Bites byEnvenomation by Loxosceles spiders, endemic to temperate and (sub)tropical regions of the Americas, Africa, and Europe, can lead to local skin injury as well as to serious systemic toxicity, including thrombus formation, vascular leakage, hemolysis, and persistent inflammation (1-3). In severe cases, the hematologic complications can lead to renal failure and death, especially in children (2, 3). Treatment is difficult; antivenoms are not very effective, and the use of corticosteroids or anti-inflammatory medication is controversial (3). The toxin responsible for the local and systemic effects of Loxosceles venom is an unusual sphingomyelinase D (SMaseD) 1 that converts sphingomyelin (SM) in the outer leaflet of the plasma membrane to ceramide 1-phosphate (N-acylsphingosine 1-phosphate) (4 -7). Strikingly, while SMaseD is not found elsewhere in the animal kingdom, a similar enzyme is produced as an exotoxin by some pathogenic bacteria, notably Corynebacterium pseudotuberculosis, Corynebacterium ulcerans, and Arcanobacterium (formerly Corynebacterium) hemolyticum (8 -10). C. pseudotuberculosis causes lymphadenitis in animals but is also pathogenic for humans, while C. ulcerans and A. hemolyticum are pathogens of pharyngitis and other human infections (11); in no case is the molecular basis for virulence known (12). The SMaseD from C. pseudotuberculosis, also named SM-specific phospholipase D (PLD), is an essential virulence determinant that contributes to the persistence and spread of the bacteria within the host (13). The Loxosceles and C. pseudotuberculosis SMases D have the same molecular mass (31-32 kDa) and share about 30% sequence similarity (see "Results"). In model systems, the spider and bacterial enzymes provoke remarkably similar pathophysiological effects, including platelet aggregation, endothelial hyperpermeability, complement-dependent hemolysis, and neutrophil-dependent skin necrosis (4 -7, 9, 14 -16).Despite decades of study it remains unclear how SMaseD can elicit such a wide variety of biological effects, particularly, since ceramide 1-phosphate is not known as a signaling molecule. In contrast to ceramide, which may reorganize lipid microdomains and associated signaling complexes (17, 18), ceramide 1-phosphate is a bilayer-preferring phospholipid that is unlikely to significantly perturb membrane structure. Furthermore, mammalian cells treated with SMaseD from either Loxosceles deserta or C. pseudotuberculosis do not convert newly formed ceramide 1-phosphate to ceramide nor does SMaseD treatment affect membrane permeability or cell viability (19,20).Given the lack of understanding of SMaseD bioactivity, we set out to re-examine the substrate specificity and cellular effects of the enzyme. Our interest was stirred by a report of more than 30 years ago, showing that partially purified SMaseD from C. pseudotuberculosis (ovis) can catalyze the release of choline from lysophosphatidylcholine (LPC) but not from phosphatidylcholine (PC) (21). LPC is an abundant plasma component and removal ...

Background: The bite of spiders belonging to the genus Loxosceles 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 Loxosceles laeta 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.

Sphingomyelinases D (SMases D) fromLoxosceles spider venom are the principal toxins responsible for the manifestation of dermonecrosis, intravascular hemolysis, and acute renal failure, which can result in death. These enzymes catalyze the hydrolysis of sphingomyelin, resulting in the formation of ceramide 1-phosphate and choline or the hydrolysis of lysophosphatidyl choline, generating the lipid mediator lysophosphatidic acid. This report represents the first crystal structure of a member of the sphingomyelinase D family from Loxosceles laeta (SMase I), which has been determined at 1.75-Å resolution using the "quick cryo-soaking" technique and phases obtained from a single iodine derivative and data collected from a conventional rotating anode x-ray source. SMase I folds as an (␣/␤) 8 barrel, the interfacial and catalytic sites encompass hydrophobic loops and a negatively charged surface. Substrate binding and/or the transition state are stabilized by a Mg 2؉

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