RESUMEN
The Loxosceles genus represents one of the main arachnid genera of medical importance in Brazil. Despite the gravity of Loxosceles-related accidents, just a handful of species are deemed medically important and only a few have undergone comprehensive venom characterization. Loxosceles amazonica is a notable example of a potentially dangerous yet understudied Loxosceles species. While there have been limited reports of accidents involving L. amazonica to date, accidents related to Loxosceles are increasing in the North and Northeast regions of Brazil, where L. amazonica has been reported. In this work, we provide a complementary biochemical and immunological characterization of L. amazonica venom, considering its most relevant enzymatic activities and its immunorecognition and neutralization by current therapeutic antivenoms. Additionally, a cDNA library enriched with phospholipase D (PLD) sequences from L. amazonica venom glands was built and subsequently sequenced. The results showed that L. amazonica venom is well immunorecognised by all the tested antibodies. Its venom also displayed proteolytic, hyaluronidase, and sphingomyelinase activities. These activities were at least partially inhibited by available antivenoms. With cDNA sequencing of PLDs, seven new putative isoforms were identified in the venom of L. amazonica. These results contribute to a better knowledge of the venom content and activities of a synanthropic, yet understudied, Loxosceles species. In vivo assays are essential to confirm the medical relevance of L. amazonica, as well as to assess its true toxic potential and elucidate its related pathophysiology.
RESUMEN
Accidents involving spiders from the genus Loxosceles cause medical emergencies in several countries of South America. The species Loxosceles laeta is ubiquitously present in Peru and is responsible for severe accidents in this country. To further characterize L. laeta venom components and to unveil possible variations in the Peruvian population, we provide an overview of the toxins-related transcripts present in the venom gland of Peruvian L. laeta. A dataset from a cDNA library previously sequenced by MiSeq sequencer (Illumina) was re-analyzed and the obtained data was compared with available sequences from Loxosceles toxins. Phospholipase-D represent the majority (69,28 %) of the transcripts related to venom toxins, followed by metalloproteases (20,72 %), sicaritoxins (6,03 %), serine-proteases (2,28 %), hyaluronidases (1,80 %) and Translationally Controlled Tumor Protein (TCTP) (0,56 %). New sequences of phospholipases D,sicaritoxins, hyaluronidase, TCTP and serine proteinases were described. Differences between the here-described toxin sequences and others, previously identified in venom glands from other spiders, were visualized upon sequence alignments. In addition, an in vitro hyaluronidase activity assay was also performed to complement comparisons between Peruvian and Brazilian L. laeta venom enzymatic activities, revealing a superior activity in the venom from Brazilian specimens. These new data provide a molecular basis that can help to explain the difference in toxicity among L. laeta venoms from different countries in South America.
Asunto(s)
Hialuronoglucosaminidasa , Venenos de Araña , Animales , Biblioteca de Genes , Hialuronoglucosaminidasa/genética , Perú , Alineación de Secuencia , Venenos de Araña/genéticaRESUMEN
Snakebites caused by Crotalus genus are the second most frequent in Brazil. Crotoxin is a beta-neurotoxin responsible for the main envenomation effects of Crotalus biting, while crotamine immobilizes the animal hind limbs, contributing to prey immobilization and to envenoming symptoms. As crotoxin and crotamine represent about 90% of Crotalus venom dry weight, these toxins are of great importance for antivenom therapy. In this sense, knowledge regarding the antigenicity/immunogenicity at the molecular level of these toxins can provide valuable information for the improvement of specific antivenoms. Therefore, the aims of this study are the identification of the B-cell epitopes from crotoxin and crotamine; and the characterization of the neutralizing potency of antibodies directed against the corresponding synthetic epitopes defined in the current study. Linear B-cell epitopes were identified using the Spot Synthesis technique probed with specific anti-C. d. terrificus venom horse IgG. One epitope of crotamine (F12PKEKICLPPSSDFGKMDCRW32) and three of crotoxin (L10LVGVEGHLLQFNKMIKFETR30; Y43CGWGGRGRPKDATDRCCFVH63 and T118YKYGYMFYPDSRCRGPSETC138) were identified. After synthesis in their soluble form, the peptides mixture correspondent to the mapped epitopes was entrapped in liposomes and used as immunogens for antibody production in rabbits. Anti-synthetic peptide antibodies were able to protect mice from the lethal activity of C. d. terrificus venom.
Asunto(s)
Crotalus/inmunología , Epítopos/inmunología , Liposomas , Venenos de Serpiente/inmunología , Secuencia de Aminoácidos , Anafilaxia/inmunología , Anafilaxia/prevención & control , Animales , Antivenenos/administración & dosificación , Antivenenos/inmunología , Crotoxina/química , Crotoxina/inmunología , Modelos Animales de Enfermedad , Mapeo Epitopo , Epítopos/administración & dosificación , Epítopos/química , Femenino , Inmunoglobulina G/inmunología , Ratones , Modelos Moleculares , Pruebas de Neutralización , Péptidos/química , Péptidos/inmunología , Conformación Proteica , Conejos , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
Loxosceles spiders are found in almost all countries of South America. In Peru, Loxosceles laeta species is the main responsible for the accidents caused by poisonous animals, being known as "killer spiders", due to the large number of fatal accidents observed. Astacin-like metalloproteases, named LALPs (Loxosceles astacin-like metalloproteases) are highly expressed in Loxosceles spiders venom gland. These proteases may be involved in hemorrhage and venom spreading, being relevant to the envenoming proccess. Thus, the aim of this work was to analyze Peruvian L. laeta venom gland transcripts using bioinformatics tools, focusing on LALPs. A cDNA library from Peruvian L. laeta venom glands was constructed and sequenced by MiSeq (Illumina) sequencer. After assembly, the resulting sequences were annotated, seeking out for similarity with previously described LALPs. Nine possible LALPs isoforms from Peruvian L. laeta venom were identified and the results were validated by in silico and in vitro experiments. This study contributes to a better understanding of the molecular diversity of Loxosceles venom and provide insights about the action of LALPs.