Role of the complement system in kidney cell death induced by Loxosceles venom Sphingomyelinases D.

Envenomation by Loxosceles spiders can result in local and systemic pathologies. Systemic loxoscelism, which can lead to death, is characterized by intravascular hemolysis, platelet aggregation, and acute kidney injury. Sphingomyelinase D (SMase D) in Loxosceles spider venom is responsible for both local and systemic pathologies, and has been shown to induce metalloprotease activity. As the complement system is involved in many renal pathologies and is involved in hemolysis in systemic loxoscelism, the aim of this study was to investigate its role and the role of complement regulators and metalloproteases in an in vitro model of Loxosceles venom induced renal pathology. We investigated the effects of the venom/SMase D and the complement system on the HK-2 kidney cell line. Using cell viability assays, western blotting, and flow cytometry, we show that human serum, as a source of complement, enhanced the venom/SMase D induced cell death and the deposition of complement components and properdin. Inhibitors for ADAM-10 and ADAM-17 prevented the venom induced release of the of the complement regulator MCP/CD46 and reduced the venom/SMase D induced cell death. Our results show that the complement system can contribute to Loxosceles venom induced renal pathology. We therefore suggest that patients experiencing systemic loxoscelism may benefit from treatment with metalloproteinase inhibitors and complement inhibitors, but this proposition should be further analyzed in future pre-clinical and clinical assays.

Sphingomyelinases D From Loxosceles Spider Venoms and Cell Membranes: Action on Lipid Rafts and Activation of Endogenous Metalloproteinases.

Loxosceles spider venom contains Sphingomyelinase D (SMase D), the key toxin causing pathology. SMase D hydrolyzes the main component of lipid rafts, sphingomyelin, which changes the membrane microenvironment resulting in the activation of endogenous metalloproteinase from the ADAMs family. Alterations in membrane microenvironment of lipid rafts contribute to the activation of several cell surface molecules. Serine proteinases convertases acting on the pro-domain of membrane metalloproteinases, such as ADAMs, increase the cleavage and the release of proteins ectodomains and receptors located at the cell surface areas containing lipid rafts. We, therefore, investigated the interaction of SMases D with these membrane microdomains (lipid rafts) in human keratinocytes, to better understand the molecular mechanism of SMases D action, and identify the ADAM(s) responsible for the cleavage of cell surface molecules. Using specific inhibitors, we observed that ADAMs 10 and 17 are activated in the cell membrane after SMase D action. Furthermore, proproteins convertases, such as furin, are involved in the SMase D induced ADAMs activation. One of the signaling pathways that may be involved in the activation of these proteases is the MAPK pathway, since phosphorylation of ERK1/2 was observed in cells treated with SMase D. Confocal analysis showed a strong colocalization between SMase D and GM1 ganglioside present in rafts. Analysis of structural components of rafts, such as caveolin-1 and flotillin-1, showed that the action of SMase D on cell membranes leads to a reduction in caveolin-1, which is possibly degraded by toxin-induced superoxide production in cells. The action of the toxin also results in flotilin-1 increased detection in the cell membrane. These results indicate that SMases D from Loxosceles venoms alter membrane rafts structure, leading to the activation of membrane bound proteases, which may explain why the lipase action of this toxin can result in proteolytic cleavage of cell surface proteins, ultimately leading to pathology.

Targeting Loxosceles spider Sphingomyelinase D with small-molecule inhibitors as a potential therapeutic approach for loxoscelism

Loxosceles spiders' venoms consist of a mixture of proteins, including the sphingomyelinases D (SMases D), which are the main toxic components responsible for local and systemic effects in human envenomation. Herein, based on the structural information of SMase D from Loxosceles laeta spider venom and virtual docking-based screening approach, three benzene sulphonate compounds (named 1, 5 and 6) were identified as potential Loxosceles SMase D inhibitors. All compounds inhibited the hydrolysis of the sphingomyelin substrate by both recombinant and native SMases D. Compounds 5 and 6 acted as SMases D uncompetitive inhibitors with Ki values of 0.49 mu M and 0.59 mu M, respectively. Compound 1 is a mixed type inhibitor, and presented a Ki value of 0.54 mu M. In addition, the three compounds inhibited the binding of SMases D to human erythrocytes and the removal of glycophorin C from the cell surface, which are important events in the complement-dependent haemolysis induced by Loxosceles venom. Moreover, compounds 5 and 6 reduced the binding of SMases to human keratinocytes membrane and the venom induced cell death. Importantly, compounds 5 and 6 also controlled the development of the necrotic lesion in an in vivo model of loxoscelism. Together, our findings indicate that the novel SMase D inhibitors presented here are able to suppress both local and systemic reactions induced by Loxosceles venoms. Since the number of Loxosceles envenomation accidents is currently growing worldwide, our results indicate that both inhibitors are promising scaffolds for the rational design of new drugs targeting SMases D from these spiders.

Targeting Loxosceles spider Sphingomyelinase D with small-molecule inhibitors as a potential therapeutic approach for loxoscelism

Loxosceles spiders' venoms consist of a mixture of proteins, including the sphingomyelinases D (SMases D), which are the main toxic components responsible for local and systemic effects in human envenomation. Herein, based on the structural information of SMase D from Loxosceles laeta spider venom and virtual docking-based screening approach, three benzene sulphonate compounds (named 1, 5 and 6) were identified as potential Loxosceles SMase D inhibitors. All compounds inhibited the hydrolysis of the sphingomyelin substrate by both recombinant and native SMases D. Compounds 5 and 6 acted as SMases D uncompetitive inhibitors with Ki values of 0.49 mu M and 0.59 mu M, respectively. Compound 1 is a mixed type inhibitor, and presented a Ki value of 0.54 mu M. In addition, the three compounds inhibited the binding of SMases D to human erythrocytes and the removal of glycophorin C from the cell surface, which are important events in the complement-dependent haemolysis induced by Loxosceles venom. Moreover, compounds 5 and 6 reduced the binding of SMases to human keratinocytes membrane and the venom induced cell death. Importantly, compounds 5 and 6 also controlled the development of the necrotic lesion in an in vivo model of loxoscelism. Together, our findings indicate that the novel SMase D inhibitors presented here are able to suppress both local and systemic reactions induced by Loxosceles venoms. Since the number of Loxosceles envenomation accidents is currently growing worldwide, our results indicate that both inhibitors are promising scaffolds for the rational design of new drugs targeting SMases D from these spiders.

Venom from Bothrops lanceolatus, a Snake Species Native to Martinique, Potently Activates the Complement System.

Bothrops lanceolatus snake venom causes systemic thrombotic syndrome but also local inflammation involving extensive oedema, pain, and haemorrhage. Systemic thrombotic syndrome may lead to fatal pulmonary embolism and myocardial and cerebral infarction. Here, we investigated the ability of B. lanceolatus venom to activate the Complement system (C) in order to improve the understanding of venom-induced local inflammation. Data presented show that B. lanceolatus venom is able to activate all C-pathways. In human serum, the venom strongly induced the generation of anaphylatoxins, such as C5a and C4a, and the Terminal Complement complex. The venom also induced cleavage of purified human components C3, C4, and C5, with the production of biologically active C5a. Furthermore, the venom enzymatically inactivated the soluble C-regulator and the C1-inhibitor (C1-INH), and significantly increased the expression of bound C-regulators, such as MCP and CD59, on the endothelial cell membrane. Our observations that B. lanceolatus venom activates the three Complement activation pathways, resulting in anaphylatoxins generation, may suggest that this could play an important role in local inflammatory reaction and systemic thrombosis caused by the venom. Inactivation of C1-INH, which is also an important inhibitor of several coagulation proteins, may also contribute to inflammation and thrombosis. Thus, further in vivo studies may support the idea that therapeutic management of systemic B. lanceolatus envenomation could include the use of Complement inhibitors as adjunct therapy.

Loxosceles venom Sphingomyelinase D activates human blood leukocytes: Role of the complement system.

Envenomation by Loxosceles spiders can result in severe systemic and local reactions, which are mainly triggered by Sphingomyelinase D (SMase D), a toxic component of Loxosceles venom. SMase D induces a systemic inflammatory condition similar to the reaction observed during an endotoxic shock. Considering the potent pro-inflammatory potential of Loxosceles venom and the SMase D, in this study we have used the whole human blood model to study the endotoxic-like shock triggered by SMase D. Recombinant purified SMase D from L. intermedia venom, similarly to LPS, induced activation of blood leukocytes, as observed by the increase in the expression of CD11b and TLR4, production of reactive oxygen and nitrogen species (superoxide anion and peroxynitrite) and release of TNF-α. Complement consumption in the plasma was also detected, and complement inhibition by compstatin decreased the SMase D and LPS-induced leukocyte activation, as demonstrated by a reduction in the expression of CD11b and TLR4 and superoxide anion production. Similar results were found for the L. intermedia venom, except for the production of TNF-α. These findings indicate that SMase D present in Loxosceles venom is able to activate leukocytes in a partially complement-dependent manner, which can contribute to the systemic inflammation that follows envenomation by this spider. Thus, future therapeutic management of systemic Loxosceles envenomation could include the use of complement inhibitors as adjunct therapy.

Venom from Bothrops lanceolatus, a snake species native to Martinique, potently activates the complement system

Bothrops lanceolatus snake venom causes systemic thrombotic syndrome but also local inflammation involving extensive oedema, pain, and haemorrhage. Systemic thrombotic syndrome may lead to fatal pulmonary embolism and myocardial and cerebral infarction. Here, we investigated the ability of B. lanceolatus venom to activate the Complement system (C) in order to improve the understanding of venom-induced local inflammation. Data presented show that B. lanceolatus venom is able to activate all C-pathways. In human serum, the venom strongly induced the generation of anaphylatoxins, such as C5a and C4a, and the Terminal Complement complex. The venom also induced cleavage of purified human components C3, C4, and C5, with the production of biologically active C5a. Furthermore, the venom enzymatically inactivated the soluble C-regulator and the C1-inhibitor (C1-INH), and significantly increased the expression of bound C-regulators, such as MCP and CD59, on the endothelial cell membrane. Our observations that B. lanceolatus venom activates the three Complement activation pathways, resulting in anaphylatoxins generation, may suggest that this could play an important role in local inflammatory reaction and systemic thrombosis caused by the venom. Inactivation of C1-INH, which is also an important inhibitor of several coagulation proteins, may also contribute to inflammation and thrombosis. Thus, further in vivo studies may support the idea that therapeutic management of systemic B. lanceolatus envenomation could include the use of Complement inhibitors as adjunct therapy.

Loxosceles venom Sphingomyelinase D activates human blood leukocytes: role of the complement system

Envenomation by Loxosceles spiders can result in severe systemic and local reactions, which are mainly triggered by Sphingomyelinase D (SMase D), a toxic component of Loxosceles venom. SMase D induces a systemic inflammatory condition similar to the reaction observed during an endotoxic shock. Considering the potent pro inflammatory potential of Loxosceles venom and the SMase D, in this study we have used the whole human blood model to study the endotoxic-like shock triggered by SMase D. Recombinant purified SMase D from L. intermedia venom, similarly to LPS, induced activation of blood leukocytes, as observed by the increase in the expression of CD11b and TLR4, production of reactive oxygen and nitrogen species (superoxide anion and peroxynitrite) and release of TNF-alpha. Complement consumption in the plasma was also detected, and complement inhibition by compstatin decreased the SMase D and LPS-induced leukocyte activation, as demonstrated by a reduction in the expression of CD11b and TLR4 and superoxide anion production. Similar results were found for the L. intermedia venom, except for the production of TNF-alpha. These findings indicate that SMase D present in Loxosceles venom is able to activate leukocytes in a partially complement-dependent manner, which can contribute to the systemic inflammation that follows envenomation by this spider. Thus, future therapeutic management of systemic Loxosceles envenomation could include the use of complement inhibitors as adjunct therapy.

Venom from Bothrops lanceolatus, a Snake Species Native to Martinique, Potently Activates the Complement System

Bothrops lanceolatus snake venom causes systemic thrombotic syndrome but also local inflammation involving extensive oedema, pain, and haemorrhage. Systemic thrombotic syndrome may lead to fatal pulmonary embolism and myocardial and cerebral infarction. Here, we investigated the ability of B. lanceolatus venom to activate the Complement system (C) in order to improve the understanding of venom-induced local inflammation. Data presented show that B. lanceolatus venom is able to activate all C-pathways. In human serum, the venom strongly induced the generation of anaphylatoxins, such as C5a and C4a, and the Terminal Complement complex. The venom also induced cleavage of purified human components C3, C4, and C5, with the production of biologically active C5a. Furthermore, the venom enzymatically inactivated the soluble C-regulator and the C1-inhibitor (C1-INH), and significantly increased the expression of bound C-regulators, such as MCP and CD59, on the endothelial cell membrane. Our observations that B. lanceolatus venom activates the three Complement activation pathways, resulting in anaphylatoxins generation, may suggest that this could play an important role in local inflammatory reaction and systemic thrombosis caused by the venom. Inactivation of C1-INH, which is also an important inhibitor of several coagulation proteins, may also contribute to inflammation and thrombosis. Thus, further in vivo studies may support the idea that therapeutic management of systemic B. lanceolatus envenomation could include the use of Complement inhibitors as adjunct therapy.

Loxosceles venom Sphingomyelinase D activates human blood leukocytes: role of the complement system

Envenomation by Loxosceles spiders can result in severe systemic and local reactions, which are mainly triggered by Sphingomyelinase D (SMase D), a toxic component of Loxosceles venom. SMase D induces a systemic inflammatory condition similar to the reaction observed during an endotoxic shock. Considering the potent pro inflammatory potential of Loxosceles venom and the SMase D, in this study we have used the whole human blood model to study the endotoxic-like shock triggered by SMase D. Recombinant purified SMase D from L. intermedia venom, similarly to LPS, induced activation of blood leukocytes, as observed by the increase in the expression of CD11b and TLR4, production of reactive oxygen and nitrogen species (superoxide anion and peroxynitrite) and release of TNF-alpha. Complement consumption in the plasma was also detected, and complement inhibition by compstatin decreased the SMase D and LPS-induced leukocyte activation, as demonstrated by a reduction in the expression of CD11b and TLR4 and superoxide anion production. Similar results were found for the L. intermedia venom, except for the production of TNF-alpha. These findings indicate that SMase D present in Loxosceles venom is able to activate leukocytes in a partially complement-dependent manner, which can contribute to the systemic inflammation that follows envenomation by this spider. Thus, future therapeutic management of systemic Loxosceles envenomation could include the use of complement inhibitors as adjunct therapy.

Tetracycline Reduces Kidney Damage Induced by Loxosceles Spider Venom.

Envenomation by Loxosceles spider can result in two clinical manifestations: cutaneous and systemic loxoscelism, the latter of which includes renal failure. Although incidence of renal failure is low, it is the main cause of death, occurring mainly in children. The sphingomyelinase D (SMase D) is the main component in Loxosceles spider venom responsible for local and systemic manifestations. This study aimed to investigate the toxicity of L. intermedia venom and SMase D on kidney cells, using both In vitro and in vivo models, and the possible involvement of endogenous metalloproteinases (MMP). Results demonstrated that venom and SMase D are able to cause death of human kidney cells by apoptosis, concomitant with activation and secretion of extracellular matrix metalloproteases, MMP-2 and MMP-9. Furthermore, cell death and MMP synthesis and secretion can be prevented by tetracycline. In a mouse model of systemic loxoscelism, Loxosceles venom-induced kidney failure was observed, which was abrogated by administration of tetracycline. These results indicate that MMPs may play an important role in Loxosceles venom-induced kidney injury and that tetracycline administration may be useful in the treatment of human systemic loxoscelism.

Sphingomyelinase D from Loxosceles laeta Venom Induces the Expression of MMP7 in Human Keratinocytes: Contribution to Dermonecrosis.

Envenomation by Loxosceles spider is characterized by the development of dermonecrosis. In previous studies, we have demonstrated that increased expression/secretion of matrix metalloproteinases 2 and 9, induced by Loxosceles intermedia venom Class 2 SMases D (the main toxin in the spider venom), contribute to the development of cutaneous loxoscelism. In the present study we show that the more potent venom containing the Class 1 SMase D from Loxosceles laeta, in addition to increasing the expression/secretion of MMP2 and MMP9, also stimulates the expression of MMP7 (Matrilysin-1), which was associated with keratinocyte cell death. Tetracycline, a matrix metalloproteinase inhibitor, prevented cell death and reduced MMPs expression. Considering that L. laeta venom is more potent at inducing dermonecrosis than L. intermedia venom, our results suggest that MMP7 may play an important role in the severity of dermonecrosis induced by L. laeta spider venom SMase D. In addition, the inhibition of MMPs by e.g. tetracyclines may be considered for the treatment of the cutaneous loxoscelism.

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.

Microcirculation abnormalities provoked by Loxosceles spiders' envenomation.

Loxoscelism is caused by envenomation by spiders from Loxosceles genus. Clinical symptoms only appear a few hours after envenomation and can evolve in local reactions, such as dermonecrosis, and systemic reactions, including intravascular haemolysis, intravascular coagulation and renal failure. Considering that alterations in the microcirculatory network are involved in the pathogenesis of different diseases, including the inflammatory process, the aim of this study was to investigate the action of venoms of males and females of Loxosceles intermedia and Loxosceles laeta on the microcirculatory network and examine the systemic production of inflammatory mediators in a murine model of loxoscelism. We observed that during systemic envenomation, the alterations in the microcirculation include increase in the number of rolling cells, which was more intense in animals injected with female Loxosceles spider venoms. This positively correlated with increase in TNF-α and NO serum levels, induction of which was higher by female venoms when compared with male venoms. The increase of leukocytes rolling was not accompanied by increase of cell adhesion. The absence of leukocyte extravasation may explain why in mice, in contrast to humans, no cutaneous loxoscelism occurs. Thus, targeting the neutrophil adhesion and extravasation in Loxosceles envenomed patients may prevent cutaneous pathology.

A serine protease isolated from the bristles of the Amazonic caterpillar, Premolis semirufa, is a potent complement system activator.

BACKGROUND: The caterpillar of the moth Premolis semirufa, commonly named pararama, is found in the Brazilian Amazon region. Accidental contact with the caterpillar bristles causes an intense itching sensation, followed by symptoms of an acute inflammation, which last for three to seven days after the first incident. After multiple accidents a chronic inflammatory reaction, called "Pararamose", characterized by articular synovial membrane thickening with joint deformities common to chronic synovitis, frequently occurs. Although complement mediated inflammation may aid the host defense, inappropriate or excessive activation of the complement system and generation of anaphylatoxins can lead to inflammatory disorder and pathologies. The aim of the present study was to evaluate, in vitro, whether the Premolis semirufa's bristles extract could interfere with the human complement system. RESULTS: The bristles extract was able to inhibit the haemolytic activity of the alternative pathway, as well as the activation of the lectin pathway, but had no effect on the classical pathway, and this inhibition seemed to be caused by activation and consumption of complement components. The extract induced the production of significant amounts of all three anaphylatoxins, C3a, C4a and C5a, promoted direct cleavage of C3, C4 and C5 and induced a significant generation of terminal complement complexes in normal human serum. By using molecular exclusion chromatography, a serine protease of 82 kDa, which activates complement, was isolated from P. semirufa bristles extract. The protease, named here as Ps82, reduced the haemolytic activity of the alternative and classical pathways and inhibited the lectin pathway. In addition, Ps82 induced the cleavage of C3, C4 and C5 and the generation of C3a and C4a in normal human serum and it was capable to cleave human purified C5 and generate C5a. The use of Phenanthroline, metalloprotease inhibitor, in the reactions did not significantly interfere with the activity of the Ps82, whereas the presence of PMSF, serine protease inhibitor, totally blocked the activity. CONCLUSION: These data show that a serine protease present in the Premolis semirufa's bristles extract has the ability to activate the complement system, which may contribute to the inflammatory process presented in humans after envenomation.

Animal venoms/toxins and the complement system.

Nature is a wealthy source of agents that have been shown to be beneficial to human health, but nature is also a rich source of potential dangerous health damaging compounds. This review will summarise and discuss the agents from the animal kingdom that have been shown to interact with the human complement (C) system. Most of these agents are toxins found in animal venoms and animal secretions. In addition to the mechanism of action of these toxins, their contribution to the field of complement, their role in human pathology and the potential benefit to the venomous animal itself will be discussed. Potential therapeutic applications will also be discussed.

Mechanism of neutrophil dysfunction: neutrophil serine proteases cleave and inactivate the C5a receptor.

Neutrophil dysfunction, resulting in inefficient bacterial clearance, is a feature of several serious medical conditions, including cystic fibrosis (CF) and sepsis. Poorly controlled neutrophil serine protease (NSP) activity and complement activation have been implicated in this phenomenon. The capacity for excess NSP secretion and complement activation to influence the expression and function of the important neutrophil-activating receptor C5aR was investigated. Purified NSPs cathepsin G (CG), neutrophil elastase (NE), and proteinase 3 cleaved C5aR to a 26- to 27-kDa membrane-bound fragment, thereby inactivating its C5a-induced signaling ability. In a supernatant transfer assay, NSPs released from neutrophils in response to C5a induced the cleavage of the C5aR on unstimulated cells. Stimulation of myeolomonocytic U937 cells and purified neutrophils with C5a resulted in downregulation of the C5aR on these cells, which, in the case of U937 cells, was largely caused by NSP-mediated cleavage of C5aR, but in the case of neutrophils, intracellular degradation was likely the main mediator in addition to a small role for NSPs. CG and NE in bronchoalveolar lavage fluid from CF patients both contributed to C5aR cleavage. We propose two converging models for C5a- and NSP-mediated neutrophil dysfunction whereby C5aR cleavage is induced by NSPs, secreted in response to: 1) excess C5a generation or other stimuli; or 2) necrosis. The consequent impairment of C5aR activity contributes to suboptimal local neutrophil priming and bacterial clearance. NSP inhibitors with specificity for both CG and NE may aid the treatment of pathologies associated with neutrophil dysfunction including sepsis and CF.

P-I snake venom metalloproteinase is able to activate the complement system by direct cleavage of central components of the cascade.

BACKGROUND: Snake Venom Metalloproteinases (SVMPs) are amongst the key enzymes that contribute to the high toxicity of snake venom. We have recently shown that snake venoms from the Bothrops genus activate the Complement system (C) by promoting direct cleavage of C-components and generating anaphylatoxins, thereby contributing to the pathology and spread of the venom. The aim of the present study was to isolate and characterize the C-activating protease from Bothrops pirajai venom. RESULTS: Using two gel-filtration chromatography steps, a metalloproteinase of 23 kDa that activates Complement was isolated from Bothrops pirajai venom. The mass spectrometric identification of this protein, named here as C-SVMP, revealed peptides that matched sequences from the P-I class of SVMPs. C-SVMP activated the alternative, classical and lectin C-pathways by cleaving the α-chain of C3, C4 and C5, thereby generating anaphylatoxins C3a, C4a and C5a. In vivo, C-SVMP induced consumption of murine complement components, most likely by activation of the pathways and/or by direct cleavage of C3, leading to a reduction of serum lytic activity. CONCLUSION: We show here that a P-I metalloproteinase from Bothrops pirajai snake venom activated the Complement system by direct cleavage of the central C-components, i.e., C3, C4 and C5, thereby generating biologically active fragments, such as anaphylatoxins, and by cleaving the C1-Inhibitor, which may affect Complement activation control. These results suggest that direct complement activation by SVMPs may play a role in the progression of symptoms that follow envenomation.

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.

Roles of promoter and 3' untranslated motifs in expression of the human C5a receptor.

The C5a receptor (C5aR) is a 7 transmembrane G-protein coupled receptor (GPCR) that mediates the powerful pro-inflammatory effect of the complement activation product C5a. Excess C5a generated under pathological conditions has been implicated in a variety of conditions including sepsis, asthma and rheumatoid arthritis, but very little is known about the regulation of expression of the C5aR. The 5' promoter region and 3' untranslated region (UTR) of the C5aR mRNA were cloned, generating enhanced green fluorescent protein (EGFP)-reporter plasmids, which were transfected into the monocytic cell line U937. Most of the cloned 2kb 5' region was dispensable for the expression of the reporter constructs and the majority of regulatory sequences are in the first 200 bp. Three motifs, a NFκB, a CCAAT and a NFAT site, were identified to be of importance by site directed mutagenesis for basal expression. Analysis of the 3'UTR of the C5aR mRNA showed that it contained two AU-rich elements (AREs), however site directed mutagenesis showed that these had no effect on basal expression. While the phorbol ester PMA and dibutyryl cAMP increased C5aR protein expression, these agents had no effect on the regulation of expression via the promoter or the 3'UTR. This is the first study to investigate the role of both the promoter and 3'UTR in regulating C5aR expression and our results show that regulation of the human C5aR is similar but not identical to that of the mouse C5aR.