Andreosso, Athena (2019) Molecular and structural insights into Chironex fleckeri venom. PhD thesis, James Cook University.

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Abstract

This thesis focuses on the characterization of the bioactivity, composition, molecular pathways and structural aspects of Chironex fleckeri venom proteins. C. fleckeri is a box jellyfish that recurrently causes minor to fatal envenomations on the beaches of the northern half of Australia. While there is an antivenom available, its effectiveness is subject to controversy and a rapidly acting treatment has not yet been found. One essential aspect in developing such a treatment is to further the current knowledge regarding the venom components and their effects.

C. fleckeri venom is composed of a complex mixture of proteins which can cause rapid cardiovascular collapse in humans and animals. Two highly cardiotoxic, haemolytic and potentially pore-forming toxins have been previously identified, CfTX-1 and -2, and are thought to be the underlying cause for the cardiovascular collapse. Each of my chapters focussed on different toxinological aspects with the overall aim of shedding some light on the complex nature of C. fleckeri venom.

Chapter 2 focussed on the intracellular effects of C. fleckeri venom on human cardiomyocytes. While the chosen method, namely fluorescence microscopy, proved inadequate for the intended analysis of C. fleckeri venom, the study provided some interesting results. All cells consistently displayed loss of adherence, nuclear condensation and loss of membrane integrity. The nuclear condensation, an event often observed in apoptosis, has not been previously reported in relation to C. fleckeri venom.

Chapter 3 focussed on the characterisation of three previously reported bioactive fractions in the venom (CTF-α, CTF-β and CTF-γ). The previously reported cardiotoxic activity of CTF-α and CTF-β, but not CTF-γ, suggested the presence of the toxins CfTX-1 and -2 in the former two fractions. Interestingly, the mass spectrometric analysis revealed the presence of these toxins in all three fractions, and further these toxins were most abundant in CTF-α, which in the present analysis displayed the least cardiotoxicity. Overall the fractions all contained CfTX-1 and -2, CfTX-A and -B as well as three other cubozoan toxins, CqTX-1, CaTX-A and CrTX-A, in differing proportions. This was reflected in the distinct bioactivity and activated molecular pathways of each of the fractions. Flow cytometry analyses revealed that neither C. fleckeri venom, nor CTF-α (top two hits: CfTX-1 and -2), induced apoptosis, whereas CTF-β (CaTX-A, CfTX-A and CfTX-B) and CTF-γ (CrTX-A and CfTX-A) treated cardiomyocytes were in early and late apoptotic stages, respectively. Overall, there was no apparent difference in bioactivity between cardiomyocytes and fibroblasts, whereas the effects of the venom on mouse erythrocytes was significantly higher than on human erythrocytes. This higher potency on mouse cells might explain why haemolysis is a symptom in laboratory animals but not in humans.

Chapter 4 represents the first structural analysis of a C. fleckeri toxin. Two predicted helical regions of CfTX-1 were synthesised to assess experimentally whether they had helical structure that may have some relevance in the putatively pore-forming activity of the venom. While complications were encountered in aqueous solutions, both peptides formed a helical structure in the membrane-mimicking solvent SDS. This data represents the first experimental structural data in favour of a pore-forming mode of action.

Overall this thesis has provided insight into the bioactivity of C. fleckeri proteins and their mechanisms of action, highlighted the complexity and the difficulty of working with animal venoms and provided some valuable insight for future studies, including those of a structural nature.

Item ID:	60494
Item Type:	Thesis (PhD)
Keywords:	Chironex fleckeri venom, CfTX-1, CTF-α, CTF-β, CTF-γ, cubozoan toxins, pore-forming toxins, jellyfish venom
Related URLs:	https://researchonline.jcu.edu.au/54593/
Copyright Information:	Copyright © 2019 Athena Andreosso.
Additional Information:	One publication arising from this thesis is available from the Related URLs field. The publication is: Chapter 4: Andreosso, Athena, Bansal, Paramjit S., Smout, Michael J., Wilson, David, Seymour, Jamie E., and Daly, Norelle L. (2018) Structural characterisation of predicted helical regions in the Chironex fleckeri CfTX-1 toxin. Marine Drugs, 16 (6). 201. pp. 1-12.
Date Deposited:	01 Oct 2019 03:53
FoR Codes:	06 BIOLOGICAL SCIENCES > 0601 Biochemistry and Cell Biology > 060101 Analytical Biochemistry @ 100%
SEO Codes:	97 EXPANDING KNOWLEDGE > 970106 Expanding Knowledge in the Biological Sciences @ 100%
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