Transcriptome analysis of Loxosceles laeta (Araneae, Sicariidae) spider venomous gland using expressed sequence tags

<p>Abstract</p> <p>Background</p> <p>The bite of spiders belonging to the genus <it>Loxosceles </it>can induce a variety of clinical symptoms, including dermonecrosis, thrombosis, vascular leakage, haemolysis, and persistent inflammation. In order to examine the transcripts expressed in venom gland of <it>Loxosceles laeta </it>spider and to unveil the potential of its products on cellular structure and functional aspects, we generated 3,008 expressed sequence tags (ESTs) from a cDNA library.</p> <p>Results</p> <p>All ESTs were clustered into 1,357 clusters, of which 16.4% of the total ESTs belong to recognized toxin-coding sequences, being the Sphingomyelinases D the most abundant transcript; 14.5% include "possible toxins", whose transcripts correspond to metalloproteinases, serinoproteinases, hyaluronidases, lipases, C-lectins, cystein peptidases and inhibitors. Thirty three percent of the ESTs are similar to cellular transcripts, being the major part represented by molecules involved in gene and protein expression, reflecting the specialization of this tissue for protein synthesis. In addition, a considerable number of sequences, 25%, has no significant similarity to any known sequence.</p> <p>Conclusion</p> <p>This study provides a first global view of the gene expression scenario of the venom gland of <it>L. laeta </it>described so far, indicating the molecular bases of its venom composition.</p

Premolis semirufa (Walker, 1856) Envenomation, Disease Affecting Rubber Tappers of the Amazon: Searching for Caterpillar-Bristles Toxic Components

Pararama, the popular name of the larval form of the moth Premolis semirufa inhabits rubber plantations in the Amazon region and the accidental contact of the skin with the caterpillar's bristles or cocoons results in immediate and intense heat, pain, edema, and itching. In many cases a chronic inflammatory reaction with immobilization of the joints occurs. The current study has evaluated the biological and immunochemical characteristics of the Pararama caterpillar bristles extract. Electrophoretic analysis showed the presence of several components, including a very intense 82 kDa band. This latter component was endowed with intense gelatinolytic activity, as observed in zymography assays. Further analysis revealed that the extract also contained hyaluronidase activity but is devoid of phospholipase A2 activity. In vivo assays, using mice, showed that the extract was not lethal, but caused significant edema and induced intense infiltration of inflammatory cells to the envenomation site. The extract also induced high specific antibody titers, but no autoantibodies were detected. The data obtained, so far, demonstrate the existence of a mixture of different enzymes in the bristles of Premolis semirufa caterpillar, which can act together in the generation and development of the clinical manifestations of the Pararama envenomation

Characterization of a gene coding for the complement system component FB from loxosceles laeta spider venom glands

The human complement system is composed of more than 30 proteins and many of these have conserved domains that allow tracing the phylogenetic evolution. The complement system seems to be initiated with the appearance of C3 and factor B (FB), the only components found in some protostomes and cnidarians, suggesting that the alternative pathway is the most ancient. Here, we present the characterization of an arachnid homologue of the human complement component FB from the spider Loxosceles laeta. This homologue, named Lox-FB, was identified from a total RNA L. laeta spider venom gland library and was amplified using RACE-PCR techniques and specific primers. Analysis of the deduced amino acid sequence and the domain structure showed significant similarity to the vertebrate and invertebrate FB/C2 family proteins. Lox-FB has a classical domain organization composed of a control complement protein domain (CCP), a von Willebrand Factor domain (vWFA), and a serine protease domain (SP). The amino acids involved in Mg2+ metal ion dependent adhesion site (MIDAS) found in the vWFA domain in the vertebrate C2/FB proteins are well conserved; however, the classic catalytic triad present in the serine protease domain is not conserved in Lox-FB. Similarity and phylogenetic analyses indicated that Lox-FB shares a major identity (43%) and has a close evolutionary relationship with the third isoform of FB-like protein (FB-3) from the jumping spider Hasarius adansoni belonging to the Family Salcitidae

C5a receptor is cleaved by metalloproteases induced by sphingomyelinase D from Loxosceles spider venom

Neutrophils are involved in numerous pathologies and are considered to be major contributors to the establishment of cutaneous loxoscelism after envenomation by the Loxosceles spider. Neutrophils are attracted to the site of envenomation by locally generated C5a and contribute to the tissue destruction. We have investigated the effects of this spider venom on the receptor for C5a: C5aR/CD88, a seven transmembrane G-protein coupled receptor. We show here that the Loxosceles venom induces the cleavage of the C5aR at two sites, resulting in the release of the extracellular N-terminus, while retaining part of the transmembrane regions. Using specific inhibitors, it was shown that the cleavage was induced by activation of an endogenous metalloprotease of the adamalysin (ADAM) family, which was activated by the sphingomyelinase D in the venom. Although it resulted in the near complete loss of the N-terminus, C5a was still able to induce a small increase in intracellular calcium and increase secretion of IL-8. The cleavage of the C5aR may well be a protective response after envenomation, rather than contributing to the pathology of Loxosceles envenomation and may represent a general mechanism for how the body protects itself against excess C5a generation in pathological circumstances such as sepsis

Effect of tetracycline on the cell viability and the expression of MMPs in human keratinocytes treated with <i>L</i>. <i>laeta</i> venom.

<p>HaCaT cell cultures were incubated with 100 μg/mL of <i>L</i>. <i>laeta</i> venom (V) or control medium (C) in the presence or absence of tetracycline (T). After 72 hours of treatment, cell viability was analyzed by MTT assay <b>(A)</b> and cell supernatants were analyzed by zymography <b>(B),</b> western blotting <b>(C)</b> and Luminex assay (tetracycline: 150 μg/mL) <b>(D).</b> Results are representative of three independent experiments and are expressed as the mean of triplicates ± standard deviation. Significant differences (*) <i>P</i><0.05; (**) <i>P</i><0.01 and (***) <i>P</i><0.001 from control or (^#) <i>P</i><0.05; (^##) <i>P</i><0.01 and (^###) <i>P</i><0.001 from venom treated cells.</p

<i>L</i>. <i>laeta</i> venom and Class 1 SMase D induce the expression of matrix metaloproteinases in human keratinocytes.

<p><i>Zymography analysis</i>: HaCaT cell culture supernatants collected after 72 hours of the treatment with <i>L</i>. <i>laeta</i> venom <b>(A)</b> or 48 hours with the SMase D Class I (SMase I) <b>(B)</b> were run on gelatin containing 10% SDS-PAGE gels under non-reducing conditions. Control supernatants were harvested after 48 and 72 hours from cells incubated with medium plus saline. <b>(C)</b> <i>Western blot analysis</i>: HaCaT cell culture supernatants, collected after 24, 48 72 hours of the treatment with medium plus saline (C), venom or SMases D from Class 1 (SMase I from <i>L</i>. <i>laeta</i>) were run on 12.5% SDS-PAGE gels, blotted and developed using MoAbs against human MMP7. Figures are representative of three independent experiments.</p

Effect of <i>L</i>. <i>laeta</i> venom and Class 1 SMase D on human keratinocytes cell viability.

<p>HaCaT cell cultures (2x10⁴ cells) were incubated with increasing concentrations (25 to 200 μg/ml) of <i>Loxosceles</i> venom <b>(A)</b> or SMase I <b>(B)</b>. After 24, 48 and 72 hours of treatment, cell viability was analyzed by MTT assay. Results are representative of three independent experiments and expressed as the mean of triplicates ± standard deviation. Significant differences (***) <i>P</i><0.001 from control.</p