Investigations into the digestive system proteases of Argiope aurantia (Araneae: Araneidae), hemolytic agents of spider venoms, and the role of both in necrotic arachnidism

Abstract

Necrotic arachnidism, or tissue necrosis following spider bites is a widespread problem. Knowledge of the exact mechanism that produces such lesions is still incomplete. Here I have examined spider digestive fluid proteases and spider venom enzymes (e.g., sphingomyelinase D and phospholipase A2) for their potential to cause necrotic lesions, as both are thought to be primary agents. The digestive fluid of Argiope aurantia was examined for its ability to cleave a variety of extra-cellular matrix proteins, including collagen, elastin, and fibronectin. Having confirmed that the fluid has collagenases, the digestive fluid was injected into the skin of rabbits to observe whether it would cause necrotic lesions. It did not. The data do not support the suggestions that spider digestive collagenases have a primary role in spider bite necrosis. In an effort to design a simple, inexpensive screening method for identifying spiders with necrotizing venoms, venom gland homogenates of a variety of spider species were examined for their ability to cause hemolysis. Those venoms that were positive were further examined for the presence of sphingomyelinase D, and the ability to evoke necrotic lesions in the skin of rabbits. Of 45 species examined, only the venom of Loxosceles reclusa and Cheiracanthium mildei caused hemolysis. Unlike L. reclusa venom, however, C. mildei venom did not possess sphingomyelinase D nor did it cause necrotic lesions in the skin of rabbits. In an effort to determine the hemolytic agent in C. mildei venom, I found evidence for a phospholipase A2. Through a combination of ion exchange chromatography, size exclusion chromatography, ultracentrifugation, and electrophoresis, an attempt was made to isolate several digestive fluid proteases, and seine protease inhibitors in milligram quantities from the digestive fluid of Argiope aurantia . The proteases, however, eluted anomalously during the size exclusion chromatography and ultracentrifugation trials. I have hypothesized that spider proteases can undergo a reversible aggregation to account for this behavior. I was able to isolate several proteases in quantities sufficient for N-terminal amino acid sequences. These data revealed that spider proteases are related to astacins and meprins, which are in the astacin family of metalloproteases