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.

Role of ErbB and IL-1 signaling pathways in the dermonecrotic lesion induced by Loxosceles sphingomyelinases D.

Sphingomyelinase D (SMase D), the main toxic component of Loxosceles venom, has a well-documented role on dermonecrotic lesion triggered by envenomation with these species; however, the intracellular mechanisms involved in this event are still poorly known. Through differential transcriptomics of human keratinocytes treated with L. laeta or L. intermedia SMases D, we identified 323 DEGs, common to both treatments, as well as upregulation of molecules involved in the IL-1 and ErbB signaling. Since these pathways are related to inflammation and wound healing, respectively, we investigated the relative expression of some molecules related to these pathways by RT-qPCR and observed different expression profiles over time. Although, after 24 h of treatment, both SMases D induced similar modulation of these pathways in keratinocytes, L. intermedia SMase D induced earlier modulation compared to L. laeta SMase D treatment. Positive expression correlations of the molecules involved in the IL-1 signaling were also observed after SMases D treatment, confirming their inflammatory action. In addition, we detected higher relative expression of the inhibitor of the ErbB signaling pathway, ERRFI1, and positive correlations between this molecule and pro-inflammatory mediators after SMases D treatment. Thus, herein, we describe the cell pathways related to the exacerbation of inflammation and to the failure of the wound healing, highlighting the contribution of the IL-1 signaling pathway and the ERRFI1 for the development of cutaneous loxoscelism.

Cytotoxic and genotoxic effects on human keratinocytes triggered by sphingomyelinase D from Loxosceles venom.

The spiders of the Loxosceles genus (called brown or violin spiders) are of medical relevance in several countries due to the many human envenomation cases reported. The main component of Loxosceles venom is the enzyme sphingomyelinase D (SMase D), which is responsible for the local and systemic effects induced by the whole venom. Here, we investigated the cytotoxic and genotoxic effects caused by Loxosceles laeta venom and SMase D on human keratinocytes to better understand the dermonecrosis development mechanism. Our findings indicate that whole venom, as well as SMase D, increases intracellular superoxide levels, leading to DNA damage. These effects appear to be dependent on the binding of SMase D to the cell surface, although the complete pathway triggered as a result of the binding still needs to be elucidated. Moreover, after SMase D treatment, we observed the presence of histone γH2AX, suggesting that the cells are undergoing DNA repair. Moreover, when ATR kinase was inhibited, the cell viability of human keratinocytes was decreased. Together, our findings strongly suggest that L. laeta venom, as well as SMase D, increases intracellular superoxide levels, leading to DNA damage in human keratinocytes. Additionally, the induced DNA damage is repaired through the activation of an apparent ATR-mediated DNA-damage response. This knowledge may contribute to a better understanding of the behaviour of human keratinocytes during cutaneous loxoscelism, a condition that affects thousands of people around the world.

Bacterial and Arachnid Sphingomyelinases D: Comparison of Biophysical and Pathological Activities.

Sphingomyelinases D have only been identified in arachnid venoms, Corynebacteria, Arcanobacterium, Photobacterium and in the fungi Aspergillus and Coccidioides. The arachnid and bacterial enzymes share very low sequence identity and do not contain the HKD sequence motif characteristic of the phospholipase D superfamily, however, molecular modeling and circular dichroism of SMases D from Loxosceles intermedia and Corynebacterium pseudotuberculosis indicate similar folds. The phospholipase, hemolytic and necrotic activities and mice vessel permeabilities were compared and both enzymes possess the ability to hydrolyze phospholipids and also promote similar pathological reactions in the host suggesting the existence of a common underlying mechanism in tissue disruption. J. Cell. Biochem. 118:2053-2063, 2017. © 2016 Wiley Periodicals, Inc.

Partial characterization of Loxosceles anomala (Mello-Leitão, 1917) venom: A brown spider of potential medical concern.

The spider's genus Loxosceles (also known as "brown spiders") is one of the few ones of medical importance in Brazil, being Loxosceles anomala a species of common occurrence in the Southeast region. This species is usually smaller in size than the other members of the Loxosceles group. A single human accident involving L. anomala was reported to date and the clinical picture shared similar characteristics with accidents caused by other Loxosceles species. Despite the potential relevance of L. anomalafor loxocelism in Minas Gerais state, its venom activity has never been characterized. In this work, we provide a preliminary characterization of L. anomala venom, considering its most relevant enzymatic activities and its venom immunorecognition by current therapeutic antivenoms. The results showed that L. anomala venom is immunorecognised by therapeutic antivenoms and by anti-phospholipase D antibodies. Its venom also shows enzymatic activities (sphingomyelinase activity, fibrinogenolytic) described for other Loxosceles venoms. This work contributes to a better knowledge on the venom content and activities of synanthropic Loxosceles species that have the potential of causing relevant human accidents.

State-of-the-art review of snake venom phosphodiesterases (svPDEs).

Phosphodiesterases (PDEs) constitute an enzyme group able to hydrolyze nucleic acids as well as some second messengers. Due to this ability and their expression in several human tissues and organs, PDEs can control a gamut of physiological processes. They are also involved in some pathological conditions, such as Alzheimer's disease and erectile dysfunction. PDEs are also expressed in snake venom glands, being called snake venoms phosphodiesterases, or simply svPDEs. The occurrence of these enzymes has already been reported in crotalid, elapid and viperid venoms, such as Crotalus, Naja and Trimeresurus, respectively, but not all of them have been characterized concerning their structure, activity and function. In this review, we are addressing general characteristics of svPDEs, in addition to their structural, biochemical and functional characteristics, and we also report some potential applications of svPDEs.

Systemic Loxoscelism, Less Frequent but More Deadly: The Involvement of Phospholipases D in the Pathophysiology of Envenomation.

Bites of Loxosceles spiders can lead to a set of clinical manifestations called loxoscelism, and are considered a public health problem in many regions. The signs and symptoms of loxoscelism are divided into cutaneous and systemic forms. The former is more frequent and includes signs of envenoming at the bite site or neighboring regions. Systemic loxoscelism, although much less frequent, is associated with complications, and can even lead to death. It may include intravascular hemolysis, acute renal failure, and thrombocytopenia. Loxosceles venoms are enriched with phospholipases D (PLDs), which are a family of isoforms found at intra-species and inter-species levels. Under experimental conditions, these enzymes reproduce the main clinical signs of loxoscelism, including an exacerbated inflammatory response at the bite site and dermonecrosis, as well as thrombocytopenia, intravascular hemolysis, and acute renal failure. The role of PLDs in cutaneous loxoscelism was described over forty years ago, when studies identified and purified toxins featured as sphingomyelinase D. More recently, the production of recombinant PLDs and discoveries about their structure and mechanism has enabled a deeper characterization of these enzymes. In this review, we describe these biochemical and functional features of Loxosceles PLDs that determine their involvement in systemic loxoscelism.

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.

Searching for the toxic potential of Loxosceles amazonica and Loxosceles willianilsoni spiders' venoms.

The Loxosceles genus belongs to the Sicariidae family and it comprises species whose venom can cause accidents with potentially fatal consequences. We have previously shown that SMase D is the enzyme responsible for the main pathological effects of Loxosceles venom. Despite the severity of accidents with Loxosceles, few species are considered to be of medical importance. Little is known about the venom of non-synanthropic species that live in natural environments. To contribute to a better understanding about the venom's toxicity of Loxosceles genus, the aim of this study was to (i) characterize the toxic properties of Loxosceles amazonica from two different localities and a recent described cave species Loxosceles willianilsoni and (ii) compare these venoms with that from Loxosceles laeta, which is among the most toxic ones. We show here that both L. amazonica venoms (from the two studied locations) and L. willianilsoni presented SMase D activity similar to that exhibited by L. laeta venom. Although L. amazonica and L. willianilsoni venoms were able to induce complement dependent human erythrocytes lysis, they were not able to induce cell death of human keratinocytes, as promoted by L. laeta venom, in the concentrations tested. These results indicate that other species of Loxosceles, in addition to those classified as medically important, have toxic potential to cause accidents in humans, despite interspecific variations that denote possible less toxicity.

Unraveling the structure and function of CdcPDE: A novel phosphodiesterase from Crotalus durissus collilineatus snake venom.

This study reports the isolation, structural, biochemical, and functional characterization of a novel phosphodiesterase from Crotalus durissus collilineatus venom (CdcPDE). CdcPDE was successfully isolated from whole venom using three chromatographic steps and represented 0.7% of total protein content. CdcPDE was inhibited by EDTA and reducing agents, demonstrating that metal ions and disulfide bonds are necessary for its enzymatic activity. The highest enzymatic activity was observed at pH 8-8.5 and 37 °C. Kinetic parameters indicated a higher affinity for the substrate bis(p-nitrophenyl) phosphate compared to others snake venom PDEs. Its structural characterization was done by the determination of the protein primary sequence by Edman degradation and mass spectrometry, and completed by the building of molecular and docking-based models. Functional in vitro assays showed that CdcPDE is capable of inhibiting platelet aggregation induced by adenosine diphosphate in a dose-dependent manner and demonstrated that CdcPDE is cytotoxic to human keratinocytes. CdcPDE was recognized by the crotalid antivenom produced by the Instituto Butantan. These findings demonstrate that the study of snake venom toxins can reveal new molecules that may be relevant in cases of snakebite envenoming, and that can be used as molecular tools to study pathophysiological processes due to their specific biological activities.

Sphingomyelinase D Activity in Sicarius tropicus Venom: Toxic Potential and Clues to the Evolution of SMases D in the Sicariidae Family.

The spider family Sicariidae includes three genera, Hexophthalma, Sicarius and Loxosceles. The three genera share a common characteristic in their venoms: the presence of Sphingomyelinases D (SMase D). SMases D are considered the toxins that cause the main pathological effects of the Loxosceles venom, that is, those responsible for the development of loxoscelism. Some studies have shown that Sicarius spiders have less or undetectable SMase D activity in their venoms, when compared to Hexophthalma. In contrast, our group has shown that Sicarius ornatus, a Brazilian species, has active SMase D and toxic potential to envenomation. However, few species of Sicarius have been characterized for their toxic potential. In order to contribute to a better understanding about the toxicity of Sicarius venoms, the aim of this study was to characterize the toxic properties of male and female venoms from Sicarius tropicus and compare them with that from Loxosceles laeta, one of the most toxic Loxosceles venoms. We show here that S. tropicus venom presents active SMases D. However, regarding hemolysis development, it seems that these toxins in this species present different molecular mechanisms of action than that described for Loxosceles venoms, whereas it is similar to those present in bacteria containing SMase D. Besides, 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 venoms from Sicarius species.

An overview on nucleases (DNase, RNase, and phosphodiesterase) in snake venoms.

In this review, we have compiled the data on pharmacological activities associated with endogenous purine release related enzymes-nucleases (DNases, RNases, and phosphodiesterases). The results of studies on toxic effects of these enzymes, emphasizing the future directions in this field, are summarized. One of the major problems facing toxicologists is the identification and characterization of specific venom nucleases since they share similar substrate specificities and biochemical properties. In this review, we have attempted to clarify some of the discrepancies about these enzymes. Further, we have tried to correlate the existence of nuclease enzymes in relation to endogenous release of purines, a multitoxin, during snake envenomation, and we also discuss the possible actions of purines. We hope that this review will stimulate renewed interest among toxicologists to biologically characterize these enzymes and elucidate their role in envenomation.

[Necrotic arachnidism in the south of France: two clinical cases of loxoscelism]. / Envenimation nécrotique dans le Sud de la France : deux observations de loxoscélisme cutané

BACKGROUND: loxosceles spiders are found throughout the world and are responsible for numerous cases of envenomation in America and Southern Europe. We describe, to our knowledge for the first time in France, two clinical cases of cutaneous loxoscelism. CASE REPORT: two cases of skin necrosis arising after supposed spider bites were grouped together because of their similar clinical presentation: an initial painless bite and rapid development of an inflammatory and painful cutaneous lesion with a central hemorrhagic bulla surrounded by a perimeter of blanched skin (the "red, white, and blue" sign). The outcome in both cases was deep skin necrosis and chronic ulceration requiring surgical treatment. DISCUSSION: loxoscelism can result in dermonecrosis. Although our cases were not documented by capture of the spider, the diagnosis of cutaneous loxoscelism was supported by the characteristic appearance of the lesion, a typical clinical course, elimination of differential diagnoses, and the confirmed presence of Loxosceles rufescens in the region. CONCLUSION: loxoscelism can occur in the south of France and although rare, must be considered in this region as a possible cause of skin necrosis.

A loxoscelism case received therapeutic apheresis and hyperbaric oxygen therapy.

Loxosceles reclusa (L.reclusa) is known to bite humans, and its venom includes several enzymes that cause clinical symptoms. Loxoscelism, a condition due to being bitten by Loxosceles spiders, commonly known as recluses, can involve a range of clinical conditions, from local cutaneous lesions to severe systemic involvement. The diagnosis of loxoscelism is usually made by anamnesis and clinical findings. Magnetic resonance imaging is recommended for patients at high risk of necrotizing fasciitis. Treatment modalities are still controversial and there is no standardized treatment approach. Reported here, our case of loxoscelism involved a 24-year-old man presenting with a Loxosceles spider bite, dermonecrotic lesion, vomiting, diarrhea, acute renal injury, and rhabdomyolysis, who was successfully treated with hyperbaric oxygen therapy, therapeutic apheresis, hemodialysis, wound debridement, and cutaneous autografting. Early diagnosis and multidisciplinary approach can be life-saving in spider bites that can cause systemic involvement. Loxoscelism should be considered in patients with skin necrosis, acute renal injury, and rhabdomyolysis.

Antigenic and Substrate Preference Differences between Scorpion and Spider Dermonecrotic Toxins, a Comparative Investigation.

The Hemiscorpius lepturus scorpion and brown spider Loxosceles intermedia represent a public health problem in Asia and America, respectively. Although distinct, these organisms contain similar toxins responsible for the principal clinical signs of envenomation. To better understand the properties of these toxins, we designed a study to compare recombinant Heminecrolysin (rHNC) and rLiD1, the major phospholipase D toxins of scorpion and spider venom, respectively. Using a competitive ELISA and a hemolytic inhibition test, we come to spot a cross reaction between scorpion and spider venoms along with an epitopic similarity between rHNC and rLiD1 associated with neutralizing antibodies. Results show that the ability of the rHNC to hydrolyze lysophosphatidylcholine (LPC) is equivalent to that of rLiD1 to hydrolyze sphingomyelin and vice-versa. rHNC exclusively catalyze transphosphatidylation of LPC producing cyclic phosphatidic acid (cPA). The in-silico analysis of hydrogen bonds between LPC and toxins provides a possible explanation for the higher transphosphatidylase activity of rHNC. Interestingly, for the first time, we reveal that lysophosphatidic acid (LPA) can be a substrate for both enzymes using cellular and enzymatic assays. The finding of the usage of LPA as a substrate as well as the formation of cPA as an end product could shed more light on the molecular basis of Hemiscorpius lepturus envenomation as well as on loxoscelism.

Toxicological Characterization and Phospholipase D Activity of the Venom of the Spider Sicarius thomisoides.

Envenomation by Loxosceles spiders (Sicariidae family) has been thoroughly documented. However, little is known about the potential toxicity of members from the Sicarius genus. Only the venom of the Brazilian Sicarius ornatus spider has been toxicologically characterized. In Chile, the Sicarius thomisoides species is widely distributed in desert and semidesert environments, and it is not considered a dangerous spider for humans. This study aimed to characterize the potential toxicity of the Chilean S. thomisoides spider. To do so, specimens of S. thomisoides were captured in the Atacama Desert, the venom was extracted, and the protein concentration was determined. Additionally, the venoms were analyzed by electrophoresis and Western blotting using anti-recombinant L. laeta PLD1 serum. Phospholipase D enzymatic activity was assessed, and the hemolytic and cytotoxic effects were evaluated and compared with those of the L. laeta venom. The S. thomisoides venom was able to hydrolyze sphingomyelin as well as induce complement-dependent hemolysis and the loss of viability of skin fibroblasts with a dermonecrotic effect of the venom in rabbits. The venom of S. thomisoides showed intraspecific variations, with a similar protein pattern as that of L. laeta venom at 32-35 kDa, recognized by serum anti-LlPLD1. In this context, we can conclude that the venom of Sicarius thomisoides is similar to Loxosceles laeta in many aspects, and the dermonecrotic toxin present in their venom could cause severe harm to humans; thus, precautions are necessary to avoid exposure to their bite.

Identification, cloning and functional characterization of a novel dermonecrotic toxin (phospholipase D) from brown spider (Loxosceles intermedia) venom.

Brown spider bites are associated with lesions including dermonecrosis, gravitational spreading and a massive inflammatory response, along with systemic problems that may include hematological disturbances and renal failure. The mechanisms by which the venom exerts its noxious effects are currently under investigation. It is known that the venom contains a major toxin (dermonecrotic toxin, biochemically a phospholipase D) that can experimentally induce dermonecrosis, inflammatory response, animal mortality and platelet aggregation. Herein, we describe cloning, heterologous expression, purification and functionality of a novel isoform of the 33 kDa dermonecrotic toxin. Circular dichroism analysis evidenced correct folding for the toxin. The recombinant toxin was recognized by whole venom serum antibodies and by a specific antibody to a previously described dermonecrotic toxin. The identified toxin was found to display phospholipase activity and dermonecrotic properties. Additionally, the toxin caused a massive inflammatory response in rabbit skin dermis, evoked platelet aggregation, increased vascular permeability, caused edema and death in mice. These characteristics in combination with functional studies for other dermonecrotic toxins illustrate that a family of dermonecrotic toxins exists, and includes a novel member with high activity that may be useful for future structural and functional studies.

Analysis of therapeutic benefits of antivenin at different time intervals after experimental envenomation in rabbits by venom of the brown spider (Loxosceles intermedia).

Bites by the brown spider (Loxosceles spp.) are an important health problem in South America, where three species predominate (Loxosceles laeta, Loxosceles gaucho, Loxosceles intermedia). Brown spider bites (loxoscelism) induce a block of cutaneous necrosis and, less commonly, may cause fatal systemic poisoning. A variety of controversial protocols are used to treat loxoscelism, while treatment with antivenin is the only venom specific treatment. Here we studied the action of the venom as well as the response to the antivenin for Loxosceles through an experimental study that simulates bites of L. intermedia (bites of this species are the most common in Brazil). Beneficial effects are known for antivenin applied quickly (within 4 h) after envenomation. Here we wished to examine the temporal development of the brown spider bite as well as the temporal patterns of the action of the antivenin to determine the time limits for beneficial use of the antivenin after envenomation. This information is important since most patients only appear for treatment several hours after being bitten. New Zealand rabbits were experimentally exposed to the venom from brown spiders by the injection of venom from L. intermedia (2x minimum necrotic dose), followed at regular time intervals by antivenin. The use of the loxoscelic antivenin--CPPI (4 mL per animal)--minimized the effects of envenomation when applied for up to 12 h after the injection of the venom, as evaluated by cutaneous (erythrema, edema, ecchymosis and necrosis) and systemic (blood cell and platelet counts, hematimetrics and fibrinogen dosage) criteria. Also, antivenin reduced the size of the necrotic area when applied up to 48 h after envenomation. Thus, therapy with loxoscelic antivenin, CPPI, may provide beneficial results by interfering with envenomation well after the bite occurred and therefore may become an important tool for medical treatment of brown spider bites.