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Viper venom botox: the molecular origin and evolution of the waglerin peptides used in anti-wrinkle skin cream

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The molecular origin of waglerin peptides has remained enigmatic despite their industrial application in skin cream products to paralyse facial muscles and thus reduce the incidence of wrinkles. Here we show that these neurotoxic peptides are the result of de novo evolution within the prepro region of the C-type natriuretic peptide gene in Tropidolaemus venoms, at a site distinct from the domain encoding for the natriuretic peptide. It is the same region that yielded the azemiopsin peptides from Azemiops feae, indicative of a close relationship of this toxin gene between these two genera. The precursor region for the molecular evolution is a biodiversity hotspot that has yielded other novel bioactive peptides with novel activities. We detail the diversity of components in this and other species in order to explore what characteristics enable it to be such a biodiscovery treasure trove. The unusual function of Tropidolaemus venoms may have been selected for due to evolutionary pressures brought about by a high likelihood of prey escape.

Three-Finger Toxin Diversification in the Venoms of Cat-Eye Snakes (Colubridae: Boiga)

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The Asian genus Boiga (Colubridae) is among the better studied non-front-fanged snake lineages, because their bites have minor, but noticeable, effects on humans. Furthermore, B. irregularis has gained worldwide notoriety for successfully invading Guam and other nearby islands with drastic impacts on the local bird populations. One of the factors thought to allow B. irregularis to become such a noxious pest is irditoxin, a dimeric neurotoxin composed of two three-finger toxins (3FTx) joined by a covalent bond between two newly evolved cysteines. Irditoxin is highly toxic to diapsid (birds and reptiles) prey, but roughly 1000 × less potent to synapsids (mammals). Venom plays an important role in the ecology of all species of Boiga, but it remains unknown if any species besides B. irregularis produce irditoxin-like dimeric toxins. In this study, we use transcriptomic analyses of venom glands from five species [B. cynodon, B. dendrophila dendrophila, B. d. gemmicincta, B. irregularis (Brisbane population), B. irregularis (Sulawesi population), B. nigriceps, B. trigonata] and proteomic analyses of B. d. dendrophila and a representative of the sister genus Toxicodryas blandingii to investigate the evolutionary history of 3FTx within Boiga and its close relative. We found that 92.5% of Boiga 3FTx belong to a single clade which we refer to as denmotoxin-like because of the close relation between these toxins and the monomeric denmotoxin according to phylogenetic, sequence clustering, and protein similarity network analyses. We show for the first time that species beyond B. irregularis secrete 3FTx with additional cysteines in the same position as both the A and B subunits of irditoxin. Transcripts with the characteristic mutations are found in B. d. dendrophila, B. d. gemmicincta, B. irregularis (Brisbane population), B. irregularis (Sulawesi population), and B. nigriceps. These results are confirmed by proteomic analyses that show direct evidence of dimerization within the venom of B. d. dendrophila, but not T. blandingii. Our results also suggest the possibility of novel dimeric toxins in other genera such as Telescopus and Trimorphodon. All together, this suggests that the origin of these peculiar 3FTx is far earlier than was appreciated and their evolutionary history has been complex.

The snake with the scorpion's sting: novel three-finger toxin sodium channel activators from the venom of the long-glanded blue coral snake (Calliophis bivirgatus)

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Millions of years of evolution have fine-tuned the ability of venom peptides to rapidly incapacitate both prey and potential predators. Toxicofera reptiles are characterized by serous-secreting mandibular or maxillary glands with heightened levels of protein expression. These glands are the core anatomical components of the toxicoferan venom system, which exists in myriad points along an evolutionary continuum. Neofunctionalisation of toxins is facilitated by positive selection at functional hotspots on the ancestral protein and venom proteins have undergone dynamic diversification in helodermatid and varanid lizards as well as advanced snakes. A spectacular point on the venom system continuum is the long-glanded blue coral snake (Calliophis bivirgatus), a specialist feeder that preys on fast moving, venomous snakes which have both a high likelihood of prey escape but also represent significant danger to the predator itself. The maxillary venom glands of C. bivirgatus extend one quarter of the snake's body length and nestle within the rib cavity. Despite the snake's notoriety its venom has remained largely unstudied. Here we show that the venom uniquely produces spastic paralysis, in contrast to the flaccid paralysis typically produced by neurotoxic snake venoms. The toxin responsible, which we have called calliotoxin (delta-elapitoxin-Cb1a), is a three-finger toxin (3FTx). Calliotoxin shifts the voltage-dependence of NaV1.4 activation to more hyperpolarised potentials, inhibits inactivation, and produces large ramp currents, consistent with its profound effects on contractile force in an isolated skeletal muscle preparation. Voltage-gated sodium channels (NaV) are a particularly attractive pharmacological target as they are involved in almost all physiological processes including action potential generation and conduction. Accordingly, venom peptides that interfere with NaV function provide a key defensive and predatory advantage to a range of invertebrate venomous species including cone snails, scorpions, spiders, and anemones. Enhanced activation or delayed inactivation of sodium channels by toxins is associated with the extremely rapid onset of tetanic/excitatory paralysis in envenomed prey animals. A strong selection pressure exists for the evolution of such toxins where there is a high chance of prey escape. However, despite their prevalence in other venomous species, toxins causing delay of sodium channel inhibition have never previously been described in vertebrate venoms. Here we show that NaV modulators, convergent with those of invertebrates, have evolved in the venom of the long-glanded coral snake. Calliotoxin represents a functionally novel class of 3FTx and a structurally novel class of NaV toxins that will provide significant insights into the pharmacology and physiology of NaV. The toxin represents a remarkable case of functional convergence between invertebrate and vertebrate venom systems in response to similar selection pressures. These results underscore the dynamic evolution of the Toxicofera reptile system and reinforces the value of using evolution as a roadmap for biodiscovery.

The evolution of fangs, venom, and mimicry systems in blenny fishes

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Venom systems have evolved on multiple occasions across the animal kingdom, and they can act as key adaptations to protect animals from predators [1]. Consequently, venomous animals serve as models for a rich source of mimicry types, as non-venomous species benefit from reductions in predation risk by mimicking the coloration, body shape, and/or movement of toxic counterparts [2–5]. The frequent evolution of such deceitful imitations provides notable examples of phenotypic convergence and are often invoked as classic exemplars of evolution by natural selection. Here, we investigate the evolution of fangs, venom, and mimetic relationships in reef fishes from the tribe Nemophini (fangblennies). Comparative morphological analyses reveal that enlarged canine teeth (fangs) originated at the base of the Nemophini radiation and have enabled a micropredatory feeding strategy in non-venomous Plagiotremus spp. Subsequently, the evolution of deep anterior grooves and their coupling to venom secretory tissue provide Meiacanthus spp. with toxic venom that they effectively employ for defense. We find that fangblenny venom contains a number of toxic components that have been independently recruited into other animal venoms, some of which cause toxicity via interactions with opioid receptors, and result in a multifunctional biochemical phenotype that exerts potent hypotensive effects. The evolution of fangblenny venom has seemingly led to phenotypic convergence via the formation of a diverse array of mimetic relationships that provide protective (Batesian mimicry) and predatory (aggressive mimicry) benefits to other fishes [2, 6]. Our results further our understanding of how novel morphological and biochemical adaptations stimulate ecological interactions in the natural world.

The cardiovascular and neurotoxic effects of the venoms of six bony and cartilaginous fish species

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Fish venoms are often poorly studied, in part due to the difficulty in obtaining, extracting, and storing them. In this study, we characterize the cardiovascular and neurotoxic effects of the venoms from the following six species of fish: the cartilaginous stingrays Neotrygon kuhlii and Himantura toshi, and the bony fish Platycephalus fucus, Girella tricuspidata, Mugil cephalus, and Dentex tumifrons. All venoms (10–100 μg/kg, i.v.), except G. tricuspidata and P. fuscus, induced a biphasic response on mean arterial pressure (MAP) in the anesthetised rat. P. fucus venom exhibited a hypotensive response, while venom from G. tricuspidata displayed a single depressor response. All venoms induced cardiovascular collapse at 200 μg/kg, i.v. The in vitro neurotoxic effects of venom were examined using the chick biventer cervicis nerve-muscle (CBCNM) preparation. N. kuhlii, H. toshi, and P. fucus venoms caused concentration-dependent inhibition of indirect twitches in the CBCNM preparation. These three venoms also inhibited responses to exogenous acetylcholine (ACh) and carbachol (CCh), but not potassium chloride (KCl), indicating a post-synaptic mode of action. Venom from G. tricuspidata, M. cephalus, and D. tumifrons had no significant effect on indirect twitches or agonist responses in the CBCNM. Our results demonstrate that envenoming by these species of fish may result in moderate cardiovascular and/or neurotoxic effects. Future studies aimed at identifying the molecules responsible for these effects could uncover potentially novel lead compounds for future pharmaceuticals, in addition to generating new knowledge about the evolutionary relationships between venomous animals.

Proteomic and functional variation within black snake venoms (Elapidae: Pseudechis)

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Pseudechis (black snakes) is an Australasian elapid snake genus that inhabits much of mainland Australia, with two representatives confined to Papua New Guinea. The present study is the first to analyse the venom of all 9 described Pseudechis species (plus one undescribed species) to investigate the evolution of venom composition and functional activity. Proteomic results demonstrated that the typical Pseudechis venom profile is dominated by phospholipase A2 toxins. Strong cytotoxicity was the dominant function for most species. P. porphyriacus, the most basal member of the genus, also exhibited the most divergent venom composition, being the only species with appreciable amounts of procoagulant toxins. The relatively high presence of factor Xa recovered in P. porphyriacus venom may be related to a predominantly amphibian diet. Results of this study provide important insights to guide future ecological and toxinological investigations.

Factor X activating atractaspis snake venoms and the relative coagulotoxicity neutralising efficacy of African antivenoms

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Atractaspis snake species are enigmatic in their natural history, and venom effects are correspondingly poorly described. Bite reports are scarce but bites have been described as causing severe hypertension, profound local tissue damage leading to amputation, and deaths are on record. Clinical descriptions have largely concentrated upon tissue effects, and research efforts have focused upon the blood-pressure affecting sarafotoxins. However, coagulation disturbances suggestive of procoagulant functions have been reported in some clinical cases, yet this aspect has been uninvestigated. We used a suite of assays to investigate the coagulotoxic effects of venoms from six different Atractaspis specimens from central Africa. The procoagulant function of factor X activation was revealed, as was the pseudo-procoagulant function of direct cleavage of fibrinogen into weak clots. The relative neutralization efficacy of South African Antivenom Producer's antivenoms on Atractaspis venoms were boomslang>polyvalent>saw-scaled viper. While the boomslang antivenom was the most effective on Atractaspis venoms, the ability to neutralize the most potent Atractaspis species in this study was up to 4-6 times less effective than boomslang antivenom neutralizes boomslang venom. Therefore, while these results suggest cross-reactivity of boomslang antivenom with the unexpectedly potent coagulotoxic effects of Atractaspis venoms, a considerable amount of this rare antivenom may be needed. This report thus reveals potent venom actions upon blood coagulation that may lead to severe clinical effects with limited management strategies.

Enter the dragon: the dynamic and multifunctional evolution of anguimorpha lizard venoms

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While snake venoms have been the subject of intense study, comparatively little work has been done on lizard venoms. In this study, we have examined the structural and functional diversification of anguimorph lizard venoms and associated toxins, and related these results to dentition and predatory ecology. Venom composition was shown to be highly variable across the 20 species of Heloderma, Lanthanotus, and Varanus included in our study. While kallikrein enzymes were ubiquitous, they were also a particularly multifunctional toxin type, with differential activities on enzyme substrates and also ability to degrade alpha or beta chains of fibrinogen that reflects structural variability. Examination of other toxin types also revealed similar variability in their presence and activity levels. The high level of venom chemistry variation in varanid lizards compared to that of helodermatid lizards suggests that venom may be subject to different selection pressures in these two families. These results not only contribute to our understanding of venom evolution but also reveal anguimorph lizard venoms to be rich sources of novel bioactive molecules with potential as drug design and development lead compounds.

Does size matter? Venom proteomic and functional comparison between night adder species (Viperidae: Causus) with short and long venom glands

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Night adders (Causus species within the Viperidae family) are amphibian specialists and a common source of snakebite in Africa. Some species are unique in that they have the longest venom glands of any viper, extending approximately 10% of the body length. Despite their potential medical importance and evolutionary novelty, their venom has received almost no research attention. In this study, venoms from a short-glanded species (C. lichtensteinii) and from a long-glanded species (C. rhombeatus) were compared using a series of proteomic and bioactivity testing techniques to investigate and compare the toxin composition and functioning of the venoms of these two species. Both C. rhombeatus and C. lichtensteinii were similar in overall venom composition and inhibition of blood coagulation through non-clotting proteolytic cleavage of fibrinogen. While the 1D gel profiles were very similar to each other in the toxin types present, 2D gel analyses revealed isoformic differences within each toxin classes. This variation was congruent with differential efficacy of South African Institute for Medical Research snake polyvalent antivenom, with C. lichtensteinii unaffected at the dose tested while C. rhombeatus was moderately but significantly neutralized. Despite the variation within toxin classes, the similarity in overall venom biochemistry suggests that the selection pressure for the evolution of long glands served to increase venom yield in order to subjugate proportionally large anurans as a unique form of niche partitioning, and is not linked to significant changes in venom function. These results not only contribute to the body of venom evolution knowledge but also highlight the limited clinical management outcomes for Causus envenomations.

Correlation between ontogenetic dietary shifts and venom variation in Australian brown snakes (Pseudonaja)

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Venom is a key evolutionary trait, as evidenced by its widespread convergent evolution across the animal kingdom. In an escalating prey-predator arms race, venoms evolve rapidly to guarantee predatory or defensive success. Variation in venom composition is ubiquitous among snakes. Here, we tested variation in venom activity on substrates relevant to blood coagulation among Pseudonaja (brown snake) species, Australian elapids responsible for the majority of medically important human envenomations in Australia. A functional approach was employed to elucidate interspecific variation in venom activity in all nine currently recognised species of Pseudonaja. Fluorometric enzymatic activity assays were performed to test variation in whole venom procoagulant activity among species. Analyses confirmed the previously documented ontogenetic shift from non-coagulopathic venom in juveniles to coagulopathic venom as adults, except for the case of P. modesta, which retains non-coagulopathic venom as an adult. These shifts in venom activity correlate with documented ontogenetic shifts in diet among brown snakes from specialisation on reptilian prey as juveniles (and throughout the life cycle of P. modesta), to a more generalised diet in adults that includes mammals. The results of this study bring to light findings relevant to both clinical and evolutionary toxinology.

Coagulating colubrids: evolutionary, pathophysiological and biodiscovery implications of venom variations between boomslang (Dispholidus typus) and twig snake (Thelotornis mossambicanus)

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Venoms can deleteriously affect any physiological system reachable by the bloodstream, including directly interfering with the coagulation cascade. Such coagulopathic toxins may be anticoagulants or procoagulants. Snake venoms are unique in their use of procoagulant toxins for predatory purposes. The boomslang (Dispholidus typus) and the twig snakes (Thelotornis species) are iconic African snakes belonging to the family Colubridae. Both species produce strikingly similar lethal procoagulant pathologies. Despite these similarities, antivenom is only produced for treating bites by D. typus, and the mechanisms of action of both venoms have been understudied. In this study, we investigated the venom of D. typus and T. mossambicanus utilising a range of proteomic and bioactivity approaches, including determining the procoagulant properties of both venoms in relation to the human coagulation pathways. In doing so, we developed a novel procoagulant assay, utilising a Stago STA-R Max analyser, to accurately detect real time clotting in plasma at varying concentrations of venom. This approach was used to assess the clotting capabilities of the two venoms both with and without calcium and phospholipid co-factors. We found that T. mossambicanus produced a significantly stronger coagulation response compared to D. typus. Functional enzyme assays showed that T. mossambicanus also exhibited a higher metalloprotease and phospholipase activity but had a much lower serine protease activity relative to D. typus venom. The neutralising capability of the available boomslang antivenom was also investigated on both species, with it being 11.3 times more effective upon D. typus venom than T. mossambicanus. In addition to being a faster clotting venom, T. mossambicanus was revealed to be a much more complex venom composition than D. typus. This is consistent with patterns seen for other snakes with venom complexity linked to dietary complexity. Consistent with the external morphological differences in head shape between the two species, CT and MRI analyses revealed significant internal structural differences in skull architecture and venom gland anatomy. This study increases our understanding of not only the biodiscovery potential of these medically important species but also increases our knowledge of the pathological relationship between venom and the human coagulation cascade.

Catch a tiger snake by its tail: differential toxicity, co-factor dependence and antivenom efficacy in a procoagulant clade of Australian venomous snakes

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A paradigm of venom research is adaptive evolution of toxins as part of a predator-prey chemical arms race. This study examined differential co-factor dependence, variations relative to dietary preference, and the impact upon relative neutralisation by antivenom of the procoagulant toxins in the venoms of a clade of Australian snakes. All genera were characterised by venoms rich in factor Xa which act upon endogenous prothrombin. Examination of toxin sequences revealed an extraordinary level of conservation, which indicates that adaptive evolution is not a feature of this toxin type. Consistent with this, the venoms did not display differences on the plasma of different taxa. Examination of the prothrombin target revealed endogenous blood proteins are under extreme negative selection pressure for diversification, this in turn puts a strong negative selection pressure upon the toxins as sequence diversification could result in a drift away from the target. Thus this study reveals that adaptive evolution is not a consistent feature in toxin evolution in cases where the target is under negative selection pressure for diversification. Consistent with this high level of toxin conservation, the antivenom showed extremely high-levels of cross-reactivity. There was however a strong statistical correlation between relative degree of phospholipid-dependence and clotting time, with the least dependent venoms producing faster clotting times than the other venoms even in the presence of phospholipid. The results of this study are not only of interest to evolutionary and ecological disciplines, but also have implications for clinical toxinology.

Canopy venom: proteomic comparison among new world arboreal pit-viper venoms

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Central and South American pitvipers, belonging to the genera Bothrops and Bothriechis, have independently evolved arboreal tendencies. Little is known regarding the composition and activity of their venoms. In order to close this knowledge gap, venom proteomics and toxin activity of species of Bothriechis, and Bothrops (including Bothriopsis) were investigated through established analytical methods. A combination of proteomics and bioactivity techniques was used to demonstrate a similar diversification of venom composition between large and small species within Bothriechis and Bothriopsis. Increasing our understanding of the evolution of complex venom cocktails may facilitate future biodiscoveries.

Vintage venoms: proteomic and pharmacological stability of snake venoms stored for up to eight decades

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For over a century, venom samples from wild snakes have been collected and stored around the world. However, the quality of storage conditions for "vintage" venoms has rarely been assessed. The goal of this study was to determine whether such historical venom samples are still biochemically and pharmacologically viable for research purposes, or if new sample efforts are needed. In total, 52 samples spanning 5 genera and 13 species with regional variants of some species (e.g., 14 different populations of Notechis scutatus) were analysed by a combined proteomic and pharmacological approach to determine protein structural stability and bioactivity. When venoms were not exposed to air during storage, the proteomic results were virtually indistinguishable from that of fresh venom and bioactivity was equivalent or only slightly reduced. By contrast, a sample of Acanthophis antarcticus venom that was exposed to air (due to a loss of integrity of the rubber stopper) suffered significant degradation as evidenced by the proteomics profile. Interestingly, the neurotoxicity of this sample was nearly the same as fresh venom, indicating that degradation may have occurred in the free N- or C-terminus chains of the proteins, rather than at the tips of loops where the functional residues are located. These results suggest that these and other vintage venom collections may be of continuing value in toxin research. This is particularly important as many snake species worldwide are declining due to habitat destruction or modification. For some venoms (such as N. scutatus from Babel Island, Flinders Island, King Island and St. Francis Island) these were the first analyses ever conducted and these vintage samples may represent the only venom ever collected from these unique island forms of tiger snakes. Such vintage venoms may therefore represent the last remaining stocks of some local populations and thus are precious resources. These venoms also have significant historical value as the Oxyuranus venoms analysed include samples from the first coastal taipan (Oxyuranus scutellatus) collected for antivenom production (the snake that killed the collector Kevin Budden), as well as samples from the first Oxyuranus microlepidotus specimen collected after the species' rediscovery in 1976. These results demonstrate that with proper storage techniques, venom samples can retain structural and pharmacological stability. This article is part of a Special Issue entitled: Proteomics of non-model organisms. Biological significance: •These results show that with proper storage venoms are useful for decades.•These results have direct implications for the use of rare venoms.

Venomous landmines: Clinical implications of extreme coagulotoxic diversification and differential neutralization by antivenom of venoms within the viperid snake genus Bitis

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The genus comprises 17 snake species that inhabit Africa and the Arabian Peninsula. They are responsible for a significant proportion of snakebites in the region. The venoms of the two independent lineages of giant and again in the common ancestor of the clade consisting of and ) induce an array of debilitating effects including anticoagulation, hemorrhagic shock and cytotoxicity, whilst the dwarf species is known to have strong neurotoxic effects. However, the venom effects of the other species within the genus have not been explored in detail. A series of coagulation assays were implemented to assess the coagulotoxic venom effects of fourteen species within the genus. This study identified procoagulant venom as the ancestral condition, retained only by the basal dwarf species suggesting anticoagulant venom is a derived trait within the genus and has been secondarily amplified on at least four occasions. A wide range of anticoagulant mechanisms were identified, such as coagulant and destructive activities upon fibrinogen in both giant and dwarf and the action of inhibiting the prothrombinase complex, which is present in a clade of dwarf . Antivenom studies revealed that while the procoagulant effects of were poorly neutralized, and thus a cause for concern, the differential mechanisms of anticoagulation in other species were all well neutralized. Thus, this study concludes there is a wide range of coagulotoxic mechanisms which have evolved within the genus and that clinical management strategies are limited for the procoagulant effects of , but that anticoagulant effects of other species are readily treated by the South African polyvalent antivenom. These results therefore have direct, real-work implications for the treatment of envenomed patients.

The sweet side of venom: glycosylated prothrombin activating metalloproteases from Dispholidus typus (boomslang) and Thelotornis mossambicanus (twig snake)

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Dispholidus typus and Thelotornis mossambicanus are closely related rear-fanged colubrid snakes that both possess strongly procoagulant venoms. However, despite similarities in overall venom biochemistry and resulting clinical manifestations, the underlying venom composition differs significantly between the two species. As a result, the only available antivenom—which is a monovalent antivenom for D. typus—has minimal cross reactivity with T. mossambicanus and is not a clinically viable option. It was hypothesised that this lack of cross reactivity is due to the additional large metalloprotease protein within T. mossambicanus venom, which may also be responsible for faster coagulation times. In this study, we found that T. mossambicanus venom is a more powerful activator of prothrombin than that of D. typus and that the SVMP transcripts from T. mossambicanus form a clade with those from D. typus. The sequences from D. typus and T. mossambicanus were highly similar in length, with the calculated molecular weights of the T. mossambicanus transcripts being significantly less than the molecular weights of some isoforms on the 1D SDS-PAGE gels. Analyses utilising degylcosylating enzymes revealed that T. mossambicanus SVMPs are glycosylated during post-translational modification, but that this does not lead to the different molecular weight bands observed in 1D SDS-PAGE gels. However, differences in glycosylation patterns may still explain some of the difference between the enzymatic activities and neutralization by antivenom that have been observed in these venoms. The results of this study provide new information regarding the treatment options for patients envenomated by T. mossambicanus as well as the evolution of these dangerous snakes.

The Evolution of Fangs, Venom, and Mimicry Systems in Blenny Fishes (vol 27, pg 1184, 2017)

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(Current Biology 27, 1184–1191; April 24, 2017) [Figure presented] In Figure 4D of this article as originally published, two labels relating to control samples were erroneously interchanged: “control buffer” was mislabeled as “forskolin alone,” and “forskolin alone” was mislabeled as “control buffer.” This error has been rectified in the article online and in the corrected figure shown below. Please note that this error is solely typographical and has no bearing on our results or conclusions. The authors apologize for any confusion that the error may have caused. In addition, we mistakenly omitted our co-author Karine Mardon from the author list of the article as originally published. This error has also been rectified in the article online. We apologize to Dr. Mardon for the omission.

Squeezers and leaf-cutters: differential diversification and degeneration of the venom system in toxicoferan reptiles

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Although it has been established that all toxicoferan squamates share a common venomous ancestor, it has remained unclear whether the maxillary and mandibular venom glands are evolving on separate gene expression trajectories or if they remain under shared genetic control. We show that identical transcripts are simultaneously expressed not only in the mandibular and maxillary glands, but also in the enigmatic snake rictal gland. Toxin molecular frameworks recovered in this study were three-finger toxin (3FTx), CRiSP, crotamine (beta-defensin), cobra venom factor, cystatin, epididymal secretory protein, kunitz, L-amino acid oxidase, lectin, renin aspartate protease, veficolin, and vespryn. We also discovered a novel low-molecular weight disulfide bridged peptide class in pythonid snake glands. In the iguanian lizards, the most highly expressed are potentially antimicrobial in nature (crotamine (beta-defensin) and cystatin), with crotamine (beta-defensin) also the most diverse. However, a number of proteins characterized from anguimorph lizards and caenophidian snakes with hemotoxic or neurotoxic activities were recruited in the common toxicoferan ancestor and remain expressed, albeit in low levels, even in the iguanian lizards. In contrast, the henophidian snakes express 3FTx and lectin toxins as the dominant transcripts. Even in the constricting pythonid and boid snakes, where the glands are predominantly mucous-secreting, low-levels of toxin transcripts can be detected. Venom thus appears to play little role in feeding behavior of most iguanian lizards or the powerful constricting snakes, and the low levels of expression argue against a defensive role. However, clearly the incipient or secondarily atrophied venom systems of these taxa may be a source of novel compounds useful in drug design and discovery.

Solenodon genome reveals convergent evolution of venom in eulipotyphlan mammals

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Venom systems are key adaptations that have evolved throughout the tree of life and typically facilitate predation or defense. Despite venoms being model systems for studying a variety of evolutionary and physiological processes, many taxonomic groups remain understudied, including venomous mammals. Within the order Eulipotyphla, multiple shrew species and solenodons have oral venom systems. Despite morphological variation of their delivery systems, it remains unclear whether venom represents the ancestral state in this group or is the result of multiple independent origins. We investigated the origin and evolution of venom in eulipotyphlans by characterizing the venom system of the endangered Hispaniolan solenodon (). We constructed a genome to underpin proteomic identifications of solenodon venom toxins, before undertaking evolutionary analyses of those constituents, and functional assessments of the secreted venom. Our findings show that solenodon venom consists of multiple paralogous kallikrein 1 () serine proteases, which cause hypotensive effects in vivo, and seem likely to have evolved to facilitate vertebrate prey capture. Comparative analyses provide convincing evidence that the oral venom systems of solenodons and shrews have evolved convergently, with the 4 independent origins of venom in eulipotyphlans outnumbering all other venom origins in mammals. We find that s have been independently coopted into the venom of shrews and solenodons following their divergence during the late Cretaceous, suggesting that evolutionary constraints may be acting on these genes. Consequently, our findings represent a striking example of convergent molecular evolution and demonstrate that distinct structural backgrounds can yield equivalent functions.

Habu coagulotoxicity: clinical implications of the functional diversification of Protobothrops snake venoms upon blood clotting factors

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Venom can affect any part of the body reachable via the bloodstream. Toxins which specifically act upon the coagulation cascade do so either by anticoagulant or procoagulant mechanisms. Here we investigated the coagulotoxic effects of six species within the medically important pit viper genus Protobothrops (Habu) from the Chinese mainland and Japanese islands, a genus known to produce hemorrhagic shock in envenomed patients. Differential coagulotoxicity was revealed: P. jerdonii and P. mangshanensis produced an overall net anticoagulant effect through the pseudo-procoagulant clotting of fibrinogen; P. flavoviridis and P. tokarensis exhibit a strong anticoagulant activity through the destructive cleavage of fibrinogen; and while P. elegans and P. mucrosquamatus both cleaved the A-alpha and B-beta chains of fibrinogen they did not exhibit strong anticoagulant activity. These variations in coagulant properties were congruent with phylogeny, with the closest relatives exhibiting similar venom effects in their action upon fibrinogen. Ancestral state reconstruction indicated that anticoagulation mediated by pseudo-procoagulant cleavage of fibrinogen is the basal state, while anticoagulation produced by destructive cleavage of fibrinogen is the derived state within this genus. This is the first in depth study of its kind highlighting extreme enzymatic variability, functional diversification and clotting diversification within one genus surrounding one target site, governed by variability in co-factor dependency. The documentation that the same net overall function, anticoagulation, is mediated by differential underlying mechanics suggests limited antivenom cross-reactivity, although this must be tested in future work. These results add to the body of knowledge necessary to inform clinical management of the envenomed patient.

Evolutionary interpretations of nicotinic acetylcholine receptor targeting venom effects by a clade of Asian viperidae snakes

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Ecological variability among closely related species provides an opportunity for evolutionary comparative studies. Therefore, to investigate the origin and evolution of neurotoxicity in Asian viperid snakes, we tested the venoms of Azemiops feae, Calloselasma rhodostoma, Deinagkistrodon acutus, Tropidolaeums subannulatus, and T. wagleri for their relative specificity and potency upon the amphibian, lizard, bird, rodent, and human α-1 (neuromuscular) nicotinic acetylcholine receptors. We utilised a biolayer interferometry assay to test the binding affinity of these pit viper venoms to orthosteric mimotopes of nicotinic acetylcholine receptors binding region from a diversity of potential prey types. The Tropidolaemus venoms were much more potent than the other species tested, which is consistent with the greater prey escape potential in arboreal niches. Intriguingly, the venom of C. rhodostoma showed neurotoxic binding to the α-1 mimotopes, a feature not known previously for this species. The lack of prior knowledge of neurotoxicity in this species is consistent with our results due to the bias in rodent studies and human bite reports, whilst this venom had a greater binding affinity toward amphibian and diapsid α-1 targets. The other large terrestrial species, D. acutus, did not display any meaningful levels of neurotoxicity. These results demonstrate that whilst small peptide neurotoxins are a basal trait of these snakes, it has been independently amplified on two separate occasions, once in Azemiops and again in Tropidolaemus, and with Calloselasma representing a third possible amplification of this trait. These results also point to broader sources of novel neuroactive peptides with the potential for use as lead compounds in drug design and discovery.

Coagulotoxic effects by brown snake (Pseudonaja) and taipan (Oxyuranus) venoms, and the efficacy of a new antivenom

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Snakebite is a neglected tropical disease that disproportionately affects the poor. Antivenom is the only specific and effective treatment for snakebite, but its distribution is severely limited by several factors, including the prohibitive cost of some products. Papua New Guinea (PNG) is a snakebite hotspot but the high costs of Australian antivenoms (thousands of dollars per treatment) makes it unaffordable in PNG. A more economical taipan antivenom has recently been developed at the Instituto Clodomiro Picado (ICP) in Costa Rica for PNG and is currently undergoing clinical trials for the treatment of envenomations by coastal taipans (Oxyuranus scutellatus). In addition to potentially having the capacity to neutralise the effects of envenomations of non-PNG taipans, this antivenom may have the capacity to neutralise coagulotoxins in venom from closely related brown snakes (Pseudonaja spp.) also found in PNG. Consequently, we investigated the cross-reactivity of taipan antivenom across the venoms of all Oxyuranus and Pseudonaja species. In addition, to ascertain differences in venom biochemistry that influence variation in antivenom efficacy, we tested for relative cofactor dependence. We found that the new ICP taipan antivenom exhibited high selectivity for Oxyuranus venoms and only low to moderate cross-reactivity with any Pseudonaja venoms. Consistent with this genus level distinction in antivenom efficacy were fundamental differences in the venom biochemistry. Not only were the Pseudonaja venoms significantly more procoagulant, but they were also much less dependent upon the cofactors calcium and phospholipid. There was a strong correlation between antivenom efficacy, clotting time and cofactor dependence. This study sheds light on the structure-function relationships of the procoagulant toxins within these venoms and may have important clinical implications including for the design of next-generation antivenoms.

Coagulating colubrids: evolutionary, pathophysiological and biodiscovery implications of venom variations between Dispholidus typus and Thelotornis mossambicanus

Clinical implications of differential antivenom efficacy in neutralising coagulotoxicity produced by venoms from species within the arboreal viperid snake genus Trimeresurus

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Snake envenomation globally is attributed to an ever-increasing human population encroaching into snake territories. Responsible for many bites in Asia is the widespread genus Trimeresurus. While bites lead to haemorrhage, only a few species have had their venoms examined in detail. We found that Trimeresurus venom causes haemorrhaging by cleaving fibrinogen in a pseudo-procoagulation manner to produce weak, unstable, short-lived fibrin clots ultimately resulting in an overall anticoagulant effect due to fibrinogen depletion. The monovalent antivenom 'Thai Red Cross Green Pit Viper antivenin', varied in efficacy ranging from excellent neutralisation of T. albolabris venom through to T. gumprechti and T. mcgregori being poorly neutralised and T. hageni being unrecognised by the antivenom. While the results showing excellent neutralisation of some non-T. albolabris venoms (such as T. flavomaculaturs, T. fucatus, and T. macrops) needs to be confirmed with in vivo tests, conversely the antivenom failure T. hageni, and the very poor results against T. gumprechti and T. mcgregori, despite being conducted in the ideal scenario of preincubation of antivenom:venom, indicates that the likelihood of clinically relevant cross-reactivity for these species is low (T. gumprechti and T. mcgregori) to non-existent (T. hageni). These same latter three species were also not inhibited by the serine protease inhibitor AEBSF, suggesting that the toxins leading to a coagulotoxic effect in these species are non-serine proteases while in contrast T. albolabris coagulotoxicity was completely impeded by AEBSF, and thus driven by kallikrein-type serine proteases. There was a conspicuous lack of phylogenetic pattern in venom variation, with the most potent venoms (T. albolabris and T. hageni) being distant to each other on the organismal tree, and with the three most divergent and poorly neutralised venoms (T. gumprechti, T. hageni, and T. mcgregori) were also not each others closest relatives. This reinforces the paradigm that the fundamental dynamic evolution of venom results in organismal phylogeny being a poor predictor of venom potency or antivenom efficacy. This study provides a robust investigation on the differential venom effects from a wide range of Trimeresurus species on coagulation, highlighting differential fibrinogenolytic effects, while also investigating the relative antivenom neutralisation capabilities of the widely available Thai Red Cross Green Pit Viper antivenom. These results therefore have immediate, real-world implications for patients envenomed by Trimeresurus species.

Clinical implications of coagulotoxic variations in Mamushi (Viperidae: Gloydius) snake venoms

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Snake bite is currently one of the most neglected tropical diseases affecting much of the developing world. Asian pit vipers are responsible for a considerable amount of envenomations annually and bites can cause a multitude of clinical complications resulting from coagulopathic and neuropathic effects. While intense research has been undertaken for some species of Asian pit viper, functional coagulopathic effects have been neglected for others. We investigated their effects upon the human clotting cascade using venoms of four species of Gloydius and Ovophis okinavensis, a species closely to Gloydius. All species of included within this investigation displayed varying fibrinogenolytic effects, resulting in a net anticoagulant outcome. Gloydius saxatilis and Gloydius ussuriensis displayed the most variable effects from differing localities, sampled from Russia and Korea. As this Gloydius investigation includes some geographical variation, notable results indicate key variations of these species that point to possible limitations in antivenom cross-reactivities, which may have implications for the clinical care of victims envenomed by these snakes.

Basal but divergent: clinical implications of differential coagulotoxicity in a clade of Asian vipers

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Envenomations by Asian pitvipers can induce multiple clinical complications resulting from coagulopathic and neuropathic effects. While intense research has been undertaken for some species, functional coagulopathic effects have been neglected. As these species’ venoms affect the blood coagulation cascade we investigated their effects upon the human clotting cascade using venoms of species from the Azemiops, Calloselasma, Deinagkistrodon and Hypnale genera. Calloselasma rhodostoma, Deinagkistrodon acutus, and Hypnale hypnale produced net anticoagulant effects through pseudo-procoagulant clotting of fibrinogen, resulting in weak, unstable, transient fibrin clots. Tropidolaemus wagleri was only weakly pseudo-procoagulant, clotting fibrinogen with only a negligible net anticoagulant effect. Azemiops feae and Tropidolaemus subannulatus did not affect clotting. This is the first study to examine in a phylogenetic context the coagulotoxic effects of related genera of basal Asiatic pit-vipers. The results reveal substantial variation between sister genera, providing crucial information about clinical effects and implications for antivenom cross-reactivity.

A taxon-specific and high-throughput method for measuring ligand binding to nicotinic acetylcholine receptors

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The binding of compounds to nicotinic acetylcholine receptors is of great interest in biomedical research. However, progress in this area is hampered by the lack of a high-throughput, cost-effective, and taxonomically flexible platform. Current methods are low-throughput, consume large quantities of sample, or are taxonomically limited in which targets can be tested. We describe a novel assay which utilizes a label-free bio-layer interferometry technology, in combination with adapted mimotope peptides, in order to measure ligand binding to the orthosteric site of nicotinic acetylcholine receptor alpha-subunits of diverse organisms. We validated the method by testing the evolutionary patterns of a generalist feeding species (), a fish specialist species (), and a snake specialist species () for comparative binding to the orthosteric site of fish, amphibian, lizard, snake, bird, marsupial, and rodent alpha-1 nicotinic acetylcholine receptors. Binding patterns corresponded with diet, with the not showing bias towards any particular lineage, while showed selectivity for fish, and a selectivity for snake. To validate the biodiscovery potential of this method, we screened and venom for binding to human alpha-1, alpha-2, alpha-3, alpha-4, alpha-5, alpha-6, alpha-7, alpha-9, and alpha-10. While was broadly potent, showed very strong but selective binding, specifically to the alpha-1 target which would be evolutionarily selected for, as well as the alpha-5 target which is of major interest for drug design and development. Thus, we have shown that our novel method is broadly applicable for studies including evolutionary patterns of venom diversification, predicting potential neurotoxic effects in human envenomed patients, and searches for novel ligands of interest for laboratory tools and in drug design and development.

A blood bond: evolutionary, pathophysiological and biodiscovery implications of coagulotoxic snake venoms

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Snakebite is a massive global burden and is a neglected area of tropical diseases. Snake venom neurotoxins have been the subject of intense research efforts, albeit significantly less is known about coagulotoxins. Venoms can deleteriously affect any physiological system reachable by the bloodstream, including being able to directly interfere with the coagulation cascade. Such coagulopathic toxins may be either anticoagulant or procoagulant and snake venoms are unique in their use of procoagulant toxins for predatory purposes. Our limited knowledge into the mechanisms of action of snake venom hinders our understanding of the clinical pathologies and also restricts our use of these powerful natural products as lead compounds in drug design and development. Two snake families which exhibit coagulant properties and lead to a range of clinical pathologies are vipers, Viperidae, and non-front-fanged snakes, Colubridae. The primary focus of this thesis was to investigate and compare the coagulotoxicity effects and mechanisms of various species of known venomous snakes that effect coagulation. Species chosen for this investigation have had limited previous research conducted, thus a knowledge gap in the literature remains. These coagulant properties were investigated through a multi-disciplinary approach, highlighting key results from both families.Data chapters 2 and 3 explore venom from the boomslang (Dispholidus typus) and the twig snakes (Thelotornis species) - iconic African snakes belonging to the family Colubridae. Both species produce strikingly similar lethal procoagulant pathologies. Despite these similarities, antivenom is only produced for treating bites by D. typus, and the mechanisms of action of both venoms have been understudied. We found that T. mossambicanus produced a significantly stronger coagulation response compared to D. typus, governed by strong prothrombin activation through PIII-SVMPs. Despite similarities in symptoms and clinical manifestations, venom composition differs widely between the two species, recovered from a combined approach of venomics and transcriptomics. The neutralising capability of the available boomslang antivenom was also investigated on both species, with it being 11.3 times more effective upon D. typus venom than T. mossambicanus. Envenomation by Crotalinae such as Asian pit vipers can induce multiple clinical complications resulting from coagulopathic and neuropathic effects. Data chapters 4 - 8 investigate the Asian Pit Viper clade. While intense research has been undertaken for some species, such as Calloselasma rhodostoma, functional coagulopathic effects have been neglected. As these species’ venoms affect the blood coagulation cascade and are known to produce haemorrhagic shock in envenomed patients, we investigated their effects upon coagulation using venoms of 33 species from the Asian pit viper clade including Azemiops, Calloselasma, Deinagkistrodon, Gloydius, Hypnale, Protobothrops, Trimeresurus and Tropidolaemus. Fibrinogen was the main coagulation target among these species with many varying coagulotoxic effects exhibited. Various species including Calloselasma rhodostoma, Deinagkistrodon acutus, Hypnale hypnale, Protobothrops mangshanensis, Ovophis okinavensis and some Trimeresurus species produced net anticoagulant effects through pseudo-procoagulant clotting of fibrinogen, resulting in weak, unstable, transient fibrin clots. Protobothrops flavoviridis, P. tokarensis, Gloydius brevicaudus, G. saxatilis, G. ussuriensis and various Trimeresurus species exhibited a strong anticoagulant activity through the destructive cleavage of fibrinogen (at varying rates) directly resulting in an overall anticoagulant effect. Tropidolaemus wagleri was only weakly pseudo-procoagulant, clotting fibrinogen with only a negligible net anticoagulant effect. Azemiops feae and Tropidolaemus subannulatus did not affect clotting and T. subannulatus was investigated for its alternatively derived neurotoxicity effects, sharing Waglerin’s peptide propeptide region. Other clotting factors, such as FX(a), thrombin, FIX(a) and FXI(a), were only mildly affected with minimal inhibition found from varying species. Cross neutralisation of the Green Pit Viper monovalent antivenom ‘Thai Red Cross Green Pit Viper antivenin’ was also investigated among the Trimeresurus species, with T. gumprechti, T. hageni and T. mcgregori being unaffected. These results indicate that anticoagulation mediated by pseudo-procoagulant cleavage of fibrinogen is the basal state among Asian pit vipers, while anticoagulation produced by destructive cleavage of fibrinogen, such as that found in Protobothrops, is the derived state. This is the first in depth study of its kind highlighting extreme enzymatic variability, functional diversification and clotting diversification within one clade (Asian pit vipers) surrounding one target site, governed by variability in co-factor dependency. This study is also the first to examine in a phylogenetic context the coagulotoxic effects of related genera of Asiatic pit vipers. The results of these various chapters reveal substantial variation between sister genera, providing crucial information about clinical effects and implications for antivenom cross-reactivity among venomous snake species. This study increases our understanding of not only the biodiscovery potential of these medically important species but also increases our knowledge of the pathological relationship between venom and the human coagulation cascade. These results add to the body of knowledge necessary to inform clinical management of the envenomed patient, and detail the diversity of components and characteristics which enable venom research to be such a biodiscovery treasure trove in unlimited diversity, leading to novel drug design surrounding this field.




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