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Sphingomyelinases D (SMases D) or dermonecrotic toxins are well characterized in Loxosceles spider venoms and have been described in some strains of pathogenic microorganisms, such as Corynebacterium sp. After spider bites, the SMase D molecules cause skin necrosis and occasional severe systemic manifestations, such as acute renal failure. In this paper, we identified new SMase D amino acid sequences from various organisms belonging to 24 distinct genera, of which, 19 are new. These SMases D share a conserved active site and a C-terminal motif. We suggest that the C-terminal tail is responsible for stabilizing the entire internal structure of the SMase D Tim barrel and that it can be considered an SMase D hallmark in combination with the amino acid residues from the active site. Most of these enzyme sequences were discovered from fungi and the SMase D activity was experimentally confirmed in the fungus Aspergillus flavus. Because most of these novel SMases D are from organisms that are endowed with pathogenic properties similar to those evoked by these enzymes alone, they might be associated with their pathogenic mechanisms.

A chimeric protein was constructed expressing three epitopes of LiD1, a dermonecrotic toxin from the venom of Loxosceles intermedia spider. This species is responsible for a large number of accidents involving spiders in Brazil. We demonstrated that the chimeric protein (rCpLi) generated is atoxic and that antibodies previously developed in rabbits against synthetic epitopes reactive with rCpLi in ELISA and immunoblot assays. The antibody response in rabbits against the rCpLi was evaluated by ELISA and we have detected an antibody response in all immunized animals. Overlapping peptides covering the amino acid sequence of the rCpLi were synthesized on a cellulose membrane, and their recognition by rabbit anti-rCpLi serum assessed. Three different antigenic regions were identified. The percentage of inhibition of the dermonecrotic, hemorrhagic and edematogenic activities caused by the recombinant protein LiD1r in naïve rabbits was assessed by pre-incubation with anti-rCpLi antibodies. Anti-rCpLi induced good dermonecrotic and hemorrhagic protection. The levels of protection were similar to the antiboides anti-LiD1r. In summary, we have developed a polyepitope recombinant chimeric protein capable of inducing multiple responses of neutralizing antibodies in a rabbit model. This engineered protein may be a promising candidate for therapeutic serum development or vaccination.

Loxosceles spider bites cause many human injuries worldwide. Injections in mice of whole Loxosceles (L.) intermedia venom or a recombinant toxin (rLiD1) produce systemic symptoms similar to those detected in envenomed humans. This animal model was used to characterize the effects of Loxosceles intermedia venom in cardiac tissues. L. intermedia antigens were detected by ELISA in kidney, heart, lung and liver of experimentally envenomed mice. In addition, rLiD1 binding to cardiomyocytes was demonstrated by immunofluorescence and confocal microscopy. Furthermore, isolated perfused heart preparations and ventricular cardiomyocytes from envenomed mice showed heart function impairment, and a significant increase of I(Ca,L) density and intracellular Ca(2+) transients, respectively. Thus, L. intermedia spider venom, as shown through the use of the recombinant toxin rLiD1, causes cardiotoxic effects and a protein from the sphingomyelinase D family plays a key role in heart dysfunction. Thus, L. intermedia spider venom and the Loxtox rLiD1 play a key role in heart dysfunction.

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