Micrurus snake venoms activate human complement system and generate anaphylatoxins

Background The genus Micrurus, coral snakes (Serpentes, Elapidae), comprises more than 120 species and subspecies distributed from the south United States to the south of South America. Micrurus snake bites can cause death by muscle paralysis and further respiratory arrest within a few hours after envenomation. Clinical observations show mainly neurotoxic symptoms, although other biological activities have also been experimentally observed, including cardiotoxicity, hemolysis, edema and myotoxicity. Results In the present study we have investigated the action of venoms from seven species of snakes from the genus Micrurus on the complement system in in vitro studies. Several of the Micrurus species could consume the classical and/or the lectin pathways, but not the alternative pathway, and C3a, C4a and C5a were generated in sera treated with the venoms as result of this complement activation. Micrurus venoms were also able to directly cleave the α chain of the component C3, but not of the C4, which was inhibited by 1,10 Phenanthroline, suggesting the presence of a C3α chain specific metalloprotease in Micrurus spp venoms. Furthermore, complement activation was in part associated with the cleavage of C1-Inhibitor by protease(s) present in the venoms, which disrupts complement activation control. Conclusion Micrurus venoms can activate the complement system, generating a significant amount of anaphylatoxins, which may assist due to their vasodilatory effects, to enhance the spreading of other venom components during the envenomation process

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

Human complement activation and anaphylatoxins generation induced by snake venom toxins from Bothrops genus

Snake venoms are a complex mixture of components, which have a wide range of actions both on prey and human victims. The genus Bothrops causes the vast majority of snakebites in Central and South America, being responsible for 80% of snake envenomations in Brazil. Envenomations are characterized by prominent local effects, including oedema, haemorrhage and necrosis, which can lead to permanent disability. Systemic manifestations such as haemorrhage, coagulopathy, shock and acute renal failure may also occur. In the present study we have investigated the action of venoms from 19 species of snakes from the genus Bothrops, occurring in Brazil, on the complement system in in vitro studies. All venoms were able to activate the classical complement pathway, in the absence of sensitizing antibody. This activation was in part associated with the cleavage of C1-Inhibitor by proteases present in these venoms, which disrupts complement activation control. No modification of the membrane bound complement regulators, such as DAF, CR1 and CD59 was detected, after treatment of human erythrocytes with the snake venoms. Some of the Bothrops venoms were also able to activate alternative and lectin pathways, as measured in haemolytic and ELISA assays. C3a, C4a and C5a were generated in sera treated with the venoms, not only through C-activation, but also by the direct cleavage of complement components, as determined using purified C3 and C4. Metallo- and/or serine-protease inhibitors prevented cleavage of C3 and C4. These results suggest that Bothrops venoms can activate the complement system, generating a large amount of anaphylatoxins, which may play an important role in the inflammatory process presented in humans after snake envenomations, and they may also assist, due to their vasodilatory effects, to enhance the spreading of other venom components

Molecular model of Lox-FB.

<p>Construction of molecular model based on structure of human factor B (PDB 2ok5.1), using the tool SWISS-Model Workspace available on <a href="http://swissmodel.expasy.org/workspace/" target="_blank">http://swissmodel.expasy.org/workspace/</a>. The further analyses were performed using the software SwissPDBViewer.</p

Multiple alignment of the vWFA and serine protease domains from Lox-FB with sequences of FB/C2 proteins from other organisms.

<p>The alignment was performed using MUSCLE algorithm available in MEGA software. Amino acids that are highlighted in bold indicate identical regions. The amino acids residues that are functionally important at Factor D or C1s cleavage site; the metal ion dependent binding site (MIDAS) and the protease active sites are indicated by dark arrows; the three amino acids residues (T⁴³¹, A⁴³³, S⁶¹⁶) that are important on stabilization of catalytic triad are indicated by sign #; the conserved cysteines residues are indicated by asterisks and the two extra cysteines present in Lox-FB, Hd-FB3 and Rd-FB are highlighted in grey. <i>Loxosceles laeta</i> (Lox-FB), <i>Hasarius adansoni</i> (Hd-FB1; HD-FB2; Hd-FB3), <i>Tachypleus tridentatus</i> (Tt-FB1;Tt-FB-2), <i>Ruditapes decussatus</i> (Rd-FB), <i>Nematostella vectensis</i> (Nv-FB), <i>Branchiostoma belcheri</i> (Bb-FB), <i>Strongylocentrotus purpuratus</i> (Sp-FB), <i>Apostichopus japonicus</i> (Aj-FB), <i>Homo sapiens</i> (Hu-C2;Hu-FB).</p

Alignment of Hu-FB and Lox-FB CCPs.

<p>The five CCPs are aligned to each other with the consensus amino acids shown in bold and at the bottom. Two disulfide bonds sustain the CCP domain and are formed between the first and third cysteine, and the second and the fourth cysteine. The alignment was done with CLUSTAL W using Bioedit v. 7.0.9.0 software.</p

Domain structure of Lox-FB deduced protein using Basic Local Alignment Tool (BLAST).

<p>Domain structure of Lox-FB deduced protein using Basic Local Alignment Tool (BLAST).</p