Modification Of Toxin Research Articles

Post-translational toxin modification by lactate controls Staphylococcus aureus virulence.

Diverse post-translational modifications have been shown to play important roles in regulating protein function in eukaryotes. By contrast, the roles of post-translational modifications in bacteria are not so well understood, particularly as they relate to pathogenesis. Here, we demonstrate post-translational protein modification by covalent addition of lactate to lysine residues (lactylation) in the human pathogen Staphylococcus aureus. Lactylation is dependent on lactate concentration and specifically affects alpha-toxin, in which a single lactylated lysine is required for full activity and virulence in infection models. Given that lactate levels typically increase during infection, our results suggest that the pathogen can use protein lactylation as a mechanism to increase toxin-mediated virulence during infection.

Pharmacological interventions for prophylaxis of vestibular migraine.

Vestibular migraine is a form of migraine where one of the main features is recurrent attacks of vertigo. These episodes are often associated with other features of migraine, including headache and sensitivity to light or sound. These unpredictable and severe attacks of vertigo can lead to a considerable reduction in quality of life. The condition is estimated to affect just under 1% of the population, although many people remain undiagnosed. A number of pharmacological interventions have been used or proposed to be used as prophylaxis for this condition, to help reduce the frequency of the attacks. These are predominantly based on treatments that are in use for headache migraine, with the belief that the underlying pathophysiology of these conditions is similar. To assess the benefits and harms of pharmacological treatments used for prophylaxis of vestibular migraine. The Cochrane ENT Information Specialist searched the Cochrane ENT Register; Central Register of Controlled Trials (CENTRAL); Ovid MEDLINE; Ovid Embase; Web of Science; ClinicalTrials.gov; ICTRP and additional sources for published and unpublished trials. The date of the search was 23 September 2022. We included randomised controlled trials (RCTs) and quasi-RCTs in adults with definite or probable vestibular migraine comparing beta-blockers, calcium channel blockers, antiepileptics, antidepressants, diuretics, monoclonal antibodies against calcitonin gene-related peptide (or its receptor), botulinum toxin or hormonal modification with either placebo or no treatment. We excluded studies with a cross-over design, unless data from the first phase of the study could be identified. We used standard Cochrane methods. Our primary outcomes were: 1) improvement in vertigo (assessed as a dichotomous outcome - improved or not improved), 2) change in vertigo (assessed as a continuous outcome, with a score on a numerical scale) and 3) serious adverse events. Our secondary outcomes were: 4) disease-specific health-related quality of life, 5) improvement in headache, 6) improvement in other migrainous symptoms and 7) other adverse effects. We considered outcomes reported at three time points: < 3 months, 3 to < 6 months, > 6 to 12 months. We used GRADE to assess the certainty of evidence for each outcome. We included three studies with a total of 209 participants. One evaluated beta-blockers and the other two evaluated calcium channel blockers. We did not identify any evidence for the remaining interventions of interest. Beta-blockers versus placebo One study (including 130 participants, 61% female) evaluated the use of 95 mg metoprolol once daily for six months, compared to placebo. The proportion of people who reported improvement in vertigo was not assessed in this study. Some data were reported on the frequency of vertigo attacks at six months and the occurrence of serious adverse effects. However, this is a single, small study and for all outcomes the certainty of evidence was low or very low. We are unable to draw meaningful conclusions from the numerical results. Calcium channel blockers versus no treatment Two studies, which included a total of 79 participants (72% female), assessed the use of 10 mg flunarizine once daily for three months, compared to no intervention. All of the evidence for this comparison was of very low certainty. Most of our outcomes were only reported by a single study, therefore we were unable to conduct any meta-analysis. Some data were reported on improvement in vertigo and change in vertigo, but no information was available regarding serious adverse events. We are unable to draw meaningful conclusions from the numerical results, as these data come from single, small studies and the certainty of the evidence was very low. There is very limited evidence from placebo-controlled randomised trials regarding the efficacy and potential harms of pharmacological interventions for prophylaxis of vestibular migraine. We only identified evidence for two of our interventions of interest (beta-blockers and calcium channel blockers) and all evidence was of low or very low certainty. Further research is necessary to identify whether these treatments are effective at improving symptoms and whether there are any harms associated with their use.

Active Expression of Genes for Protein Modification Enzymes in Habu Venom Glands.

Genes encoding snake venom toxins have been studied extensively. However, genes involved in the modification and functioning of venom proteins are little known. Protobothrops is a genus of pit vipers, which are venomous and inhabit the Nansei (Southwest) islands of Japan, Taiwan China, Vietnam, Thailand, Myanmar, Nepal, Bhutan, and India. Our previous study decoded the genome of Protobothrops flavoviridis, a species endemic to the Nansei Islands, Japan, and revealed unique evolutionary processes of some venom genes. In this study, we analyzed genes that are highly expressed in venom glands to survey genes for candidate enzymes or chaperone proteins involved in toxin folding and modification. We found that, in addition to genes that encode venom proteins and ribosomal proteins, genes that encode protein disulfide isomerase (PDI) family members (orthologs of human P4HB and PDIA3), Selenoprotein M (SELENOM), and Calreticulin (CALR) are highly expressed in venom glands. Since these enzymes or chaperones are involved in protein modification and potentially possess protein folding functions, we propose that P4HB, SELENOM, CALR, and PDIA3 encode candidate enzymes or chaperones to confer toxic functions upon the venom transcriptome.

Introduction of a cold sensitivity-conferring mutation into the RTA-Bddsx hybrid system of Bactrocera dorsalis for establishment of a thermally controllable homozygous line.

For efficient control of the economically important fruit pest Bactrocera dorsalis, a hybrid system combining ricin toxicity and sex-related alternative splicing of the doublesex gene has been developed. This system exhibits the expected female-specific lethal effect; however, the transgenic females do not survive, making it difficult to raise stable homozygous lines. Since modification of ricin toxin A chain (RTA) through a single-residue change (Gly212 > Arg212 ) leads to cold-sensitive posttranslational repression of its toxicity, we utilized this unique property to obtain RTA-Bddsx females that survive at low temperature for line maintenance. In transient expression experiments using embryonic injection, two groups treated with RTAcs-derived DNA (LERQcs and RTAcs) exhibited temperature-dependent effects. The toxicity was higher at 29 °C than at 18 °C. The proportion of males was close to 50% at 18 °C in all the tested groups except LERQcs-treated flies, which exhibited a high proportion of males (over 70%) at 29 °C. The results indicate the cold-sensitive responses of RTA and further suggest a female-specific lethal effect. Subsequently, 14 putative RTAcs-Bddsx transgenic Ds-Red+ G1 males were identified, and female-specific lethal effects were observed in Ds-Red+ G2 and G3 lines under cultivation at 29 °C but not at 18 °C. The male ratio can be increased to up to 95% in G3 line 001, indicating that RTAcs functions well in B. dorsalis. The improved RTAcs-Bddsx system with conditional toxicity represents a novel and promising step toward the practical control of B. dorsalis. © 2021 Society of Chemical Industry.

Recruitment of toxin-like proteins with ancestral venom function supports endoparasitic lifestyles of Myxozoa

Cnidarians are the oldest lineage of venomous animals and use nematocysts to discharge toxins. Whether venom toxins have been recruited to support parasitic lifestyles in the Endocnidozoa (Myxozoa + Polypodium) is, however, unknown. To examine this issue we variously employed transcriptomic, proteomic, associated molecular phylogenies, and localisation studies on representative primitive and derived myxozoans (Malacosporea and Myxosporea, respectively), Polypodium hydriforme, and the free-living staurozoan Calvadosia cruxmelitensis. Our transcriptomics and proteomics analyses provide evidence for expression and translation of venom toxin homologs in myxozoans. Phylogenetic placement of Kunitz type serine protease inhibitors and phospholipase A2 enzymes reveals modification of toxins inherited from ancestral free-living cnidarian toxins, and that venom diversity is reduced in myxozoans concordant with their reduced genome sizes. Various phylogenetic analyses of the Kunitz-type toxin family in Endocnidozoa suggested lineage-specific gene duplications, which offers a possible mechanism for enhancing toxin diversification. Toxin localisation in the malacosporean Buddenbrockia plumatellae substantiates toxin translation and thus illustrates a repurposing of toxin function for endoparasite development and interactions with hosts, rather than for prey capture or defence. Whether myxozoan venom candidates are expressed in transmission stages (e.g. in nematocysts or secretory vesicles) requires further investigation.

Advances in Molecular Understanding of α-Helical Membrane-Active Peptides.

Biological membranes separate the interior of cells or cellular compartments from their outer environments. This barrier function of membranes can be disrupted by membrane-active peptides, some of which can spontaneously penetrate through the membranes or open leaky transmembrane pores. However, the origin of their activity/toxicity is not sufficiently understood for the development of more potent peptides. To this day, there are no design rules that would be generally valid, and the role of individual amino acids tends to be sequence-specific.In this Account, we describe recent progress in understanding the design principles that govern the activity of membrane-active peptides. We focus on α-helical amphiphilic peptides and their ability to (1) translocate across phospholipid bilayers, (2) form transmembrane pores, or (3) act synergistically, i.e., to produce a significantly more potent effect in a mixture than the individual components.We refined the description of peptide translocation using computer simulations and demonstrated the effect of selected residues. Our simulations showed the necessity to explicitly include charged residues in the translocation description to correctly sample the membrane perturbations they can cause. Using this description, we calculated the translocation of helical peptides with and without the kink induced by the proline/glycine residue. The presence of the kink had no effect on the translocation barrier, but it decreased the peptide affinity to the membrane and reduced the peptide stability inside the membrane. Interestingly, the effects were mainly caused by the peptide's increased polarity, not the higher flexibility of the kink.Flexibility plays a crucial role in pore formation and affects distinct pore structures in different ways. The presence of a kink destabilizes barrel-stave pores, because the kink prevents the tight packing of peptides in the bundle, which is characteristic of the barrel-stave structure. In contrast, the kink facilitates the formation of toroidal pores, where the peptides are only loosely arranged and do not need to closely assemble. The exact position of the kink in the sequence further determines the preferred arrangement of peptides in the pore, i.e., an hourglass or U-shaped structure. In addition, we demonstrated that two self-associated (via termini) helical peptides could mimic the behavior of peptides with a helix-kink-helix motif.Finally, we review the recent findings on the peptide synergism of the archetypal mixture of Magainin 2 and PGLa peptides. We focused on a bacterial plasma membrane mimic that contains negatively charged lipids and lipids with negative intrinsic curvature. We showed that the synergistic action of peptides was highly dependent on the lipid composition. When the lipid composition and peptide/lipid ratios were changed, the systems exhibited more complex behavior than just the previously reported pore formation. We observed membrane adhesion, fusion, and even the formation of the sponge phase in this regime. Furthermore, enhanced adhesion/partitioning to the membrane was reported to be caused by lipid-induced peptide aggregation.In conclusion, the provided molecular insight into the complex behavior of membrane-active peptides provides clues for the design and modification of antimicrobial peptides or toxins.

Integrating Top-Down and Bottom-Up Mass Spectrometric Strategies for Proteomic Profiling of Iranian Saw-Scaled Viper, Echis carinatus sochureki, Venom.

Saw-scaled or carpet vipers (genus Echis) are considered to cause a higher global snakebite mortality than any other snake. Echis carinatus sochureki (ECS) is a widely distributed snake species, also found across the thirteen provinces of Iran, where it is assumed to be responsible for the most snakebite envenomings. Here, we collected the Iranian specimens of ECS from three different geographically distinct populations, investigated food habits, and performed toxicity assessment and venom proteome profiling to better understand saw-scaled viper life. Our results show that the prey items most commonly found in all populations were arthropods, with scorpions from the family Buthidae particularly well represented. LD50 (median lethal dose) values of the crude venom demonstrate highly comparable venom toxicities in mammals. Consistent with this finding, venom characterization via top-down and bottom-up proteomics, applied to both crude venoms and size-exclusion chromatographic fractions, revealed highly comparable venom compositions among the different populations. By combining all proteomics data, we identified 22 protein families from 102 liquid chromatography and tandem mass spectrometry (LC-MS/MS) raw files, including the most abundant snake venom metalloproteinases (SVMPs, 29-34%); phospholipase A2 (PLA2s, 26-31%); snake venom serine proteinases (SVSPs, 11-12%); l-amino acid oxidases (LAOs, 8-11%), C-type lectins/lectin-like (CTLs, 7-9%) protein families, and many newly detected ones, e.g., renin-like aspartic proteases (RLAPs), fibroblast growth factors (FGFs), peptidyl-prolyl cis-trans isomerases (PPIs), and venom vasodilator peptides (VVPs). Furthermore, we identified and characterized methylated, acetylated, and oxidized proteoforms relating to the PLA2 and disintegrin toxin families and the site of their modifications. It thus seems that post-translational modifications (PTMs) of toxins, particularly target lysine residues, may play an essential role in the structural and functional properties of venom proteins and might be able to influence the therapeutic response of antivenoms, to be investigated in future studies.

Methionine 58 is a key residue in the modulation of BmK scorpion toxin AGP-SYPU2 activity through in silico and in vivo studies

Protein dynamic networks play an important role in the regulation of many protein systems. Some residues that are far away from the interface between proteins and their targets have a critical role in modulating the activity of some scorpion toxins. Here, conservation analysis combined with an in vivo experiment has reveals that Met58 is a key residue of BmK scorpion toxin AGP-SYPU2 in the modulation of analgesic activity. Molecular dynamics simulations clearly reveal the conformational changes that allow the loop between the β2 and β3 sheets to be exposed on the toxin surface to interact with its targets. Our results emphasize specific roles for the residue Met58 in the NC domain and our work gives valuable information for further modification of scorpion toxins to obtain new analgesic peptides with enhanced activity. Communicated by Ramaswamy H. Sarma

Discovery of a selective, state-independent inhibitor of NaV1.7 by modification of guanidinium toxins

The voltage-gated sodium channel isoform NaV1.7 is highly expressed in dorsal root ganglion neurons and is obligatory for nociceptive signal transmission. Genetic gain-of-function and loss-of-function NaV1.7 mutations have been identified in select individuals, and are associated with episodic extreme pain disorders and insensitivity to pain, respectively. These findings implicate NaV1.7 as a key pharmacotherapeutic target for the treatment of pain. While several small molecules targeting NaV1.7 have been advanced to clinical development, no NaV1.7-selective compound has shown convincing efficacy in clinical pain applications. Here we describe the discovery and characterization of ST-2262, a NaV1.7 inhibitor that blocks the extracellular vestibule of the channel with an IC50 of 72 nM and greater than 200-fold selectivity over off-target sodium channel isoforms, NaV1.1–1.6 and NaV1.8. In contrast to other NaV1.7 inhibitors that preferentially inhibit the inactivated state of the channel, ST-2262 is equipotent in a protocol that favors the resting state of the channel, a protocol that favors the inactivated state, and a high frequency protocol. In a non-human primate study, animals treated with ST-2262 exhibited reduced sensitivity to noxious heat. These findings establish the extracellular vestibule of the sodium channel as a viable receptor site for the design of selective ligands targeting NaV1.7.

Effect of helical kink in antimicrobial peptides on membrane pore formation.

Every cell is protected by a semipermeable membrane. Peptides with the right properties, for example Antimicrobial peptides (AMPs), can disrupt this protective barrier by formation of leaky pores. Unfortunately, matching peptide properties with their ability to selectively form pores in bacterial membranes remains elusive. In particular, the proline/glycine kink in helical peptides was reported to both increase and decrease antimicrobial activity. We used computer simulations and fluorescence experiments to show that a kink in helices affects the formation of membrane pores by stabilizing toroidal pores but disrupting barrel-stave pores. The position of the proline/glycine kink in the sequence further controls the specific structure of toroidal pore. Moreover, we demonstrate that two helical peptides can form a kink-like connection with similar behavior as one long helical peptide with a kink. The provided molecular-level insight can be utilized for design and modification of pore-forming antibacterial peptides or toxins.

Reciprocal transplantation of the heterotrophic coral Tubastraea coccinea (Scleractinia: Dendrophylliidae) between distinct habitats did not alter its venom toxin composition.

Tubastraea coccinea is an azooxanthellate coral species recorded in the Indian and Atlantic oceans and is presently widespread in the southwestern Atlantic with an alien status for Brazil. T. coccinea outcompete other native coral species by using a varied repertoire of biological traits. For example, T. coccinea has evolved potent venom capable of immobilizing and digesting zooplankton prey. Diversification and modification of venom toxins can provide potential adaptive benefits to individual fitness, yet acquired alteration of venom composition in cnidarians is poorly understood as the adaptive flexibility affecting toxin composition in these ancient lineages has been largely ignored. We used quantitative high‐throughput proteomics to detect changes in toxin expression in clonal fragments of specimens collected and interchanged from two environmentally distinct and geographically separate study sites. Unexpectedly, despite global changes in protein expression, there were no changes in the composition and abundance of toxins from coral fragments recovered from either site, and following clonal transplantation between sites. There were also no apparent changes to the cnidome (cnidae) and gross skeletal or soft tissue morphologies of the specimens. These results suggest that the conserved toxin complexity of T. coccinea co‐evolved with innovation of the venom delivery system, and its morphological development and phenotypic expression are not modulated by habitat pressures over short periods of time. The adaptive response of the venom trait to specific predatory regimes, however, necessitates further consideration.

Removal of the translocation domain and the furin cleavage site decreases the relative hepatotoxicity of the targeted antitumor toxins

Targeted toxins are promising anticancer agents that allow selectively destroying cancer cells due to the increased content of onco-specific markers on their surface. The use of such anti-cancer toxins in medicine is mainly hampered by their high non-specific toxicity, in particular, hepatotoxicity. In our work on human cell line, we have shown that the removal of the DARPin-PE40 translocation toxin domain leads to a decrease in hepatoto-xicity. The same effect is also observed when inactivation of the furin cleavage site in the DARPin-PE40 molecule was done. Simultaneous removal of both the translocation domain and the furin cleavage site showed the best results. This toxin modification can be used to create more selective anti-cancer toxins.

Removal of the Translocation Domain and the Furin Cleavage Site Decreases the Relative Hepatotoxicity of the Targeted Antitumor Toxins.

Targeted toxins are promising anticancer agents that allow selectively destroying cancer cells due to the increased content of onco-specific markers on their surface. The use of such anti-cancer toxins in medicine is mainly hampered by their high non-specific toxicity, in particular, hepatotoxicity. In our work on human cell line, we have shown that the removal of the DARPin-PE40 translocation toxin domain leads to a decrease in hepatotoxicity. The same effect is also observed when inactivation of the furin cleavage site in the DARPin-PE40 molecule was done. Simultaneous removal of both the translocation domain and the furin cleavage site showed the best results. This toxin modification can be used to create more selective anti-cancer toxins.

Intein-mediated site-specific synthesis of tumor-targeting protein delivery system: Turning PEG dilemma into prodrug-like feature.

Poor tumor-targeted and cytoplasmic delivery is a bottleneck for protein toxin-based cancer therapy. Ideally, a protein toxin drug should remain stealthy in circulation for prolonged half-life and reduced side toxicity, but turn activated at tumor. PEGylation is a solution to achieve the first goal, but creates a hurdle for the second because PEG rejects interaction between the drugs and tumor cells therein. Such PEG dilemma is an unsolved problem in protein delivery. Herein proposed is a concept of turning PEG dilemma into prodrug-like feature. A site-selectively PEGylated, gelatinase-triggered cell-penetrating trichosanthin protein delivery system is developed with three specific aims. The first is to develop an intein-based ligation method for achieving site-specific modification of protein toxins. The second is to develop a prodrug feature that renders protein toxins remaining stealthy in blood for reduced side toxicity and improved EPR effect. The third is to develop a gelatinase activatable cell-penetration strategy for enhanced tumor targeting and cytoplasmic delivery. Of note, site-specific modification is a big challenge in protein drug research, especially for such a complicated, multifunctional protein delivery system. We successfully develop a protocol for constructing a macromolecular prodrug system with intein-mediated ligation synthesis. With an on-column process of purification and intein-mediated cleavage, the site-specific PEGylation then can be readily achieved by conjugation with the activated C-terminus, thus constructing a PEG-capped, cell-penetrating trichosanthin system with a gelatinase-cleavable linker that enables tumor-specific activation of cytoplasmic delivery. It provides a promising method to address the PEG dilemma for enhanced protein drug delivery, and importantly, a facile protocol for site-specific modification of such a class of protein drugs for improving their druggability and industrial translation.

Proteomic and Glycoproteomic Profilings Reveal That Post-translational Modifications of Toxins Contribute to Venom Phenotype in Snakes.

Snake venoms are biological weapon systems composed of secreted proteins and peptides that are used for immobilizing or killing prey. Although post-translational modifications are widely investigated because of their importance in many biological phenomena, we currently still have little understanding of how protein glycosylation impacts the variation and stability of venom proteomes. To address these issues, here we characterized the venom proteomes of seven Bothrops snakes using a shotgun proteomics strategy. Moreover, we compared the electrophoretic profiles of native and deglycosylated venoms and, in order to assess their subproteomes of glycoproteins, we identified the proteins with affinity for three lectins with different saccharide specificities and their putative glycosylation sites. As proteinases are abundant glycosylated toxins, we examined the effect of N-deglycosylation on their catalytic activities and show that the proteinases of the seven venoms were similarly affected by removal of N-glycans. Moreover, we prospected putative glycosylation sites of transcripts of a B. jararaca venom gland data set and detected toxin family related patterns of glycosylation. Based on our global analysis, we report that Bothrops venom proteomes and glycoproteomes contain a core of components that markedly define their composition, which is conserved upon evolution in parallel to other molecular markers that determine their phylogenetic classification.

TO THE PROBLEM OF NEW BIOPATHOGEN TYPES

Perspective directions of work on development of new types of biopathogens are presented in the paper. Convention on prohibition of biological weapon does not prevent development of new biopathogens. Success in gene engineering and molecular biology opens huge opportunities for receiving new biological agents and modification of the known ones. The basic directions in development of new types of biopathogens are the following: modification of especially dangerous infectious agents; modification of slow infectious agents; modification of uncontrolled infectious agents; synthesis of regulatory genes on the basis of viral vectors; modification of latent viruses; modification of oncogenes and oncogenic viruses; development of movable genetic elements based on phages, plasmids et c; synthesis of genes coding immunoprivileged organ and tissue proteins; modification of animal and plant toxins with elevated resistance to environmental factors; synthesis of genes coding formation of chemical and low concentration biological compounds with toxic or other regulatory effect. Development of new biopathogens will be accompanied by working out new methods for delivering such agents to the target. It is possible to develop a weapon possessing a high degree of selectivity and damaging genome of organism-target of a certain race, ethnos, nation.

Of tracking channel activity, modulating AMPA receptor function, permeating potassium ions, and the plant response to osmotic stress.

This month’s installment of Generally Physiological considers modification of tarantula toxins to track channel activation state, how the prototypical TARP stargazin regulates AMPA receptor signaling, how potassium ions make it through the selectivity filter, and identification of a calcium-

Bt toxin modification for enhanced efficacy.

Insect-specific toxins derived from Bacillus thuringiensis (Bt) provide a valuable resource for pest suppression. Here we review the different strategies that have been employed to enhance toxicity against specific target species including those that have evolved resistance to Bt, or to modify the host range of Bt crystal (Cry) and cytolytic (Cyt) toxins. These strategies include toxin truncation, modification of protease cleavage sites, domain swapping, site-directed mutagenesis, peptide addition, and phage display screens for mutated toxins with enhanced activity. Toxin optimization provides a useful approach to extend the utility of these proteins for suppression of pests that exhibit low susceptibility to native Bt toxins, and to overcome field resistance.

Isolation and characterization of a presynaptic neurotoxin, P-elapitoxin-Bf1a from Malaysian Bungarus fasciatus venom

Presynaptic neurotoxins are one of the major components in Bungarus venom. Unlike other Bungarus species that have been studied, β-bungarotoxin has never been isolated from Bungarus fasciatus venom. It was hypothesized that the absence of β-bungarotoxin in this species was due to divergence during evolution prior to evolution of β-bungarotoxin. In this study, we have isolated a β-bungarotoxin isoform we named P-elapitoxin-Bf1a by using gel filtration, cation-exchange and reverse-phase chromatography from Malaysian B. fasciatus venom. The toxin consists of two heterogeneous subunits, subunit A and subunit B. LCMS/MS data showed that subunit A was homologous to acidic phospholipase A2 subunit A3 from Bungarus candidus and B. multicinctus venoms, whereas subunit B was homologous with subunit B1 from B. fasciatus venom that was previously detected by cDNA cloning. The toxin showed concentration- and time-dependent reduction of indirect-twitches without affecting contractile responses to ACh, CCh or KCl at the end of experiment in the chick biventer preparation. Toxin modification with 4-BPB inhibited the neurotoxic effect suggesting the importance of His-48. Tissue pre-incubation with monovalent B. fasciatus (BFAV) or neuro-polyvalent antivenom (NPV), at the recommended titer, was unable to inhibit the twitch reduction induced by the toxin. This study indicates that Malaysian B. fasciatus venom has a unique β-bungarotoxin isoform which was not neutralized by antivenoms. This suggests that there might be other presynaptic neurotoxins present in the venom and there is a variation in the enzymatic neurotoxin composition in venoms from different localities.

Medically important differences in snake venom composition are dictated by distinct postgenomic mechanisms.

Variation in venom composition is a ubiquitous phenomenon in snakes and occurs both interspecifically and intraspecifically. Venom variation can have severe outcomes for snakebite victims by rendering the specific antibodies found in antivenoms ineffective against heterologous toxins found in different venoms. The rapid evolutionary expansion of different toxin-encoding gene families in different snake lineages is widely perceived as the main cause of venom variation. However, this view is simplistic and disregards the understudied influence that processes acting on gene transcription and translation may have on the production of the venom proteome. Here, we assess the venom composition of six related viperid snakes and compare interspecific changes in the number of toxin genes, their transcription in the venom gland, and their translation into proteins secreted in venom. Our results reveal that multiple levels of regulation are responsible for generating variation in venom composition between related snake species. We demonstrate that differential levels of toxin transcription, translation, and their posttranslational modification have a substantial impact upon the resulting venom protein mixture. Notably, these processes act to varying extents on different toxin paralogs found in different snakes and are therefore likely to be as important as ancestral gene duplication events for generating compositionally distinct venom proteomes. Our results suggest that these processes may also contribute to altering the toxicity of snake venoms, and we demonstrate how this variability can undermine the treatment of a neglected tropical disease, snakebite.