnerability to ammonia toxicity, particularly due to reduced glutamate uptake activity and BDNF content, contribute to our understanding of the neuropsychiatric alterations in HE.
Assuntos
Fator Neurotrófico Derivado do Encéfalo/metabolismo , Ácido Glutâmico/metabolismo , Hipocampo/metabolismo , Hiperamonemia/patologia , Amônia/sangue , Animais , Animais Recém-Nascidos , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Células Cultivadas , Modelos Animais de Doenças , Feminino , Proteína Glial Fibrilar Ácida/metabolismo , Glutamato-Amônia Ligase/metabolismo , Glutationa/metabolismo , Hipocampo/efeitos dos fármacos , Hiperamonemia/induzido quimicamente , Técnicas In Vitro , L-Lactato Desidrogenase/metabolismo , Transportadores de Ânions Orgânicos/metabolismo , Ratos , Ratos Wistar , Subunidade beta da Proteína Ligante de Cálcio S100/metabolismo , Simportadores/metabolismo , Urease/toxicidadeRESUMO
Ureases are metalloenzymes that hydrolyze urea into ammonia and carbon dioxide. They were the first enzymes to be crystallized and, with them, the notion that enzymes are proteins became accepted. Novel toxic properties of ureases that are independent of their enzyme activity have been discovered in the last three decades. Since our first description of the neurotoxic properties of canatoxin, an isoform of the jack bean urease, which appeared in Toxicon in 1981, about one hundred articles have been published on "new" properties of plant and microbial ureases. Here we review the present knowledge on the non-enzymatic properties of ureases. Plant ureases and microbial ureases are fungitoxic to filamentous fungi and yeasts by a mechanism involving fungal membrane permeabilization. Plant and at least some bacterial ureases have potent insecticidal effects. This entomotoxicity relies partly on an internal peptide released upon proteolysis of ingested urease by insect digestive enzymes. The intact protein and its derived peptide(s) are neurotoxic to insects and affect a number of other physiological functions, such as diuresis, muscle contraction and immunity. In mammal models some ureases are acutely neurotoxic upon injection, at least partially by enzyme-independent effects. For a long time bacterial ureases have been recognized as important virulence factors of diseases by urease-producing microorganisms. Ureases activate exocytosis in different mammalian cells recruiting eicosanoids and Ca(2+)-dependent pathways, even when their ureolytic activity is blocked by an irreversible inhibitor. Ureases are chemotactic factors recognized by neutrophils (and some bacteria), activating them and also platelets into a pro-inflammatory "status". Secretion-induction by ureases may play a role in fungal and bacterial diseases in humans and other animals. The now recognized "moonlighting" properties of these proteins have renewed interest in ureases for their biotechnological potential to improve plant defense against pests and as potential targets to ameliorate diseases due to pathogenic urease-producing microorganisms.
Assuntos
Metaloproteínas/toxicidade , Neurotoxinas/toxicidade , Urease/toxicidade , Animais , Apoenzimas/genética , Apoenzimas/metabolismo , Apoenzimas/farmacologia , Apoenzimas/toxicidade , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/farmacologia , Proteínas de Bactérias/toxicidade , Fungicidas Industriais/farmacologia , Fungicidas Industriais/toxicidade , Humanos , Inseticidas/metabolismo , Inseticidas/farmacologia , Inseticidas/toxicidade , Isoenzimas/genética , Isoenzimas/metabolismo , Isoenzimas/farmacologia , Isoenzimas/toxicidade , Metaloproteínas/genética , Metaloproteínas/metabolismo , Metaloproteínas/farmacologia , Neurotoxinas/genética , Neurotoxinas/metabolismo , Neurotoxinas/farmacologia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Proteínas de Plantas/farmacologia , Proteínas de Plantas/toxicidade , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Proteínas Recombinantes/toxicidade , Urease/genética , Urease/metabolismo , Urease/farmacologiaRESUMO
Ureases are multifunctional proteins that display biological activities independently of their enzymatic function, such as induction of exocytosis and insecticidal effects. Rhodnius prolixus, a major vector of Chagas' disease, is a model for studies on the entomotoxicity of jack bean urease (JBU). We have previously shown that JBU induces the production of eicosanoids in isolated tissues of R. prolixus. In insects, the immune response comprises cellular and humoral reactions, and is centrally modulated by eicosanoids. Cyclooxygenase products signal immunity in insects, mainly cellular reactions, such as hemocyte aggregation. In searching for a link between JBU's toxic effects and immune reactions in insects, we have studied the effects of this toxin on R. prolixus hemocytes. JBU triggers aggregation of hemocytes after injection into the hemocoel and when applied to isolated cells. On in vitro assays, the eicosanoid synthesis inhibitors dexamethasone (phospholipase A2 indirect inhibitor) and indomethacin (cyclooxygenase inhibitor) counteracted JBU's effect, indicating that eicosanoids, more specifically cyclooxygenase products, are likely to mediate the aggregation response. Contrarily, the inhibitors esculetin and baicalein were inactive, suggesting that lipoxygenase products are not involved in JBU's effect. Extracellular calcium was also necessary for JBU's effect, in agreement to other cell models responsive to ureases. A progressive darkening of the medium of JBU-treated hemocytes was observed, suggestive of a humoral response. JBU was immunolocalized in the cultured cells upon treatment along with cytoskeleton damage. The highest concentration of JBU tested on cultured cells also led to nuclei aggregation of adherent hemocytes. This is the first time urease has been shown to affect insect hemocytes, contributing to our understanding of the entomotoxic mechanisms of action of this protein.
Assuntos
Vetores Artrópodes/fisiologia , Canavalia/química , Doença de Chagas/transmissão , Eicosanoides/toxicidade , Hemócitos/efeitos dos fármacos , Rhodnius/fisiologia , Urease/toxicidade , Animais , Canavalia/toxicidade , Agregação Celular/efeitos dos fármacos , Eicosanoides/biossíntese , Larva , Cultura Primária de CélulasRESUMO
BACKGROUND: Ureases are multifunctional enzymes that display biological activities independent of their enzymatic function, including exocytosis induction and insecticidal effects. The hemipteran Rhodnius prolixus is one of the known susceptible models for this toxicity. It has been shown that Jack bean urease (JBU) has deleterious effects on R. prolixus, and these effects are modulated by eicosanoids, which are synthesized in a cascade involving phospholipase A2 (PLA2) enzymes. METHODS: R. prolixus genome was screened for putative PLA2s and matching transcripts were cloned. Predicted amino acid sequences were analyzed and transcript distribution among tissues was determined by qPCR. RNAi techniques were used and subsequent JBU toxicity assays were performed. RESULTS: Two PLA2 genes were identified, Rhopr-PLA2III and Rhopr-PLA2XII. The transcripts are widely distributed in the tissues but at different levels. The analyses fit the putative proteins into groups III and XII of secretory PLA2s. After 70% of Rhopr-PLA2XII expression was knocked down, JBU's toxicity was decreased by more than 50% on 5th instars R. prolixus. CONCLUSIONS: Rhopr-PLA2XII gene is linked to JBU's toxic effect in R. prolixus and our findings support previous studies demonstrating that eicosanoids modulate this toxicity. GENERAL SIGNIFICANCE: Besides identifying and characterizing two PLA2 genes in the major Chagas' disease vector R. prolixus, we have shown that the potent toxicity of JBU is linked to one of these genes. Our results contribute to the general comprehension of urease's mechanisms of action in insects, and, potentially, to studies on the control of the Chagas' disease parasite transmission.
Assuntos
Canavalia/enzimologia , Insetos Vetores , Metionina/química , Fosfolipases A2/metabolismo , Rhodnius/fisiologia , Urease/toxicidade , Sequência de Aminoácidos , Animais , Sequência de Bases , Doença de Chagas/transmissão , Clonagem Molecular , Biblioteca Gênica , Isoenzimas , Dados de Sequência Molecular , Oxirredução , Fosfolipases A2/química , Fosfolipases A2/genética , Filogenia , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase em Tempo Real , Homologia de Sequência de AminoácidosRESUMO
Recently, ureases were included in the arsenal of plant defense proteins, alongside many other proteins with biotechnological potential such as insecticides. Isoforms of Canavalia ensiformis urease (canatoxin-CNTX and jack bean urease-JBURE-I) are toxic to insects of different orders. This toxicity is due in part to the release of a 10 kDa peptide from the native protein, by cathepsin-like enzymes present in the insect digestive tract. The entomotoxic peptide, Jaburetox-2Ec, exhibits potent insecticidal activity against several insects, including many resistant to the native ureases. JBURE-I and Jaburetox-2Ec cause major alterations of post-feeding physiological processes in insects, which contribute to, or can be the cause of, their entomotoxic effect. An overview of the current knowledge on plant urease processing and mechanisms of action in insects is presented in this review.
Assuntos
Canavalia/enzimologia , Inseticidas/metabolismo , Controle Biológico de Vetores , Proteínas de Plantas/metabolismo , Toxinas Biológicas/metabolismo , Urease/metabolismo , Animais , Insetos/efeitos dos fármacos , Insetos/fisiologia , Inseticidas/toxicidade , Estágios do Ciclo de Vida/efeitos dos fármacos , Proteínas de Plantas/toxicidade , Isoformas de Proteínas , Toxinas Biológicas/toxicidade , Urease/toxicidadeRESUMO
Jackbean (Canavalia ensiformis) ureases are entomotoxic upon the release of internal peptides by insect's digestive enzymes. Here we studied the digestive peptidases of Oncopeltus fasciatus (milkweed bug) and its susceptibility to jackbean urease (JBU). O. fasciatus nymphs fed urease showed a mortality rate higher than 80% after two weeks. Homogenates of midguts dissected from fourth instars were used to perform proteolytic activity assays. The homogenates hydrolyzed JBU in vitro, yielding a fragment similar in size to known entomotoxic peptides. The major proteolytic activity at pH 4.0 upon protein substrates was blocked by specific inhibitors of aspartic and cysteine peptidases, but not significantly affected by inhibitors of metallopeptidases or serine peptidases. The optimal activity upon N-Cbz-Phe-Arg-MCA was at pH 5.0, with complete blockage by E-64 in all pH tested. Optimal activity upon Abz-AIAFFSRQ-EDDnp (a substrate for aspartic peptidases) was detected at pH 5.0, with partial inhibition by Pepstatin A in the pH range 2-8. Fluorogenic substrates corresponding to the N- and C-terminal regions flanking a known entomotoxic peptide within urease sequence were also tested. While the midgut homogenate did not hydrolyze the N-terminal peptide, it cleaved the C-terminal peptide maximally at pH 4.0-5.0, and this activity was inhibited by E-64 (10 µM). The midgut homogenate was submitted to ion-exchange chromatography followed by gel filtration. A 22 kDa active fraction was obtained, resolved in SDS-PAGE (12%), the corresponding band was in-gel digested by trypsin, the peptides were analyzed by mass spectrometry, retrieving a cathepsin L protein. The purified cathepsin L was shown to have at least two possible cleavage sites within the urease sequence, and might be able to release a known insecticidal peptide in a single or cascade event. The results suggest that susceptibility of O. fasciatus nymphs to jackbean urease is, like in other insect models, due mostly to limited proteolysis of ingested protein and subsequent release of entomotoxic peptide(s) by cathepsin-like digestive enzymes.
Assuntos
Canavalia/toxicidade , Inseticidas/metabolismo , Ninfa/efeitos dos fármacos , Proteínas de Plantas/metabolismo , Precursores de Proteínas/metabolismo , Urease/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Canavalia/enzimologia , Catepsina L/química , Catepsina L/metabolismo , Cromatografia por Troca Iônica , Cumarínicos/farmacologia , Inibidores de Cisteína Proteinase/farmacologia , Digestão , Dipeptídeos/farmacologia , Eletroforese em Gel de Poliacrilamida , Heterópteros/efeitos dos fármacos , Heterópteros/enzimologia , Concentração de Íons de Hidrogênio , Hidrólise/efeitos dos fármacos , Inseticidas/química , Inseticidas/isolamento & purificação , Inseticidas/toxicidade , Leucina/análogos & derivados , Leucina/farmacologia , Dados de Sequência Molecular , Ninfa/enzimologia , Fragmentos de Peptídeos/análise , Proteínas de Plantas/química , Proteínas de Plantas/isolamento & purificação , Proteínas de Plantas/toxicidade , Precursores de Proteínas/química , Precursores de Proteínas/isolamento & purificação , Precursores de Proteínas/toxicidade , Urease/química , Urease/isolamento & purificação , Urease/toxicidadeRESUMO
Ureases are metalloenzymes that are widespread among plants, fungi and bacteria. Urease isoforms (jack bean urease-JBU and canatoxin) from Canavalia ensiformis seeds are toxic to insects and fungi, suggesting a role in plant defense. The entomotoxic effect is due to the release of a 10-kDa peptide by cathepsin-like enzymes in the insect's midgut. Urease causes a decrease in post-feeding weight loss in Rhodnius prolixus, suggesting an effect on water balance. To investigate how this impairment occurs, we have evaluated the action of JBU and the urease-derivated peptide Jaburetox-2Ec on R. prolixus Malpighian tubules and also investigated the involvement of second messengers. JBU and Jaburetox-2Ec affect serotonin-induced secretion from Malpighian tubules. This effect is not cAMP-dependent, but the Jaburetox-2Ec effect is cGMP-dependent. Eicosanoid metabolites and calcium ions appear to be involved in JBU effect on diuresis, but are not involved in the action of Jaburetox-2Ec. Jaburetox-2Ec, but not JBU, causes a change in the transepithelial potential of the tubules. Canatoxin has a similar effect on tubules secretion, decreasing the secretion rate, but the urease from Helicobacter pylori has no significant effect. These data are helpful in our understanding of the actions of ureases and derived peptides on insects, and also reinforces the potential use of these proteins as biopesticides.
Assuntos
Túbulos de Malpighi/efeitos dos fármacos , Rhodnius/efeitos dos fármacos , Sistemas do Segundo Mensageiro/efeitos dos fármacos , Urease/toxicidade , Análise de Variância , Animais , Larva/efeitos dos fármacos , Túbulos de Malpighi/metabolismo , Proteínas de Plantas , Radioimunoensaio , Serotonina/metabolismoRESUMO
The broad distribution of ureases in leguminous seeds, as well as the accumulation pattern of the protein during seed maturation, are suggestive of an important physiological role for this enzyme. Since the isolation and characterization of jack bean urease by Sumner in 1926, many investigations have been dedicated to the structural and biological features of this enzyme; nevertheless, many questions still remain. It has been reported that ureases from plants (jack bean and soybean seeds) display biological properties unrelated to their ureolytic activity, notably a high insecticidal activity against Coleoptera (beetles) and Hemiptera (bugs), suggesting that ureases might be involved in plant defense. Besides the insecticidal activity, canatoxin, a jack bean urease isoform, causes convulsions and death in mice and rats, induces indirect hemagglutination (hemilectin activity) and promotes exocytosis in several cell types. Not only plant ureases but also some microbial ureases (found in Bacillus pasteurii and Helicobacter pylori) are able to induce activation of platelets in a process mediated by lipoxygenase-derived metabolites. This review summarizes the biological and structural properties of plant ureases, compares them with those displayed by bacterial ureases, and discusses the significance of these findings.
Assuntos
Plantas/enzimologia , Urease/química , Urease/metabolismo , Sequência de Aminoácidos , Animais , Biotecnologia , Insetos/efeitos dos fármacos , Dados de Sequência Molecular , Nitrogênio/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/toxicidade , Toxinas Biológicas/química , Toxinas Biológicas/toxicidade , Urease/toxicidadeRESUMO
Canatoxin, a urease isoform from Canavalia ensiformis seeds, shows insecticidal activity against different insect species. Its toxicity relies on an internal 10 kDa peptide (pepcanatox), released by hydrolysis of Canatoxin by cathepsins in the digestive system of susceptible insects. In the present work, based on the N-terminal sequence of pepcanatox, we have designed primers to amplify by PCR a 270-bp fragment corresponding to pepcanatox using JBURE-II cDNA (one of the urease isoforms cloned from C. ensiformis, with high identity to JBURE-I, the classical urease) as a template. This amplicon named jaburetox-2 was cloned into pET 101 vector to obtain heterologous expression in Escherichia coli of the recombinant protein in C-terminal fusion with V-5 epitope and 6-His tag. Jaburetox-2Ec was purified on Nickel-NTA resin and bioassayed in insect models. Dysdercus peruvianus larvae were fed on cotton seed meal diets containing 0.01% (w/w) Jaburetox-2Ec and, after 11 days, all individuals were dead. Jaburetox-2Ec was also tested against Spodoptera frugiperda larvae and caused 100% mortality. In contrast, high doses of Jaburetox-2Ec were innocuous when injected or ingested by mice and neonate rats. Modeling of Jaburetox-2Ec, in comparison with other peptide structures, revealed a prominent beta-hairpin motif consistent with an insecticidal activity based on either neurotoxicity or cell permeation.
Assuntos
Canavalia/enzimologia , Inseticidas/isolamento & purificação , Urease/química , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Primers do DNA , Eletroforese em Gel de Poliacrilamida , Inseticidas/química , Inseticidas/toxicidade , Dados de Sequência Molecular , Proteínas de Plantas , Reação em Cadeia da Polimerase , Homologia de Sequência de Aminoácidos , Urease/genética , Urease/isolamento & purificação , Urease/toxicidadeRESUMO
Urease and the cytotoxin VacA are two major virulence factors of the human pathogen Helicobacter pylori, which is responsible for severe gastroduodenal diseases. Diffusion of urea, the substrate of urease, into the stomach is critically required for the survival of infecting H. pylori. We now show that VacA increases the transepithelial flux of urea across model epithelia by inducing an unsaturable permeation pathway. This transcellular pathway is selective, as it conducts thiourea, but not glycerol and mannitol, demonstrating that it is not due to a loosening of intercellular junctions. Experiments performed with different cell lines, grown in a nonpolarized state, confirm that VacA permeabilizes the cell plasma membrane to urea. Inhibition studies indicate that transmembrane pores formed by VacA act as passive urea transporters. Thus, their inhibition by the anion channel blocker 5-nitro-2-(3-phenylpropylamino) benzoic acid significantly decreases toxin-induced urea fluxes in both polarized and nonpolarized cells. Moreover, phloretin, a well-known inhibitor of eukaryotic urea transporters, blocks VacA-mediated urea and ion transport and the toxin's main biologic effects. These data show that VacA behaves as a low-pH activated, passive urea transporter potentially capable of permeabilizing the gastric epithelium to urea. This opens the novel possibility that in vivo VacA may favor H. pylori infectivity by optimizing urease activity.
Assuntos
Proteínas de Bactérias/toxicidade , Toxinas Bacterianas/toxicidade , Helicobacter pylori/enzimologia , Helicobacter pylori/patogenicidade , Proteínas de Membrana Transportadoras , Ureia/metabolismo , Animais , Transporte Biológico Ativo , Células CACO-2 , Proteínas de Transporte/metabolismo , Permeabilidade da Membrana Celular/efeitos dos fármacos , Células Cultivadas , Cães , Epitélio/efeitos dos fármacos , Epitélio/metabolismo , Mucosa Gástrica/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Glicoproteínas de Membrana/metabolismo , Floretina/farmacologia , Estômago/efeitos dos fármacos , Urease/toxicidade , Virulência , Transportadores de UreiaRESUMO
Urease catalyzes the hydrolysis of urea to ammonia and carbamate and has been found to be an important pathogenic factor for certain bacteria. Cryptococcus neoformans is a significant human pathogenic fungus that produces large amounts of urease; thus we wanted to investigate the importance of urease in the pathogenesis of cryptococcosis. We cloned and sequenced the genomic locus containing the single-copy C. neoformans urease gene (URE1) and used this to disrupt the native URE1 in the serotype A strain H99. The ure1 mutant strains were found to have in vitro growth characteristics, phenoloxidase activity, and capsule size similar to those of the wild type. Comparison of a ure1 mutant with H99 after intracisternal inoculation into corticosteroid-treated rabbits revealed no significant differences in colony counts recovered from the cerebrospinal fluid. However, when these two strains were compared in both the murine intravenous and inhalational infection models, there were significant differences in survival. Mice infected with a ure1 strain lived longer than mice infected with H99 in both models. The ure1 strain was restored to urease positivity by complementation with URE1, and two resulting transformants were significantly more pathogenic than the ure1 strain. Our results suggest that urease activity is involved in the pathogenesis of cryptococcosis but that the importance may be species and/or infection site specific.
Assuntos
Criptococose/etiologia , Cryptococcus neoformans/enzimologia , Urease/toxicidade , Animais , Criptococose/mortalidade , Cryptococcus neoformans/patogenicidade , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Reação em Cadeia da Polimerase , Coelhos , Especificidade da Espécie , Urease/genética , VirulênciaRESUMO
The pathogenic role of Helicobacter pylori virulence factors has been studied with a mouse model of gastric disease. BALB/c mice were treated orally with different amounts of sonic extracts of cytotoxic H. pylori strains (NCTC 11637, 60190, 84-183, and 87A300 [CagA+/Tox+]). The pathological effects on histological sections of gastric mucosae were assessed and were compared with the effects of treatments with extracts from noncytotoxic strains (G21 and G50 [CagA-/Tox-]) and from strains that express either CagA alone (D931 [CagA+/Tox-]) or the cytotoxin alone (G104 [CagA-/Tox+]). The treatment with extracts from cytotoxic strains induced various epithelial lesions (vacuolation, erosions, and ulcerations), recruitment of inflammatory cells in the lamina propria, and a marked reduction of the mucin layer. Extracts of noncytotoxic strains induced mucin depletion but no other significant pathology. Crude extracts of strain D931, expressing CagA alone, caused only mild infiltration of inflammatory cells, whereas extracts of strain G104, expressing cytotoxin alone, induced extensive epithelial damage but little inflammatory reaction. Loss of the mucin layer was not associated with a cytotoxic phenotype, since this loss was observed in mice treated with crude extracts of all strains. The pathogenic roles of CagA, cytotoxin, and urease were further assessed by using extracts of mutant strains of H. pylori defective in the expression of each of these virulence factors. The results obtained suggest that (i) urease activity does not play a significant role in inducing the observed gastric damage, (ii) cytotoxin has an important role in the induction of gastric epithelial cell lesions but not in eliciting inflammation, and (iii) other components present in strains which carry the cagA gene, but distinct from CagA itself, are involved in eliciting the inflammatory response.
Assuntos
Antígenos de Bactérias , Proteínas de Bactérias/toxicidade , Citotoxinas/toxicidade , Helicobacter pylori/patogenicidade , Urease/toxicidade , Animais , Mucosa Gástrica/patologia , Camundongos , Camundongos Endogâmicos BALB C , VirulênciaRESUMO
Recent studies have indicated that ammonia is involved in the pathophysiology of Helicobacter pylori-associated gastric mucosal damage. Helicobacter pylori-associated chronic active gastritis is characterized by an invasion of neutrophils. We investigated the interrelationship among hypochlorous acid (oxidant produced by neutrophil), ammonia (product of Helicobacter pylori urease), and monochloramine (product of ammonia and hypochlorous acid) in the development of gastric mucosal damage in rats. Gastric mucosal lesions were produced by exposure of the gastric mucosa to ammonia, urea with urease, or urea with Helicobacter pylori in rats subjected to ischemia. Pretreatment with taurine (scavenger of hypochlorous acid) or antineutrophil serum significantly attenuated gastric mucosal lesions induced by the above test agents. Ammonia-induced gastric mucosal lesions were exacerbated in the presence of hypochlorous acid with concomitant generation of monochloramine. These results suggest that the ammonia, hypochlorous acid, and monochloramine triad may be important in Helicobacter pylori-mediated gastric mucosal damage.
Assuntos
Amônia/toxicidade , Mucosa Gástrica/efeitos dos fármacos , Neutrófilos/metabolismo , Animais , Cloraminas/toxicidade , Relação Dose-Resposta a Droga , Interações Medicamentosas , Mucosa Gástrica/enzimologia , Gastrite/etiologia , Gastrite/metabolismo , Infecções por Helicobacter/etiologia , Infecções por Helicobacter/metabolismo , Helicobacter pylori/patogenicidade , Ácido Hipocloroso/toxicidade , Soros Imunes/farmacologia , Masculino , Neutrófilos/imunologia , Peroxidase/metabolismo , Ratos , Ratos Sprague-Dawley , Taurina/farmacologia , Ureia/toxicidade , Urease/toxicidadeRESUMO
The effects of urea-urease-ammonia on the rat gastric mucosa were examined and compared with those of NH4OH and NH4Cl. The mucosal application of urea with urease produced a reduction in potential difference (PD) in a dose-related manner for urea, and a significant drop was observed by > 0.1% urea in the presence of 100 units urease. Such PD reduction was also observed when the mucosa was exposed to either NH4OH (> 0.03%) or NH4Cl (> 1%); delta PD (20 mV) caused by 0.3% NH4OH and 3% NH4Cl was equivalent to that induced by 0.5% urea+urease (100 units). The combined oral administration of urea (approximately 6%) and urease (100 units) did not induce any macroscopic damage in the gastric mucosa. NH4Cl given orally had no or little effect on the mucosa at any dose levels even at 10%, while NH4OH given orally caused hemorrhagic lesions in the mucosa at the dose of > 0.3%. In contrast, both urea+urease and NH4Cl given prior to HCl/ethanol protected the gastric mucosa against damage in a dose-related manner, and a significant effect was obtained by urea at > 0.5% and by NH4Cl at > 1%. NH4OH was also effective in reducing the severity of HCl/ethanol-induced gastric lesions at lower dose (0.3%). The protective effect of urea+urease was attenuated significantly by prior administration of indomethacin or coadministration of hydroxyurea, while that of NH4Cl or NH4OH was mitigated by indomethacin.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Amônia/toxicidade , Amônia/uso terapêutico , Mucosa Gástrica/efeitos dos fármacos , Compostos de Amônio Quaternário/toxicidade , Compostos de Amônio Quaternário/uso terapêutico , Ureia/toxicidade , Ureia/uso terapêutico , Urease/toxicidade , Urease/uso terapêutico , Animais , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Interações Medicamentosas , Determinação da Acidez Gástrica , Mucosa Gástrica/fisiologia , Concentração de Íons de Hidrogênio , Potenciais da Membrana/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Úlcera Gástrica/induzido quimicamente , Úlcera Gástrica/prevenção & controleRESUMO
BACKGROUND: The pathophysiological mechanism by which Helicobacter pylori induces mucosal injury has not been clarified. The aim of this study was to investigate the role of urea, urease, and ammonia in rat gastric mucosal lesions using an ex vivo chamber model. METHODS: Two groups of rats, normotensive rats and those subjected to ischemia, were studied. The gastric mucosa was examined histologically and macroscopically, and the transmucosal potential difference was measured. RESULTS: Instillation of urea into the stomach generated ammonia in the presence of urease. The amount of ammonia was increased depending on the concentration of urea and was closely associated with the severity of the histological lesions. The exposure of the stomach to 15-60 mmol/L ammonium hydroxide induced both a reduction in transmucosal potential difference and microscopic damage to the gastric mucosa in normotensive rats. Moreover, 15-60 mmol/L ammonium hydroxide produced severe macroscopic gastric lesions in the rats subjected to ischemia. CONCLUSIONS: These results show that ammonia is deleterious to the gastric mucosa and suggest the importance of urea, urease, and ammonia in the pathophysiology of gastric diseases in H. pylori-infected patients.