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Neurotoxicity of Malaysian Bungarus candidus and Bungarus fasciatus venoms
thesisposted on 21.02.2017, 23:44 by Ahmad Rusmili, Muhamad Rusdi
The current study examined the neurotoxicity of venoms from two species of krait found in Malaysia, i.e. Bungarus candidus (Malayan krait) and Bungarus fasciatus (banded krait). The aim of the study was to examine the in vitro neurotoxic effects of Malaysian B. candidus and B. fasciatus venoms and to determine the effectiveness of the commercially available krait monovalent and neuro polyvalent antivenoms, produced in Thailand, in neutralizing the neurotoxicity in a skeletal muscle preparation. B. candidus and B. fasciatus are geographically distributed in several countries in South East Asia and cause neurotoxicity in systemically envenomed victims. Case reports indicate that high antivenom doses are usually needed before toxicity is reversed. With increasing evidence that intraspecific variations in the composition of snake venoms affects the neutralizing capability of antivenoms, there is a need to study the neutralizing capability of the antivenoms used to treat envenoming by Malaysian Bungarus spp., as these antivenoms are developed from animals immunized with venom from different localities. In addition, we were interested in identifying the toxins in the venom that may affect antivenom efficacy. In vitro neurotoxicity of venoms from Malaysian B. candidus and B. fasciatus were evaluated using the chick biventer cervicis nerve-muscle preparation to obtain t90 values (i.e. the time for 90% reduction of initial twitch height). Although several toxins have been isolated from these venoms, and assayed using the same preparation, t90 values for these venoms have not been reported. Both venoms (1-10 µg/ml) caused complete inhibition of indirect twitches in the preparation without obvious myotoxicity. The monovalent antivenoms and neuro polyvalent antivenom were effective in neutralizing the in vitro toxicity, when added prior to the venom, at higher concentrations than the recommended titer. No cross-reactivity was seen when the monovalent antivenom raised against the venom from the different species was used. Reversal of neurotoxicity was only achieved when polyvalent antivenom was added at a higher antivenom titre at the t90 time point after B. fasciatus venom. Western blotting of crude venoms against both monovalent antivenoms and the neuro polyvalent antivenom revealed that there are differences in the antigenicity of components in the venom. These findings showed that early antivenom administration of high doses of Thailand's krait monovalent and neuro polyvalent antivenom may be beneficial in the treatment of systemic envenoming. These findings also indicate that there are differences between the composition of the venoms used in this study and the venoms that were used for antivenom production. Differences in activity and neutralization capability of Thailand's antivenoms prompted the need to study the venoms in further detail. A proteomics study was conducted to determine the composition of Malaysian B. candidus and B. fasciatus venoms. Intact protein analysis by LCMS and reverse-phase HPLC showed that there were significant differences in chromatographic elution patterns between Malaysian B. candidus and B. fasciatus venoms. LCMS/MS analysis of the fractions and gel bands from the venoms indicated marked differences in the number and composition of identified venom proteins between the venoms. Eighteen different protein families were detected in the venoms, with 103 different venom proteins in Malaysian B. candidus venom and 86 different venom proteins in Malaysian B. fasciatus venom. In both venoms, phospholipase A₂ (PLA₂), three finger toxins, and Kunitz-type inhibitors were found to be the major families in the venoms. Venom protein families that have not previously been reported in these venoms including natriuretic peptides, vespryn, and thrombin-like enzymes were also detected. Several presynaptic and postsynaptic neurotoxins that previously have only been detected at the transcript level in venoms from other localities were also detected in the venoms. An interesting discovery was the detection of subunits A and B of β-bungarotoxins and α-bungarotoxin in Malaysian B. fasciatus venom. Detection of these toxins suggests that Malaysian B. fasciatus venom is unique compared to venoms from other localities as these toxins were thought not to be present in B. fasciatus venom. With the availability of venom proteomics data, the targeted isolation and characterisation of neurotoxins was conducted to determine whether the effects of these toxins could be reversed by the antivenoms. A new isoform of a short-chain neurotoxin from Malaysian B. fasciatus, which we named α-elapitoxin-Bf1b, was isolated using mass-spectrometry and a bioassay guided approach. α-Elapitoxin-Bf1b was found to be a monomer with a molecular weight of 6.9 kDa and highly homologous to a toxin that has been previously detected at the transcript level in the B. fasciatus venom gland, Neurotoxin 3FTx-RI. The in vitro neurotoxicity of α-elapitoxin-Bf1b was concentration- and time-dependent, and reversible by repetitive washing. The pA₂ of the toxin was calculated to be 9.17 ± 0.64 using the Lew-Angus method. This value is higher than the pA₂’s for tubocurarine and α-bungarotoxin. Both B. fasciatus monovalent and neuro polyvalent antivenoms failed to neutralize the neurotoxicity of α-elapitoxin-Bf1b. The presence of α-elapitoxin-Bf1b and the incapability of the antivenoms to reverse its neurotoxicity indicated that α-elapitoxin-Bf1b is probably unique to Malaysian B. fasciatus venom and variations in three finger toxins in the venoms could be a crucial factor to explain the lack of effectiveness of the antivenoms. It also possible that there are other uncharacterized postsynaptic neurotoxins in the venom. A β-bungarotoxin-like presynaptic neurotoxin, which we named P-elapitoxin-Bf1a, was also isolated from Malaysian B. fasciatus venom. P-elapitoxin-Bf1a was found to be a heterodimer with an intact protein weight of 20.75 kDa. The heavy subunit, labelled as subunit A, was found to be highly homologous with β-bungarotoxin subunit A3 from B. candidus and B. multicinctus venom, whereas the light subunit was found to be highly homologous with β-bungarotoxin subunit B1 from B. fasciatus venom. P-elapitoxin-Bf1a displayed concentration- and time- dependent neurotoxicity without significantly affecting contractile response to ACh, CCh or KCl. P-elapitoxin-Bf1a was irreversible upon repetitive washing. Modification at His-48, with 4-BPB, prevented the neurotoxic effect and PLA₂ activity of P-elapitoxin-Bf1a. At the recommended titre, B. fasciatus monovalent and neuro polyvalent antivenoms failed to neutralize the neurotoxicity caused by P-elapitoxin-Bf1a. Isolation of P-elapitoxin-Bf1a showed that β-bungarotoxin-like toxins are present in Malaysian B. fasciatus venom and variations in enzymatic composition may occur in the venom. Similar to postsynaptic toxins, variability in the enzymatic composition may play an important role in antivenom effectiveness. In conclusion, we have examined the in vitro neurotoxicity of Malaysian B. candidus and B. fasciatus venoms, mapped the venom proteome of both venoms, isolated and characterized two new isoforms of neurotoxins, i.e. a postsynaptic and a presynaptic neurotoxin, and examined their susceptibility to the commercially available antivenoms used to treat envenoming by these species. These data will provide useful insights for the management of systemic envenoming and may assist in the future development of an antivenom(s) for B. candidus and B. fasciatus from different localities.