Ontogenetic change in the venom composition of one Mexican black-tailed rattlesnake (Crotalus molossus nigrescens) from Durango, Mexico.

To corroborate the ontogenetic shift in the venom composition of the Mexican Black-tailed Rattlesnake (Crotalus molossus nigrescens) previously reported through the census approach, we evaluated the shift in the protein profile, lethality, and proteolytic and phospholipase activities of four venom samples obtained in 2015, 2018, 2019, and 2021 from one C. m. nigrescens individual (CMN06) collected in Durango, Mexico. We demonstrated that the venom of C. m. nigrescens changed from a myotoxin-rich venom to a phospholipase A2 and snake venom metalloproteinase-rich venom. Additionally, the proteolytic and phospholipase activities increased with age, but the lethality decreased approximately three times.

Mitogen-Activated Protein Kinases SvPmk1 and SvMps1 Are Critical for Abiotic Stress Resistance, Development and Pathogenesis of Sclerotiophoma versabilis.

Mitogen-activated protein kinase (MAPK) signaling pathways are evolutionarily conserved in eukaryotes and modulate responses to both internal and external stimuli. Pmk1 and Mps MAPK pathways regulate stress tolerance, vegetative growth and cell wall integrity in Saccharomyces cerevisiae and Pyricularia oryzae. Here, we deployed genetic and cell biology strategies to investigate the roles of the orthologs of Pmk1 and Mps1 in Sclerotiophoma versabilis (herein referred to as SvPmk1 and SvMps1, respectively). Our results showed that SvPmk1 and SvMps1 are involved in hyphal development, asexual reproduction and pathogenesis in S. versabilis. We found that ∆Svpmk1 and ∆Svmps1 mutants have significantly reduced vegetative growths on PDA supplemented with osmotic stress-inducing agents, compared to the wild type, with ∆Svpmps1 being hypersensitive to hydrogen peroxide. The two mutants failed to produce pycnidia and have reduced pathogenicity on Pseudostellaria heterophylla. Unlike SvPmk1, SvMps1 was found to be indispensable for the fungal cell wall integrity. Confocal microscopic analyses revealed that SvPmk1 and SvMps1 are ubiquitously expressed in the cytosol and nucleus. Taken together, we demonstrate here that SvPmk1 and SvMps1 play critical roles in the stress resistance, development and pathogenesis of S. versabilis.

Basement membrane degradation and inflammation play a role in the pulmonary hemorrhage induced by a P-III snake venom metalloproteinase.

Snakebite envenoming is a neglected tropical disease affecting millions of people every year, especially in vulnerable rural populations in the developing world. Viperid snakes cause envenomings characterized by a complex pathophysiology which includes local and systemic hemorrhage due to the action of snake venom metalloproteinases (SVMPs). The pathogenesis of SVMP-induced systemic hemorrhage has not been investigated in detail. This study explored the pulmonary hemorrhage induced in a murine model by a P-III SVMP from the venom of Crotalus simus. Histological analysis revealed extravasation in the lungs as early as 15 min after intravenous injection of the toxin, and hemorrhage increased at 360 min. Western blot analysis demonstrated the cleavage of basement membrane (BM) proteins in lung homogenates and in bronchoalveolar lavage fluid, implying an enzymatic disruption of this extracellular matrix structure at the capillary-alveolar barrier. Likewise, alveolar edema was observed, with an increment in protein concentration in the bronchoalveolar lavage fluid, and a neutrophil-rich inflammatory infiltrate was present in the parenchyma of the lungs as part of the inflammatory reaction. Pretreatment of mice with indomethacin, pentoxifylline and an anti-neutrophil antibody resulted in a significant decrease in pulmonary hemorrhage at 360 min. These findings suggest that this P-III SVMP induces acute lung injury through the direct action of this enzyme in the capillary-alveolar barrier integrity, as revealed by BM degradation, and as a consequence of the inflammatory reaction that develops in lung tissue. Our findings provide novel clues to understand the mechanism of action of hemorrhagic SVMPs in the lungs.

Differential coagulotoxicity of metalloprotease isoforms from Bothrops neuwiedi snake venom and consequent variations in antivenom efficacy.

Bothrops (lance-head pit vipers) venoms are rich in weaponised metalloprotease enzymes (SVMP). These toxic enzymes are structurally diverse and functionally versatile. Potent coagulotoxicity is particularly important for prey capture (via stroke-induction) and relevant to human clinical cases (due to consumption of clotting factors including the critical depletion of fibrinogen). In this study, three distinct isoforms of P-III class SVMPs (IC, IIB and IIC), isolated from Bothrops neuwiedi venom, were evaluated for their differential capacities to affect hemostasis of prey and human plasma. Furthermore, we tested the relative antivenom neutralisation of effects upon human plasma. The toxic enzymes displayed differential procoagulant potency between plasma types, and clinically relevant antivenom efficacy variations were observed. Of particular importance was the confirmation the antivenom performed better against prothrombin activating toxins than Factor X activating toxins, which is likely due to the greater prevalence of the former in the immunising venoms used for antivenom production. This is clinically relevant as the enzymes displayed differential potency in this regard, with one (IC) in particular being extremely potent in activating Factor X and thus was correspondingly poorly neutralised. This study broadens the current understanding about the adaptive role of the SVMPs, as well as highlights how the functional diversity of SVMP isoforms can influence clinical outcomes. Key Contribution: Our findings shed light upon the hemorrhagic and coagulotoxic effects of three SVMPs of the P-III class, as well as the coagulotoxic effects of SVMPs on human, avian and amphibian plasmas. Antivenom neutralised prothrombin-activating isoforms better than Factor X activating isoforms.

Hemorrhagic and procoagulant P-III snake venom metalloproteinases differ in their binding to the microvasculature of mouse cremaster muscle.

Binding of two P-III snake venom metalloproteinase (SVMPs), one procoagulant and one hemorrhagic, to microvessels was compared in an ex vivo model. The procoagulant SVMP did not bind to the microvasculature, in contrast to the clear localization on microvessels of the hemorrhagic SVMP. Deglycosylation of the procoagulant enzyme did not enable this toxin to bind to microvessels, suggesting that glycosylation is not interfering with binding. These observations suggest that procoagulant SVMPs lack exosites for interaction with microvessels components.

Inflammatory Reaction Induced by Two Metalloproteinases Isolated from Bothrops atrox Venom and by Fragments Generated from the Hydrolysis of Basement Membrane Components.

Snake venom metalloproteinases (SVMPs) play an important role in local tissue damage of snakebite patients, mostly by hydrolysis of basement membrane (BM) components. We evaluated the proinflammatory activity of SVMPs Atroxlysin-Ia (ATXL) and Batroxrhagin (BATXH) from Bothrops atrox venom and their hydrolysis products of Matrigel. BALB/c mice were injected with SVMPs (2 µg), for assessment of paw edema and peritoneal leukocyte accumulation. Both SVMPs induced edema, representing an increase of ~70% of the paw size. Leukocyte infiltrates reached levels of 6 × 106 with ATXL and 5 × 106 with BATXH. TNF-α was identified in the supernatant of BATXH-or venom-stimulated MPAC cells. Incubation of Matrigel with the SVMPs generated fragments, including peptides from Laminin, identified by LC-MS/MS. The Matrigel hydrolysis peptides caused edema that increased 30% the paw size and promoted leukocyte accumulation (4-5 × 106) to the peritoneal cavity, significantly higher than Matrigel control peptides 1 and 4 h after injection. Our findings suggest that ATXL and BATXH are involved in the inflammatory reaction observed in B. atrox envenomings by direct action on inflammatory cells or by releasing proinflammatory peptides from BM proteins that may amplify the direct action of SVMPs through activation of endogenous signaling pathways.

Differential coagulotoxicity of metalloprotease isoforms from Bothrops neuwiedi snake venom and consequent variations in antivenom efficacy

Bothrops (lance-head pit vipers) venoms are rich in weaponised metalloprotease enzymes (SVMP). These toxic enzymes are structurally diverse and functionally versatile. Potent coagulotoxicity is particularly important for prey capture (via stroke-induction) and relevant to human clinical cases (due to consumption of clotting factors including the critical depletion of fibrinogen). In this study, three distinct isoforms of P-III class SVMPs (IC, IIB and IIC), isolated from Bothrops neuwiedi venom, were evaluated for their differential capacities to affect hemostasis of prey and human plasma. Furthermore, we tested the relative antivenom neutralisation of effects upon human plasma. The toxic enzymes displayed differential procoagulant potency between plasma types, and clinically relevant antivenom efficacy variations were observed. Of particular importance was the confirmation the antivenom performed better against prothrombin activating toxins than Factor X activating toxins, which is likely due to the greater prevalence of the former in the immunising venoms used for antivenom production. This is clinically relevant as the enzymes displayed differential potency in this regard, with one (IC) in particular being extremely potent in activating Factor X and thus was correspondingly poorly neutralised. This study broadens the current understanding about the adaptive role of the SVMPs, as well as highlights how the functional diversity of SVMP isoforms can influence clinical outcomes.

Inflammatory reaction induced by two metalloproteinases isolated from bothrops atrox venom and by fragments generated from the hydrolysis of basement membrane components

Snake venom metalloproteinases (SVMPs) play an important role in local tissue damage of snakebite patients, mostly by hydrolysis of basement membrane (BM) components. We evaluated the proinflammatory activity of SVMPs Atroxlysin-Ia (ATXL) and Batroxrhagin (BATXH) from Bothrops atrox venom and their hydrolysis products of Matrigel. BALB/c mice were injected with SVMPs (2 µg), for assessment of paw edema and peritoneal leukocyte accumulation. Both SVMPs induced edema, representing an increase of ~70% of the paw size. Leukocyte infiltrates reached levels of 6 × 106 with ATXL and 5 × 106 with BATXH. TNF-a was identified in the supernatant of BATXH—or venom-stimulated MPAC cells. Incubation of Matrigel with the SVMPs generated fragments, including peptides from Laminin, identified by LC–MS/MS. The Matrigel hydrolysis peptides caused edema that increased 30% the paw size and promoted leukocyte accumulation (4–5 × 106) to the peritoneal cavity, significantly higher than Matrigel control peptides 1 and 4 h after injection. Our findings suggest that ATXL and BATXH are involved in the inflammatory reaction observed in B. atrox envenomings by direct action on inflammatory cells or by releasing proinflammatory peptides from BM proteins that may amplify the direct action of SVMPs through activation of endogenous signaling pathways

Biological and Proteolytic Variation in the Venom of Crotalus scutulatus scutulatus from Mexico.

Rattlesnake venoms may be classified according to the presence/absence and relative abundance of the neurotoxic phospholipases A 2 s (PLA 2 s), such as Mojave toxin, and snake venom metalloproteinases (SVMPs). In Mexico, studies to determine venom variation in Mojave Rattlesnakes (Crotalus scutulatus scutulatus) are limited and little is known about the biological and proteolytic activities in this species. Tissue (34) and venom (29) samples were obtained from C. s. scutulatus from different locations within their distribution in Mexico. Mojave toxin detection was carried out at the genomic (by PCR) and protein (by ELISA) levels for all tissue and venom samples. Biological activity was tested on representative venoms by measuring LD 50 and hemorrhagic activity. To determine the approximate amount of SVMPs, 15 venoms were separated by RP-HPLC and variation in protein profile and proteolytic activity was evaluated by SDS-PAGE (n = 28) and Hide Powder Azure proteolytic analysis (n = 27). Three types of venom were identified in Mexico which is comparable to the intraspecific venom diversity observed in the Sonoran Desert of Arizona, USA: Venom Type A (∼Type II), with Mojave toxin, highly toxic, lacking hemorrhagic activity, and with scarce proteolytic activity; Type B (∼Type I), without Mojave toxin, less toxic than Type A, highly hemorrhagic and proteolytic; and Type A + B, containing Mojave toxin, as toxic as venom Type A, variable in hemorrhagic activity and with intermediate proteolytic activity. We also detected a positive correlation between SVMP abundance and hemorrhagic and proteolytic activities. Although more sampling is necessary, our results suggest that venoms containing Mojave toxin and venom lacking this toxin are distributed in the northwest and southeast portions of the distribution in Mexico, respectively, while an intergradation in the middle of both zones is present.

Peptidomimetic hydroxamate metalloproteinase inhibitors abrogate local and systemic toxicity induced by Echis ocellatus (saw-scaled) snake venom.

The ability of two peptidomimetic hydroxamate metalloproteinase inhibitors, Batimastat and Marimastat, to abrogate toxic and proteinase activities of the venom of Echis ocellatus from Cameroon and Ghana was assessed. Since this venom largely relies for its toxicity on the action of zinc-dependent metalloproteinases (SVMPs), the hypothesis was raised that toxicity could be largely eliminated by using SVMP inhibitors. Both hydroxamate molecules inhibited local and pulmonary hemorrhagic, in vitro coagulant, defibrinogenating, and proteinase activities of the venoms in conditions in which venom and inhibitors were incubated prior to the test. In addition, the inhibitors prolonged the time of death of mice receiving 4 LD50s of venom by the intravenous route. Lower values of IC50 were observed for in vitro and local hemorrhagic activities than for systemic effects. When experiments were performed in conditions that simulated the actual circumstances of snakebite, i.e. by administering the inhibitor after envenoming, Batimastat completely abrogated local hemorrhage if injected immediately after venom. Moreover, it was also effective at inhibiting lethality and defibrinogenation when venom and inhibitor were injected by the intraperitoneal route. Results suggest that these, and possibly other, metalloproteinase inhibitors may become an effective adjunct therapy in envenomings by E. ocellatus when administered at the anatomic site of venom injection rapidly after the bite.

Viperid Envenomation Wound Exudate Contributes to Increased Vascular Permeability via a DAMPs/TLR-4 Mediated Pathway.

Viperid snakebite envenomation is characterized by inflammatory events including increase in vascular permeability. A copious exudate is generated in tissue injected with venom, whose proteomics analysis has provided insights into the mechanisms of venom-induced tissue damage. Hereby it is reported that wound exudate itself has the ability to induce increase in vascular permeability in the skin of mice. Proteomics analysis of exudate revealed the presence of cytokines and chemokines, together with abundant damage associated molecular pattern molecules (DAMPs) resulting from both proteolysis of extracellular matrix and cellular lysis. Moreover, significant differences in the amounts of cytokines/chemokines and DAMPs were detected between exudates collected 1 h and 24 h after envenomation, thus highlighting a complex temporal dynamic in the composition of exudate. Pretreatment of mice with Eritoran, an antagonist of Toll-like receptor 4 (TLR4), significantly reduced the exudate-induced increase in vascular permeability, thus suggesting that DAMPs might be acting through this receptor. It is hypothesized that an "Envenomation-induced DAMPs cycle of tissue damage" may be operating in viperid snakebite envenomation through which venom-induced tissue damage generates a variety of DAMPs which may further expand tissue alterations.

Purification and Immunoprotection Evaluation of AaHIV from Complex Venom Metalloproteinases of Deinagkistrodon acutus.

The aim of this study was to investigate the immunoprotective effects of AaHIV in mice. After purification, a 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed. Bicinchoninic acid was used to determine the molecular weight and concentration of AaHIV. AaHIV, venom complex (VC), and phosphate buffered saline (PBS) were subsequently used to immunize the mice three times, and the blood was sampled 1 week after the third immunization to determine the serum immunoglobulin G (IgG) antibody titer. A skin-bleeding inhibition assay and toxin-eliminating assay were performed on the immunized mice. The purity and concentration of AaHIV were 86.6% and 1.20 mg/mL, respectively. The AaHIV group exhibited higher antibody titers than the VC group. The survival rate of the AaHIV group (7/10) was significantly higher than that of the PBS group (0/10) (P = 0.0031). The high titer of antibodies induced by AaHIV partially neutralized the bleeding activity of the Deinagkistrodon acutus venom complex.

Distal M domain of cobra ADAM-like metalloproteinase mediates the binding of positively charged cysteine-rich domain to αvß3 integrin in the suppression of cell migration.

We have previously identified two new P-III type ADAM-like snake venom metalloproteinases (SVMPs), i.e., atragin and kaouthiagin-like, from Taiwan cobra venom and determined their 3D structures with a distinct C- and I-shaped metalloproteinase/disintegrin-like/cysteine-rich (MDC) modular architecture. Herein, we investigated their functional targets to elucidate the role of cobra SVMPs in perturbing wound healing in snakebite victims. We showed that the non-RGD (Arg-Gly-Asp) C-shaped SVMP atragin binds about ten-fold stronger than the RGD-containing I-shaped SVMP kaouthiagin-like to αvß3 integrin in the surface-immobilized form. Atragin binds to αvß3 integrin through a novel interaction mode involving distal M and C domains via the RRN sequence motif in the hyper variable loop. In a cell adhesion assay, the adhesion of fibroblasts to atragin was mediated by αvß3 integrin. Furthermore, atragin inhibited wound healing and suppressed cell migration in a αvß3 integrin-dependent manner. These results, together with our previous demonstration of non-cytotoxic cobra CTX A5 in targeting αvß3 integrin, suggest that cobra venom consists of several non-RGD toxins with integrin-binding specificity that could perturb wound healing in snakebite victims.

Inflammation and oxidative stress in viper bite: an insight within and beyond.

Though systemic and local manifestations of snakebite are considered serious, the relevance of oxidative stress in viper bite pathology is largely denied. However, over the past decade, studies have provided substantial evidence for the presence of persistent oxidative stress in viper bite victims. This review aims at highlighting the disturbances in redox homeostasis soon after viper envenomation and its implications in the pathomechanism of secondary/long term complications including thrombocytopenia, hypopituitarism, infertility, renal abnormalities and persistent local tissue degradation. Both enzymatic and non-enzymatic components of viper venom play a pivotal role in bringing redox turbulence in victims. Venom-induced hemorrhage and necrosis with subsequent release of damage associated molecular pattern (DAMPs) molecules also contribute to sustained oxidative stress and inflammation. Studies have demonstrated that along with anti-venom therapy an antioxidant treatment during the early stages of viper bite and also long term treatment could help to reduce the occurrence of secondary/long term complications. Further, proper knowledge regarding the pathophysiology will allow for exploration of new avenues in the treatment of viper bite.

High resolution analysis of snake venom metalloproteinase (SVMP) peptide bond cleavage specificity using proteome based peptide libraries and mass spectrometry. (AU)

Both serine and metalloproteinases have been shown to play the role of toxins in the venoms of many snakes. Determination of the natural protein substrates of these toxins is an important feature in the toxinological characterization of these proteinases. Furthermore, characterization of their peptide bond specificity is of value for understanding active site preference of the proteinase associated with effective proteolysis as well as of use in the design of peptide substrates and inhibitor lead compounds. Typically the determination of peptide bond cleavage specificity of snake venom serine proteinases (SVSPs) and snake venom metalloproteinases (SVMPs) has been performed using limited sets of peptides or small oligopeptides as experimental substrates. Although this approach has yielded valuable data it is generally limited in scope due to the relatively small sets of substrates used to generate the consensus specificity sequences for these proteinases. In this study we use a large, plasma based, proteome-derived peptide library as substrates along with mass spectrometry to explore the peptide bond specificity of three PI SVMPs and one PIII SVMP to determine their individual peptide cleavage consensus sequences. All of the proteinases assayed displayed a clear preference for a leucine residue in the P1Œ site. Careful analysis of the specificity profiles of the SVMPs examined showed interesting differences in the preferences at the other P and PŒ sites suggesting functional differences between these proteinases.