An examination of the potential role of spider digestive proteases as a causative factor in spider bite necrosis

Tissue necrosis following spider bites is a widespread problem. In the continental United States, the brown recluse (Loxosceles reclusa), hobo spider (Tegenaria agrestis), garden spider (Argiope aurantia) and Chiracanthium species, among others, reportedly cause such lesions. The exact mechanism producing such lesions is controversial. There is evidence for both venom sphingomyelinase and spider digestive collagenases. We have examined the role of spider digestive proteases in spider bite necrosis. The digestive fluid of A. aurantia was assayed for its ability to cleave a variety of connective tissue proteins, including collagen. 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.     

Screening of Necrotizing Spiders in Korea Using Nonradioactive Sphingomyelinase Assay (I) Wandering Spiders

There are now more than 40,000 identified spider species in the world, and considered about 100 species as actually dangerous to human. Spider bites cause a range of symptoms from simple swellings to disfiguring necrotic lesions, and occasionally death. While spider bites are not a major medical problem in Korea, it would be of great value to know which species of spiders pose a threat to human health. A middle molecular weight protein, sphingomyelinase D, has been identified in the venom of the brown recluse spider and strong evidence suggests that they have a major role in spider bite necrosis. For the identification of necrotizing species, we have investigated using recently developed non‐radioactive assay of sphingomyelinase for rapidly screening the necrotizing venoms. Here, we demonstrate the fetal toxicity of total 57 species (32 genera, 9 families) of the wandering spiders among 622 identified spider species in Korea. It has been revealed that two species of the Thomisidae spider, Ozyptila nongae (0.2467) and Diaea subdola (0.2020) have the strongest sphingomyelinase activities among themselves. In addition one species of the family Pisauridae, Dolomedes sulfureus (0.2341) has also relatively higher value comparing to other wandering spiders. However comparing to that of the brown recluse spider, Loxosceles reclusa (1.814) in North America the necrotizing activities of these Korean wandering species are still very low state, so there seems to be little possibilities to create serious medical problems by the necrotizing arachnidism in Korean peninsula.     

Screening of Necrotizing Spiders in Korea Using Nonradioactive Sphingomyelinase Assay(II) Web-building Spiders

Spider bites cause a range of symptoms from simple swellings to disfiguring necrotic lesions, and occasionally death. While spider bites are not a major medical problem in Korea, it would be of great value to know which species of spiders pose a threat to human health. A middle molecular weight protein, sphingomyelinase D, has been identified in the venom of the brown recluse spider and strong evidence suggests that they have a major role in spider bite necrosis. For the identification of necrotizing species, we have investigated using recently developed non‐radioactive assay of sphingomyelinase for rapidly screening the necrotizing venoms. Here, we demonstrate the fetal toxicity of total 65 species (24 genera, 9 families) of the web‐building spiders among 622 identified spider species in Korea. It has been revealed that four species of the orb‐weaving spider, Araneus ventricosus (family Araneidae, 0.3509), Dipoena castrata (0.2413, family Theridiidae), Argiope minuta (0.1836, family Araneidae), and Paracoelotes spinivulva (0.1760, family Amaurobiidae) have relatively strong activities among themselves. However, comparing to that of the brown recluse spider, Loxosceles recluse (1.814) in North America, the necrotizing shpingomyelinase activities of these Korean web‐building spiders are still very low. Based on our results, we may thus conclude that there would be little possibilities in South Korea to create serious medical problems caused by necrotizing arachnidism.     

Red blood cell lysis induced by the venom of the brown recluse spider: the role of sphingomyelinase D.

Red blood cell lysis induced by the venom of Loxosceles reclusa, the brown recluse spider, may be related to the hemolytic anemia observed in several cases of spider envenomation. These investigations demonstrate that the venom of the brown recluse spider contains a calcium-dependent, heat-labile hemolysin of molecular weight approximately 19,000. The pH optimum for the hemolytic reaction was 7.1, and the optimum calcium concentration for venom-induced lysis was observed within the range of 6 to 10 mm. Sheep red blood cells were more susceptible to the spider hemolysin than human red blood cells, although both types exhibited appreciable lysis. Digestion of sheep red blood cell membranes with partially purified venom lysin resulted in degradation of the sphingomyelin component. However, reaction of the membranes with the venom lysin produced no release of water-soluble phosphate, and no free fatty acids were generated. These results indicate that the sphingomyelin-degrading activity of the venom is not a phospholipase C- or a phospholipase A₂-type activity. Sphingomyelin was employed as substrate for the venom hemolysin, and the organic and aqueous fractions of the reaction mixtures were analyzed by thin-layer chromatography. Analysis of the organic fraction revealed a phosphate-containing product with the solubility and chromatographic characteristics of N-acylsphingosine phosphate (ceramide phosphate), and analysis of the aqueous fraction demonstrated the presence of choline. The isolation and identification of these products indicate that the sphingomyelin of the red cell membrane is hydrolyzed by a sphingomyelinase D-type activity expressed by the partially purified venom hemolysin. A close correspondence between the hemolytic and sphingomyelinase D activities was observed when the partially purified hemolysin was further characterized in polyacrylamide gel electrophoresis at pH 8.3 and pH 4.9. The hemolytic and sphingomyelinase activities were coincident within the electrophoretic pattern at both pHs. The results presented demonstrate conclusively a direct lytic action of brown recluse venom upon red blood cells and report for the first time the presence of sphingomyelinase D in spider venom.     

Lateral gene transfer of a dermonecrotic toxin between spiders and bacteria

MOTIVATION: Spiders in the genus Loxosceles, including the notoriously toxic brown recluse, cause severe necrotic skin lesions owing to the presence of a venom enzyme called sphingomyelinase D (SMaseD). This enzyme activity is unknown elsewhere in the animal kingdom but is shared with strains of pathogenic Corynebacteria that cause various illnesses in farm animals. The presence of the same toxic activity only in distantly related organisms poses an interesting and medically important question in molecular evolution. Results: We use superpositions of recently determined structures and sequence comparisons to infer that both bacterial and spider SMaseDs originated from a common, broadly conserved domain family, the glycerophosphoryl diester phosphodiesterases. We also identify a unique sequence/structure motif present in both SMaseDs but not in the ancestral family, supporting SMaseD origin through a single divergence event in either bacteria or spiders, followed by lateral gene transfer from one lineage to the other.     

Venom of the Brazilian Spider Sicarius ornatus (Araneae,Sicariidae) Contains Active Sphingomyelinase D: Potential for Toxicity after Envenomation

Background

The spider family Sicariidae includes two genera, Sicarius and Loxosceles. Bites by Sicarius are uncommon in humans and, in Brazil, a single report is known of a 17-year old man bitten by a Sicarius species that developed a necrotic lesion similar to that caused by Loxosceles. Envenomation by Loxosceles spiders can result in dermonecrosis and severe ulceration. Sicarius and Loxosceles spider venoms share a common characteristic, i.e., the presence of Sphingomyelinases D (SMase D). We have previously shown that Loxosceles SMase D is the enzyme responsible for the main pathological effects of the venom. Recently, it was demonstrated that Sicarius species from Africa, like Loxosceles spiders from the Americas, present high venom SMase D activity. However, despite the presence of SMase D like proteins in venoms of several New World Sicarius species, they had reduced or no detectable SMase D activity. In order to contribute to a better understanding about the toxicity of New World Sicarius venoms, the aim of this study was to characterize the toxic properties of male and female venoms from the Brazilian Sicarius ornatus spider and compare these with venoms from Loxosceles species of medical importance in Brazil.

Methodology/Principal Findings

SDS-PAGE analysis showed variations in the composition of Loxosceles spp. and Sicarius ornatus venoms. Differences in the electrophoretic profiles of male and female venoms were also observed, indicating a possible intraspecific variation in the composition of the venom of Sicarius spider. The major component in all tested venoms had a Mr of 32–35 kDa, which was recognized by antiserum raised against Loxosceles SMases D. Moreover, male and female Sicarius ornatus spiders'' venoms were able to hydrolyze sphingomyelin, thus showing an enzymatic activity similar to that determined for Loxosceles venoms. Sicarius ornatus venoms, as well as Loxosceles venoms, were able to render erythrocytes susceptible to lysis by autologous serum and to induce a significant loss of human keratinocyte cell viability; the female Sicarius ornatus venom was more efficient than male.

Conclusion

We show here, for the first time, that the Brazilian Sicarius ornatus spider contains active Sphingomyelinase D and is able to cause haemolysis and keratinocyte cell death similar to the South American Loxosceles species, harmful effects that are associated with the presence of active SMases D. These results may suggest that envenomation by this Sicarius spider has the potential to cause similar pathological events as that caused by Loxosceles envenomation. Our results also suggest that, in addition to the interspecific differences, intraspecific variations in the venoms composition may play a role in the toxic potential of the New World Sicarius venoms species.     

Predation by cosmopolitan spiders upon the medically significant pest species Loxosceles reclusa (Araneae: Sicariidae): limited possibilities for biological control

Interspecific predation of three cosmopolitan house spiders, Achearanea tepidariorum (Kock 1841) (Theridiidae), Steotoda triangulosa (Walckenaer 1802) (Theridiidae), and Pholcus phalangioides (Doleschall 1859) (Pholcidae), and the medically significant brown recluse spider, Loxosceles reclusa (Sicariidae) were examined to evaluate transitive predatory relationships and to explore the potential use of cosmopolitan spiders as effective biological control agents on L. reclusa. Fifty houses from northeastern Kansas were visually inspected from May to December 2002 for cosmopolitan spiders and L. reclusa. In 25 houses, insect monitoring traps were used to sample spider diversity and abundance. The remaining 25 houses were monitored to examine intraguild predation and spider behavior. If cosmopolitan spiders have the ability to regulate or decrease L. reclusa populations, houses with large cosmopolitan spider populations are expected to have significantly fewer L. reclusa than houses without cosmopolitan spiders. Predation and/or evidence of predation by all three cosmopolitan spiders on L. reclusa was detected in 68% of houses. Spearman's rank correlation analysis showed overall positive relationships between population densities of cosmopolitan spiders and L. reclusa. When evaluated independently, the presence of both A. tepidariorum and S. triangulosa showed negative, yet nonsignificant, relationships with L. reclusa densities, whereas P. phalangioides showed a positive nonsignificant relationship. Although statistical tests showed a decrease in L. reclusa population densities with increased population densities of two cosmopolitan species, alluding to a potential beneficial interaction for biological control, observations of spider behavior, web positioning (niche partitioning), and predation showed little possibility of biological control capabilities.     

The phylogenetic distribution of sphingomyelinase D activity in venoms of Haplogyne spiders

The venoms of Loxosceles spiders cause severe dermonecrotic lesions in human tissues. The venom component sphingomyelinase D (SMD) is a contributor to lesion formation and is unknown elsewhere in the animal kingdom. This study reports comparative analyses of SMD activity and venom composition of select Loxosceles species and representatives of closely related Haplogyne genera. The goal was to identify the phylogenetic group of spiders with SMD and infer the timing of evolutionary origin of this toxin. We also preliminarily characterized variation in molecular masses of venom components in the size range of SMD. SMD activity was detected in all (10) Loxosceles species sampled and two species representing their sister taxon, Sicarius, but not in any other venoms or tissues surveyed. Mass spectrometry analyses indicated that all Loxosceles and Sicarius species surveyed had multiple (at least four to six) molecules in the size range corresponding to known SMD proteins (31-35 kDa), whereas other Haplogynes analyzed had no molecules in this mass range in their venom. This suggests SMD originated in the ancestors of the Loxosceles/Sicarius lineage. These groups of proteins varied in molecular mass across species with North American Loxosceles having 31-32 kDa, African Loxosceles having 32-33.5 kDa and Sicarius having 32-33 kDa molecules.     

Hemolytic potency and phospholipase activity of some bee and wasp venoms

1. The action of crude venoms of four aculeate species: Apis mellifera, Vespa crabro, Vespula germanica and Vespula vulgaris on human erythrocytes was investigated in order to determine the lytic and phospholipase activity of different aculeate venoms and their ability to induce red blood cell hemolysis. 2. Bee venom was the only extract to completely lyse red blood cells at the concentration of 2-3 micrograms/ml. 3. Phospholipase activity in all of the examined vespid venoms was similar and the highest value was recorded in V. germanica. 4. Vespid venoms exhibited phospholipase B activity, which is lacking in honeybee venom. 5. In all membrane phospholipids but lecithin, lysophospholipase activity of vespid venoms was 2-6 times lower than the relevant phospholipase activity. 6. The incubation of red blood cells with purified bee venom phospholipase A2 was not accompanied by lysis and, when supplemented with purified melittin, the increase of red blood cell lysis was approximately 30%.     

Studies of the necrotic actions of the venoms of several Australian spiders.

1. Raw venoms from a number of Australian Araneomorph spiders were found to cause epidermal disruption in cultured skin from both mice and humans. 2. The more potent ones also caused loss of epidermal cell-cell adhesion of mouse skin in vivo. 3. Raw venoms from three Mygalomorph species did not have these actions. 4. Venom gland extracts from the Araneomorph species were also ineffective. 5. It was concluded that where spider venoms appear to possess necrogenic activity the most likely reason for this is contamination of the venoms with digestive tract secretions.     

Platelet aggregation and sphingomyelinase D activity of a purified toxin from the venom of Loxosceles reclusa

A facile and quantitative assay for measuring the activity of sphingomyelinase D in recluse spider venom has been developed using L-alpha-[palmitoyl-1-14C]lysophosphatidylcholine as substrate. This assay avoids the problem of substrate insolubility that occurs when sphingomyelin and other insoluble lipids are used as substrates. This assay has been employed in gel filtration and isoelectric focusing isolation techniques to purify sphingomyelinase D from spider venom. The purified sphingomyelinase exhibits four active enzyme forms in isoelectric focusing with pI values of 8.7, 8.4, 8.2, and 7.8. Each active form when examined in SDS-polyacrylamide gel electrophoresis gave an estimated molecular weight of 32 000. The four active enzyme forms were immunologically cross-reactive with each other as demonstrated with radioimmune assays using an antiserum developed to one of the active forms. Each active form hydrolysed sphingomyelin to release choline and produce N-acylsphingosine phosphate. One of the active enzyme forms was characterized further in dermonecrosis and platelet aggregation measurements. This purified sphingomyelinase D was identified as a poisonous toxin that can developed typical dermonecrotic spider lesions when injected into experimental animals at levels expected to be delivered in a normal bite. Furthermore, the purified toxin acts to aggregate human blood platelets. The toxin-induced platelet aggregation has been related to serotonin release as aggregation occurs, and it has been shown to be inhibited by EDTA over the range of 0.6 yo 3.0 mM EDTA. It is suggested that spider-induced dermonecrosis could result in part from platelet aggregation at and near the site of envenomation.     

Molecular cloning and functional characterization of two isoforms of dermonecrotic toxin from Loxosceles intermedia (brown spider) venom gland

Brown spider (Genus Loxosceles) bites are normally associated with necrotic skin degeneration, gravitational spreading, massive inflammatory response at injured region, platelet aggregation causing thrombocytopenia and renal disturbances. Brown spider venom has a complex composition containing many different toxins, of which a well-studied component is the dermonecrotic toxin. This toxin alone may produce necrotic lesions, inflammatory response and platelet aggregation. Biochemically, dermonecrotic toxin belongs to a family of toxins with 30-35 kDa characterized as sphingomyelinase-D. Here, employing a cDNA library of Loxosceles intermedia venom gland, we cloned and expressed two recombinant isoforms of the dermonecrotic toxin LiRecDT2 (1062 bp cDNA) and LiRecDT3 (1007 bp cDNA) that encode for signal peptides and complete mature proteins. Phylogenetic tree analysis revealed a structural relationship for these toxins compared to other members of family. Recombinant molecules were expressed as N-terminal His-tag fusion proteins in Escherichia coli and were purified to homogeneity from cell lysates by Ni(2+) chelating chromatography, resulting in proteins of 33.8 kDa for LiRecDT2 and 34.0 kDa for LiRecDT3. Additional evidence for related toxins containing sequence/epitopes identity comes from antigenic cross-reactivity using antibodies against crude venom toxins and antibodies raised with a purified dermonecrotic toxin. Recombinant toxins showed differential functionality in rabbits: LiRecDT2 caused a macroscopic lesion with gravitational spreading upon intradermal injection, while LiRecDT3 evoked transient swelling and erythema upon injection site. Light microscopic analysis of skin biopsies revealed edema, a collection of inflammatory cells in and around blood vessels and a proteinaceous network at the dermis. Moreover, differential functionality for recombinant toxins was also demonstrated by a high sphingomyelinase activity for LiRecDT2 and low activity for LiRecDT3 as well as greater in vitro platelet aggregation and blood vessel permeability induced by LiRecDT2 and residual activity for LiRecDT3. Cloning and expression of two recombinant dermonecrotic toxins demonstrate an intraspecific family of homologous toxins that act in synergism for deleterious activities of the venom and open possibilities for biotechnological applications for recombinant toxins as research tools for understanding the inflammatory response, vascular integrity and platelet aggregation modulators.     

Isotachophoretic and immunological analysis of venoms from sea snakes (Laticauda semifasciata) and brown recluse spiders (Loxosceles reclusa) of different morphology, locality, sex, and developmental stages

Sea snake venom: The venom compositions of sea snakes, Laticauda semifasciata, with different scale patterns were analyzed by isotachophoresis. The comparison showed quantitative rather than qualitative differences. Similarly, L. semifasciata venoms of Philippine and Japanese origins differed only in the quantity of certain proteins. Spider venom: 3. Loxosceles reclusa venom apparatus extract is rich in neutral and acidic proteins but contains relatively small quantities of basic proteins. Differences in venom apparatus extract composition between nymph and adult (male or female) were detected by isotachophoresis. The extracts of male and female venom apparatus were very similar. Extracts of venom apparatus of spiders collected in locations separated by 100 miles were the same.     

Tracking a medically important spider: climate change, ecological niche modeling, and the brown recluse (Loxosceles reclusa)

Most spiders use venom to paralyze their prey and are commonly feared for their potential to cause injury to humans. In North America, one species in particular, Loxosceles reclusa (brown recluse spider, Sicariidae), causes the majority of necrotic wounds induced by the Araneae. However, its distributional limitations are poorly understood and, as a result, medical professionals routinely misdiagnose brown recluse bites outside endemic areas, confusing putative spider bites for other serious conditions. To address the issue of brown recluse distribution, we employ ecological niche modeling to investigate the present and future distributional potential of this species. We delineate range boundaries and demonstrate that under future climate change scenarios, the spider's distribution may expand northward, invading previously unaffected regions of the USA. At present, the spider's range is centered in the USA, from Kansas east to Kentucky and from southern Iowa south to Louisiana. Newly influenced areas may include parts of Nebraska, Minnesota, Wisconsin, Michigan, South Dakota, Ohio, and Pennsylvania. These results illustrate a potential negative consequence of climate change on humans and will aid medical professionals in proper bite identification/treatment, potentially reducing bite misdiagnoses.     

Two new phospholipase D isoforms of Loxosceles laeta: cloning, heterologous expression, functional characterization, and potential biotechnological application

Toxin phospholipases-D present in the venom of Loxosceles spiders is the principal responsible for local and systemic effects observed in the loxoscelism. In this study, we describe the cloning, expression, functional evaluation, and potential biotechnological application of cDNAs, which code for two new phospholipase D isoforms, LIPLD1 and LIPLD2, of the spider Loxosceles laeta. The recombinant protein rLIPLD1 had hydrolytic activity on sphingomyelin and in vitro hemolytic activity on human red blood cells, whereas rLIPLD2 was inactive. The purified recombinant proteins and the venom are recognized by polyclonal anti-rLIPLD1 and rLIPLD2 sera produced in animals and conferred immunoprotection against the venom. These new isoforms reinforce the importance of the multigene family of phospholipases-D present in Loxosceles spiders. A highly immunogenic inactive isoform such as rLIPLD2 raises important expectation for its use as a potential immunogenic inducer of the immunoprotective response to the toxic action of the venom of Loxosceles laeta.     

Platelet aggregation and sphingomyelinase D activity of a purified toxin from the venom of loxosceles reclusa

A facile and quantitative assay for measuring the activity of sphingomyelinase D in recluse spider venom has been developed using L-α-[palmitoyl-1-¹⁴C]lysophosphatidylcholine as substrate. This assay avoids the problem of substrate insolubility that occurs when sphingomyelin and other lipids are used as subtrates. This assay has been employed in gel filtration and isoelectric focusing isolation techniques to purify sphingomyelinase D from spider venom. The purified sphingomyelinase exhibits four active enzyme forms in isoelectric focusing with pI values of 8.7, 8.4., 8.2, and 7.8. Each active form when examined in SDS-polyacrylamide gel electrophoresis gave an estimated molecular weight of 32 000. The four active enzyme forms were immunologically cross-reactive with each other as demonstrated with radioimmune assays using an antiserum developed to one of the active forms. Each active form hydrolysed sphingomyelin to release choline and produce N-acylsphingosine phosphate. One of the active enzyme forms was characterized further in dermonecrosis and platelet aggregation measurements. This purified sphingomyelinase D was identified as a poisonous toxin that can develop the typical dermonecrotic spider lesion when injected into experimental animals at levels expected to be delivered in a normal bite. Furthermore, the purified toxin acts to aggregate human blood platelets. The toxin-induced platelet aggregation has been related to serotonin release as aggregation occurs, and it has been shown to be inhibited by EDTA over the range of 0.6 to 3.0 mM EDTA. It is suggested that spider-induced dermonecrosis could result in part from platelet aggregation at and near the site of envenomation.     

A comparison of polypeptide compositions of individual <Emphasis Type="Italic">Agelena orientalis</Emphasis> spider venoms

Spider venoms are peculiar combinatory libraries of polypeptide molecules that specifically affect various cell targets. However, the question has remained up to now regarding whether the observed diversity of the polypeptides results from the synthesis of the complete library of these molecules by each individual spider or is due to the peculiarity of each zooid producing a limited set of components. We studied the composition of the mixed venom taken from several dozens of zooids of the Central Asian species of the Agelena orientalis and compared it with the venoms of 20 individual spiders of this species. The venoms were qualitatively and quantitatively analyzed by HPLC, mass spectrometry, and amino acid sequencing. It was shown that the individual venoms contain a lesser number of polypeptide components in comparison with the mixed venom and, in addition, differ from each other by the component composition. The set of components produced by single zooids is relatively narrow, and on the whole is a set identical to that of the mixed venom. The polypeptides with a high content in the venom were structurally characterized and compared with the amino acid sequences deduced from the cDNA library of this species.     

Venom from spiders of the genus Hippasa: biochemical and pharmacological studies

The venoms from female spiders of the genus Hippasa namely H. partita, H. agelenoides and H. lycosina are compared for biochemical and pharmacological properties. SDS-PAGE pattern revealed varied protein composition. Marked variability is seen with casein hydrolyzing enzymes in SDS-PAGE zymogram. H. partita venom was the only venom that hydrolyzed gelatin while the other two venoms did not. The venoms shared similar hyaluronidase activity, showing a single activity band in SDS-PAGE zymogram. The PLA2 activity varied as H. partita>H. agelenoides>H. lycosina venoms. Marked differences were noted in the ability to induce edema, cytotoxicity, myotoxicity and neurotoxicity, while hemorrhage was associated exclusively with H. partita venom.     

Molecular cloning and expression of a functional dermonecrotic and haemolytic factor from Loxosceles laeta venom

The bite of spiders of the genus Loxosceles can induce a variety of biological effects, including dermonecrosis and complement-dependent haemolysis. The aim of this study was to generate recombinant proteins from the Loxosceles spider gland to facilitate structural and functional studies in the mechanisms of loxoscelism. Using "Expressed Sequencing Tag" strategy of aleatory clones from, L. laeta venom gland cDNA library we have identified clones containing inserts coding for proteins with significant similarity with previously obtained N-terminus of sphingomyelinases from Loxosceles intermedia venom [1]. Clone H17 was expressed as a fusion protein containing a 6x His-tag at its N-terminus and yielded a 33kDa protein. The recombinant protein was endowed with all biological properties ascribed to the whole L. laeta venom and sphingomyelinases from L. intermedia, including dermonecrotic and complement-dependent haemolytic activities. Antiserum raised against the recombinant protein recognised a 32-kDa protein in crude L. laeta venom and was able to block the dermonecrotic reaction caused by whole L. laeta venom. This study demonstrates conclusively that the sphingomyelinase activity in the whole venom is responsible for the major pathological effects of Loxosceles spider envenomation.