RESUMEN
Micrurus dumerilii is a coral snake of clinic interest in Colombia. Its venom is mainly composed of phospholipases A2 being MdumPLA2 the most abundant protein. Nevertheless, Micrurus species produce a low quantity of venom, which makes it difficult to produce anticoral antivenoms. Therefore, in this work, we present the recombinant expression of MdumPLA2 to evaluate its biological activities and its immunogenic potential to produce antivenoms. For this, a genetic construct rMdumPLA2 was cloned into the pET28a vector and expressed heterologously in bacteria. His-rMdumPLA2 was extracted from inclusion bodies, refolded in vitro, and isolated using affinity and RP-HPLC chromatography. His-rMdumPLA2 was shown to have phospholipase A2 activity, a weak anticoagulant effect, and induced myonecrosis and edema. The anti-His-rMdumPLA2 antibodies produced in rabbits recognized native PLA2, the complete venom of M. dumerilii, and a phospholipase from another species of the Micrurus genus. Antibodies neutralized 100% of the in vitro phospholipase activity of the recombinant toxin and a moderate percentage of the myotoxic activity of M. dumerilii venom in mice. These results indicate that His-rMdumPLA2 could be used as an immunogen to improve anticoral antivenoms development. This work is the first report of an M. dumerilii functional recombinant PLA2.
Asunto(s)
Antivenenos , Serpientes de Coral , Venenos Elapídicos , Fosfolipasas A2 , Animales , Ratones , Conejos , Antivenenos/biosíntesis , Antivenenos/genética , Antivenenos/inmunología , Venenos Elapídicos/enzimología , Fosfolipasas A2/biosíntesis , Fosfolipasas A2/genética , Fosfolipasas A2/inmunología , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunologíaRESUMEN
Bothrops snakes cause around 80% of snakebites in Brazil, with muscle tissue damage as an important consequence, which may cause dysfunction on the affected limb. Bothropstoxin-I (BthTX-I) from Bothrops jararacussu is a K49-phospholipase A2, involved in the injury and envenomation's inflammatory response. Immune system components act in the resolution of tissue damage and regeneration. Thus, macrophages exert a crucial role in the elimination of dead tissue and muscle repair. Here, we studied the cellular influx and presence of classical and alternative macrophages (M1 and M2) during muscle injury induced by BthTX-I and the regeneration process. BthTX-I elicited intense inflammatory response characterized by neutrophil migration, then increased influx of M1 macrophages followed by M2 population that declined, resulting in tissue regeneration. The high expressions of TNF-α and IL6 were changed by increased TGF-ß expression after BthTX-I injection, coinciding with the iNOs and arginase expression and the peaks of M1 and M2 macrophages in muscle tissue. A coordinated sequence of PAX7, MyoD, and myogenin expression involved in muscle regenerative process appeared after BthTX-I injection. Together, these results demonstrate a direct correlation between the macrophage subsets, cytokine microenvironment, and the myogenesis process. This information may be useful for new envenomation and muscular dysfunction therapies.
Asunto(s)
Bothrops , Venenos de Crotálidos/toxicidad , Macrófagos/fisiología , Enfermedades Musculares/inducido químicamente , Fosfolipasas A2/toxicidad , Regeneración/fisiología , Animales , Brasil , Venenos de Crotálidos/enzimología , Citocinas/metabolismo , Desarrollo de Músculos/inmunología , Fosfolipasas A2/inmunología , Regeneración/inmunología , Mordeduras de Serpientes/complicaciones , Mordeduras de Serpientes/inmunología , Factores de TiempoRESUMEN
Insect venom phospholipases have been identified in nearly all clinically relevant social Hymenoptera, including bees, wasps and ants. Among other biological roles, during the envenoming process these enzymes cause the disruption of cellular membranes and induce hypersensitive reactions, including life threatening anaphylaxis. While phospholipase A2 (PLA2) is a predominant component of bee venoms, phospholipase A1 (PLA1) is highly abundant in wasps and ants. The pronounced prevalence of IgE-mediated reactivity to these allergens in sensitized patients emphasizes their important role as major elicitors of Hymenoptera venom allergy (HVA). PLA1 and -A2 represent valuable marker allergens for differentiation of genuine sensitizations to bee and/or wasp venoms from cross-reactivity. Moreover, in massive attacks, insect venom phospholipases often cause several pathologies that can lead to fatalities. This review summarizes the available data related to structure, model of enzymatic activity and pathophysiological roles during envenoming process of insect venom phospholipases A1 and -A2.
Asunto(s)
Venenos de Artrópodos/enzimología , Himenópteros/enzimología , Mordeduras y Picaduras de Insectos/inmunología , Fosfolipasas A1/inmunología , Fosfolipasas A2/inmunología , Secuencia de Aminoácidos , Animales , Venenos de Artrópodos/inmunología , Humanos , Mordeduras y Picaduras de Insectos/enzimología , Fosfolipasas A1/química , Fosfolipasas A1/metabolismo , Fosfolipasas A2/química , Fosfolipasas A2/metabolismoRESUMEN
Polyclonal antibodies raised in Balb-c mice against BnSP-7, a Lys-49 phospholipase A2, were used to measure cross reactivity against other snake venoms. Using ELISA, these antibodies were able to recognize PLA2s isoforms present in venoms of botropic snakes at 1:6400, 1:3200 and 1:100 ratios (w/w). These antibodies highly recognized proteins of low molecular weight (â¼14,000) from crude snake venom Bp and Bm by Western Blotting. PLA2 these venoms, by alignment of primary structures demonstrated high identity with BnSP-7 PLA2, especially in the C-terminal region. However, the crude snake venom Bd and Bj, showed low recognition. The PLA2 activity of Bothrops pauloensis, Bothrops moojeni venoms or BpPLA2-TXI was inhibited significantly when anti-BnSP-7 antibodies were incubated at 1:10 and 1:20 ratios (venoms or toxin:anti-BnSP-7, w/w), respectively. The myotoxic effect induced by the same venoms was also reduced significantly at 1:1, 1:10 and 1:20 ratios, by decreased creatine kinase levels. The anti-PLA2 polyclonal antibodies effectively recognized PLA2s from Bothrops pauloensis and Bothrops moojeni venoms, and neutralized specific catalytic and myotoxic activity.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Bothrops/inmunología , Reacciones Cruzadas/inmunología , Venenos de Crotálidos/inmunología , Fosfolipasas A2/inmunología , Venenos de Serpiente/inmunología , Secuencia de Aminoácidos , Animales , Western Blotting , Bothrops/clasificación , Bothrops/metabolismo , Venenos de Crotálidos/metabolismo , Ensayo de Inmunoadsorción Enzimática , Masculino , Ratones Endogámicos BALB C , Pruebas de Neutralización , Fosfolipasas A2/genética , Fosfolipasas A2/metabolismo , Homología de Secuencia de Aminoácido , Venenos de Serpiente/metabolismo , Especificidad de la EspecieRESUMEN
Micrurus clarki is an uncommon coral snake distributed from the Southeastern Pacific of Costa Rica to Western Colombia, for which no information on its venom could be found in the literature. Using a 'venomics' approach, proteins of at least nine families were identified, with a moderate predominance of three-finger toxins (3FTx; 48.2%) over phospholipase A2 (PLA2; 36.5%). Comparison of this venom profile with those of other Micrurus species suggests that it may represent a more balanced, 'intermediate' type within the dichotomy between 3FTx- and PLA2-predominant venoms. M. clarki venom was strongly cross-recognized and, accordingly, efficiently neutralized by an equine therapeutic antivenom against M. nigrocinctus, revealing their high antigenic similarity. Lethal activity for mice could be reproduced by a PLA2 venom fraction, but, unexpectedly, not by fractions corresponding to 3FTxs. The most abundant venom component, hereby named clarkitoxin-I, was identified as a short-chain (type I) 3FTx, devoid of lethal effect in mice, whose target remains to be defined. Its amino acid sequence of 66 residues shows high similarity with predicted sequences of venom gland transcripts described for M. fulvius, M. browni, and M. diastema.
Asunto(s)
Antivenenos/inmunología , Reacciones Cruzadas/inmunología , Venenos Elapídicos , Fosfolipasas A2 , Proteoma/análisis , Toxinas Biológicas , Secuencia de Aminoácidos , Animales , Cromatografía Líquida de Alta Presión , Venenos Elapídicos/química , Venenos Elapídicos/inmunología , Venenos Elapídicos/toxicidad , Elapidae , Electroforesis en Gel de Poliacrilamida , Dosificación Letal Mediana , Ratones , Pruebas de Neutralización , Fosfolipasas A2/química , Fosfolipasas A2/inmunología , Fosfolipasas A2/toxicidad , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Toxinas Biológicas/química , Toxinas Biológicas/inmunología , Toxinas Biológicas/toxicidadRESUMEN
Four proteins with phospholipase A2 (PLA2) activity, designated P9a(Cdt-PLA2), P9b(Cdt-PLA2), P10a(Cdt-PLA2) and P10b(Cdt-PLA2) were purified from the venom of Crotalus durissus terrificus by two chromatographic steps: a gel filtration and reversed phase HPLC. The profile obtained clearly shows that three of them have a similar abundance. The molecular mass, 14193.8340Da for P9a(Cdt-PLA2), 14134.9102Da for P9b(Cdt-PLA2), 14242.6289Da for P10a(Cdt-PLA2) and 14183.8730Da for P10b(Cdt-PLA2), were initially evaluated by SDS-PAGE and confirmed by ESI-Q-TOF spectrometry, and all of them displayed a monomeric conformation. Also, partial amino acid sequence of each protein was obtained and their alignments with other crotalic PLA2 revealed a high degree of identity among them. Additionally, we studied some pharmacological activities like neurotoxicity, myotoxicity and lethality, which prompted us to pick two of them, P9a(Cdt-PLA2) and P10a(Cdt-PLA2) that resulted to be less toxic that the others, and further characterize them to be used as immunogen. We next injected these last proteins in mice to produce antitoxins against them and ELISA and dot blots reveled that both toxins do not show immunogenic differences, unlike those other pharmacologic activities tested. Furthermore, the antibodies produced cross-reacted with all the isoforms purified demonstrating the feasibility of using only one of them and ensuring the cross-reaction of all. The results obtained show that P9a(Cdt-PLA2) isoform has the lowest toxicity and also a good purification performance; thus this protein may be a promising candidate to be employed in the production of crotalic antitoxins.
Asunto(s)
Antivenenos/inmunología , Crotalus , Crotoxina/inmunología , Inmunoglobulina G/inmunología , Fosfolipasas A2/inmunología , Animales , Antivenenos/farmacología , Pollos , Cromatografía en Gel , Cromatografía de Fase Inversa , Venenos de Crotálidos/enzimología , Venenos de Crotálidos/inmunología , Venenos de Crotálidos/toxicidad , Crotoxina/antagonistas & inhibidores , Crotoxina/toxicidad , Ensayo de Inmunoadsorción Enzimática , Sueros Inmunes/inmunología , Immunoblotting , Inmunoglobulina G/aislamiento & purificación , Inmunoglobulina G/farmacología , Isoenzimas , Dosificación Letal Mediana , Masculino , Ratones , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/patología , Fosfolipasas A2/química , Fosfolipasas A2/toxicidadRESUMEN
The benefits from treatment with antivenom sera are indubitable. However, the mechanism for toxin neutralization has not been completely elucidated. A mixture of anti-bothropic and anti-crotalic horse antivenom has been reported to be more effective in neutralizing the effects of Bothrops jararacussu snake venom than anti-bothropic antivenom alone. This study determined which regions in the three PLA2s from B. jararacussu snake venom are bound by antibodies in tetravalent anti-bothropic and monovalent anti-crotalic commercial horse antivenom. Mapping experiments of BthTX-I, BthTX-II and BthA-I using two small libraries of 69 peptides each revealed six major IgG-binding epitopes that were recognized by both anti-bothropic and anti-crotalic horse antivenom. Two epitopes in BthTX-I were only recognized by the anti-bothropic horse antivenom, while anti-crotalic horse antivenom recognized four unique epitopes across the three PLA2s. Our studies suggest that the harmful activities of the PLA2s present in the venom of B. jararacussu are neutralized by the combinatorial treatment with both antivenom sera through their complementary binding sites, which provides a wide coverage on the PLA2s. This is the first peptide microarray of PLA2s from B. jararacussu snake venom to survey the performance of commercial horse antiophidic antivenom. Regions recognized by the protective antivenom sera are prime candidates for improved venom cocktails or a chimeric protein encoding the multiple epitopes to immunize animals as well as for designing future synthetic vaccines.
Asunto(s)
Venenos de Crotálidos/inmunología , Epítopos de Linfocito B/inmunología , Fosfolipasas A2 Grupo II/inmunología , Caballos/inmunología , Fosfolipasas A2/inmunología , Secuencia de Aminoácidos , Animales , Antivenenos/química , Bothrops , Reacciones Cruzadas , Venenos de Crotálidos/química , Venenos de Crotálidos/enzimología , Ensayo de Inmunoadsorción Enzimática , Fosfolipasas A2 Grupo II/química , Datos de Secuencia Molecular , Pruebas de Neutralización , Biblioteca de Péptidos , Fosfolipasas A2/química , Estructura Terciaria de Proteína , Alineación de SecuenciaRESUMEN
Crotalus durissus terrificus, C. d. collilineatus, C. d. cascavella and C. d. marajoensis are responsible minor but severe snake bites in Brazil. The venoms of these snakes share the presence of crotoxin, a neurotoxin comprising of two associated components, crotapotin and phospholipase A2 (PLA2). Treatment of the victims with specific antiserum is the unique effective therapeutic measure. The ability of anti-Crotalus antisera produced by the routine using crude venom to immunize horses or purified crotoxin and PLA2 as individual immunogens was compared. Antisera obtained from horses immunized with C. durissus terrificus crude venom were able to recognize and neutralize not only the toxins presents in C. durissus terrificus, but also the ones present in the venoms from C. d. collilineatus, C. d. cascavella and C. d. marajoensis. Antisera from horses immunized with individual crotoxin or PLA2, although in lesser titers, were also able of recognizing the toxins in all four Crotalus species and neutralize the lethality of the C. d. terrificus venom.
Asunto(s)
Antivenenos/biosíntesis , Antivenenos/farmacología , Venenos de Crotálidos/toxicidad , Mordeduras de Serpientes/tratamiento farmacológico , Animales , Antivenenos/inmunología , Bioensayo , Venenos de Crotálidos/inmunología , Crotoxina/inmunología , Modelos Animales de Enfermedad , Caballos/inmunología , Dosificación Letal Mediana , Masculino , Ratones , Neurotoxinas/inmunología , Pruebas de Neutralización , Fosfolipasas A2/inmunología , Mordeduras de Serpientes/inmunología , Mordeduras de Serpientes/mortalidad , Análisis de SupervivenciaRESUMEN
The hybrid created from the crossbreeding of European and African bees, known as the Africanised bee, has provided numerous advantages for current beekeeping. However, this new species exhibits undesirable behaviours, such as colony defence instinct and a propensity to attack en masse, which can result in serious accidents. To date, there is no effective treatment for cases of Africanised bee envenomation. One promising technique for developing an efficient antivenom is the use of phage display technology, which enables the production of human antibodies, thus avoiding the complications of serum therapy, such as anaphylaxis and serum sickness. The aim of this study was to produce human monoclonal single-chain Fv (scFv) antibody fragments capable of inhibiting the toxic effects of Africanised bee venom. We conducted four rounds of selection of antibodies against the venom and three rounds of selection of antibodies against purified melittin. Three clones were selected and tested by enzyme-linked immunosorbent assay to verify their specificity for melittin and phospholipase A2. Two clones (C5 and C12) were specific for melittin, and one (A7) was specific for phospholipase A2. In a kinetic haemolytic assay, these clones were evaluated individually and in pairs. The A7-C12 combination had the best synergistic effect and was chosen to be used in the assays of myotoxicity inhibition and lethality. The A7-C12 combination inhibited the in vivo myotoxic effect of the venom and increased the survival of treated animals.
Asunto(s)
Antivenenos/inmunología , Venenos de Abeja/toxicidad , Meliteno/inmunología , Fosfolipasas A2/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Venenos de Abeja/inmunología , Abejas , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Mordeduras y Picaduras de Insectos/inmunología , Mordeduras y Picaduras de Insectos/terapia , Ratones , Anticuerpos de Cadena Única/inmunología , SobrevidaAsunto(s)
Antivenenos/inmunología , Crotalus/inmunología , Crotoxina/antagonistas & inhibidores , Fosfolipasas A2/inmunología , Animales , Antivenenos/biosíntesis , Dextranos , Indicadores y Reactivos , Masculino , Ratones , Pruebas de Neutralización/métodos , Fosfolipasas A2/aislamiento & purificación , ConejosRESUMEN
The toxicity of crotoxin, the major toxin of Crotalus durissus terrificus (South American rattlesnake) venom, is mediated by its basic phospholipase A(2) (PLA(2)) subunit. This PLA(2) is non-covalently associated with crotapotin, an acidic, enzymatically inactive subunit of the crotoxin complex. In this work, rabbit antiserum raised against crotapotin purified from Crotalus durissus cascavella venom was tested for its ability to neutralize the neurotoxicity of this venom and its crotoxin in vitro. The ability of this antiserum to inhibit the enzymatic activity of the crotoxin complex and PLA(2) alone was also assessed, and its potency in preventing myotoxicity was compared with that of antisera raised against crotoxin and PLA(2). Antiserum to crotapotin partially neutralized the neuromuscular blockade caused by venom and crotoxin in electrically stimulated mouse phrenic nerve-hemidiaphragm preparations and prevented the venom-induced myotoxicity, but did not inhibit the enzymatic activity of crotoxin and purified PLA(2). In contrast, previous findings showed that antisera against crotoxin and PLA(2) from C. d. cascavella effectively neutralized the neuromuscular blockade and PLA(2) activity of this venom and its crotoxin. The partial neutralization of crotoxin-mediated neurotoxicity by antiserum to crotapotin probably reduced the binding of crotoxin to its receptor following interaction of the antiserum with the crotapotin moiety of the complex.