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.

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.

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.

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.

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.

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.

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.

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.

Diversity of astacin-like metalloproteases identified by transcriptomic analysis in Peruvian Loxosceles laeta spider venom and in vitro activity characterization.

Loxosceles spiders are found in almost all countries of South America. In Peru, Loxosceles laeta species is the main responsible for the accidents caused by poisonous animals, being known as "killer spiders", due to the large number of fatal accidents observed. Astacin-like metalloproteases, named LALPs (Loxosceles astacin-like metalloproteases) are highly expressed in Loxosceles spiders venom gland. These proteases may be involved in hemorrhage and venom spreading, being relevant to the envenoming proccess. Thus, the aim of this work was to analyze Peruvian L. laeta venom gland transcripts using bioinformatics tools, focusing on LALPs. A cDNA library from Peruvian L. laeta venom glands was constructed and sequenced by MiSeq (Illumina) sequencer. After assembly, the resulting sequences were annotated, seeking out for similarity with previously described LALPs. Nine possible LALPs isoforms from Peruvian L. laeta venom were identified and the results were validated by in silico and in vitro experiments. This study contributes to a better understanding of the molecular diversity of Loxosceles venom and provide insights about the action of LALPs.

Acanthocytosis and brain damage in area postrema and choroid plexus: Description of novel signs of Loxosceles apachea envenomation in rats.

Loxocelism is a neglected medical problem that depends on its severity, can cause a cutaneous or viscero-cutaneous syndrome. This syndrome is characterized by hemostatic effects and necrosis, and the severity of the loxoscelism depends on the amount of venom injected, the zone of inoculation, and the species. In the Chihuahuan desert, the most abundant species is L. apachea. Its venom and biological effects are understudied, including neurological effects. Thus, our aim is to explore the effect of this regional species of medical interest in the United States-Mexico border community, using rat blood and central nervous system (CNS), particularly, two brain structures involved in brain homeostasis, Area postrema (AP) and Choroid plexus (PC). L. apachea specimens were collected and venom was obtained. Different venom concentrations (0, 0.178 and 0.87 µg/g) were inoculated into Sprague-Dawley rats (intraperitoneal injection). Subsequently, blood was extracted and stained with Wright staining; coronal sections of AP were obtained and stained with Hematoxylin-Eosin (HE) staining and laminin γ immunolabelling, the same was done with CP sections. Blood, AP and CP were observed under the microscope and abnormalities in erythrocytes and fluctuation in leukocyte types were described and quantified in blood. Capillaries were also quantified in AP and damage was described in CP. L. apachea venom produced a segmented neutrophil increment (neutrophilia), lymphocyte diminishment (leukopenia) and erythrocytes presented membrane abnormalities (acanthocytosis). Extravasated erythrocytes were observed in HE stained sections from both, AP and CP, which suggest that near to this section a hemorrhage is present; through immunohistofluorescence, a diminishment of laminin γ was observed in AP endothelial cells and in CP ependymal cells when these structures were exposed to L. apachea venom. In conclusion, L. apachea venom produced leukopenia, netrophilia and acanthocytosis in rat peripheral blood, and also generated hemorrhages on AP and CP through degradation of laminin γ.

Design and Production of a Recombinant Hybrid Toxin to Raise Protective Antibodies Against Loxosceles Spider Venom.

Human accidents with spiders of the genus Loxosceles are an important health problem affecting thousands of people worldwide. Patients evolve to severe local injuries and, in many cases, to systemic disturbances as acute renal failure, in which cases antivenoms are considered to be the most effective treatment. However, for antivenom production, the extraction of the venom used in the immunization process is laborious and the yield is very low. Thus, many groups have been exploring the use of recombinant Loxosceles toxins, particularly phospholipases D (PLDs), to produce the antivenom. Nonetheless, some important venom activities are not neutralized by anti-PLD antibodies. Astacin-like metalloproteases (ALMPs) are the second most expressed toxin acting on the extracellular matrix, indicating the importance of its inclusion in the antigen's formulation to provide a better antivenom. Here we show the construction of a hybrid recombinant immunogen, called LgRec1ALP1, composed of hydrophilic regions of the PLD and the ALMP toxins from Loxosceles gaucho. Although the LgRec1ALP1 was expressed as inclusion bodies, it resulted in good yields and it was effective to produce neutralizing antibodies in mice. The antiserum neutralized fibrinogenolytic, platelet aggregation and dermonecrotic activities elicited by L. gaucho, L. laeta, and L. intermedia venoms, indicating that the hybrid recombinant antigen may be a valuable source for the production of protective antibodies against Loxosceles ssp. venoms. In addition, the hybrid recombinant toxin approach may enrich and expand the alternative antigens for antisera production for other venoms.

Acute kidney injury due to systemic Loxoscelism: a cross-sectional study in Northeast Brazil.

INTRODUCTION: Loxoscelism is a clinical condition involving spiders of the genus Loxosceles. One of the most severe complications is acute kidney injury (AKI). This study aimed to investigate AKI and other complications associated with loxoscelism. METHODS: We analyzed cases diagnosed with loxoscelism in an area where most accidents were caused by Loxosceles amazonica from January 2010 to December 2015. AKI was defined according to the KDIGO criteria. RESULTS: Forty-five patients were recorded: 95.6% presented characteristic necrotic skin lesions and 13.3% AKI. CONCLUSIONS: Loxoscelism could cause kidney involvement which is uncommon and could lead to the death of these patients.

Acute kidney injury due to systemic Loxoscelism: a cross-sectional study in Northeast Brazil

Abstract INTRODUCTION: Loxoscelism is a clinical condition involving spiders of the genus Loxosceles. One of the most severe complications is acute kidney injury (AKI). This study aimed to investigate AKI and other complications associated with loxoscelism. METHODS: We analyzed cases diagnosed with loxoscelism in an area where most accidents were caused by Loxosceles amazonica from January 2010 to December 2015. AKI was defined according to the KDIGO criteria. RESULTS: Forty-five patients were recorded: 95.6% presented characteristic necrotic skin lesions and 13.3% AKI. CONCLUSIONS: Loxoscelism could cause kidney involvement which is uncommon and could lead to the death of these patients.

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.

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.

Platelet participation in the pathogenesis of dermonecrosis induced by Loxosceles gaucho venom.

Loxosceles gaucho spider venom induces in vitro platelet activation and marked thrombocytopenia in rabbits. Herein, we investigated the involvement of platelets in the development of the dermonecrosis induced by L. gaucho venom, using thrombocytopenic rabbits as a model. L. gaucho venom evoked a drop in platelet and neutrophil counts 4 h after venom injection. Ecchymotic areas at the site of venom inoculation were noticed as soon as 4 h in thrombocytopenic animals but not in animals with initial normal platelet counts. After 5 days, areas of scars in thrombocytopenic animals were also larger, evidencing the marked development of lesions in the condition of thrombocytopenia. Histologically, local hemorrhage, collagen fiber disorganization, and edema were more severe in thrombocytopenic animals. Leukocyte infiltration, predominantly due to polymorphonuclears, was observed in the presence or not of thrombocytopenia. Thrombus formation was demonstrated by immunohistochemistry at the microvasculature, and it occurred even under marked thrombocytopenia. Taken together, platelets have an important role in minimizing not only the hemorrhagic phenomena but also the inflammatory and wound-healing processes, suggesting that cutaneous loxoscelism may be aggravated under thrombocytopenic conditions.