School of Biological Sciences, The University of Queensland, St Lucia QLD 4072, AUSTRALIA.
The phylum Cnidaria (corals, sea pens, sea anemones, jellyfish and hydroids) is the oldest venomous animal lineage (~750 million years old), making it an ideal phylum to understand the origin and diversification of venom. Cnidarians are characterised by specialised cellular structures called cnidae, which they utilise to inject mixtures of bioactive compounds or venom for predation and defence. In recent years cnidarian venoms have begun to be investigated as a potential source of novel bioactive therapeutics. However, in comparison with several venomous lineages that are relatively younger, such as snakes, cone snails and spiders, the diversification and molecular evolutionary regimes of toxins encoded by these fascinating and ancient animals remain poorly understood.
The first experimental part of this thesis (Chapter 2) is comprised of the phylogenetic and molecular evolutionary histories of pharmacologically characterised cnidarian toxin families. These include peptide neurotoxins (voltage-gated Na+ and K+ channel-targeting toxins: NaTxs and KTxs, respectively), pore-forming toxins (actinoporins, aerolysin-related toxins, and jellyfish toxins) and small cysteine-rich peptides (SCRiPs). Our analyses show that most cnidarian toxins remain conserved under the strong influence of negative selection. A deeper analysis of the molecular evolutionary histories of neurotoxins demonstrates that type III KTxs have evolved from NaTxs under the regime of positive selection and likely represent a unique evolutionary innovation of the Actinioidea lineage. We also identify a few functionally important sites in other classes of neurotoxins and pore-forming toxins that experienced episodic adaptation. In addition, we describe the first family of neurotoxic peptides (SCRiPs) in reef-building corals, Acropora millepora, that are likely involved in the envenoming function.
The third chapter describes the development of a novel venom extraction technique which is rapid, repeatable and cost effective. This technique involves using ethanol to both induce cnidae discharge and to recover venom contents in one step. Our model species is the notorious Australian box jellyfish (Chironex fleckeri) which has a notable impact on public health. By using the complementary approaches of transcriptomics, proteomics, mass spectrometry, histology, and scanning electron microscopy, this study compares the proteome profile of the new ethanol recovery based method to a previously reported protocol, based upon density purified intact cnidae and pressure induced disruption. This work not only results in the expansion of identified Australian box jellyfish venom components but also illustrates that ethanol extraction method could greatly augment future cnidarian venom proteomics research efforts.
The forth chapter is constituted by previously described venom extraction technique, ethanol- induced discharge of cnidae, to rapidly and efficiently characterise venom components of the Jelly Blubber or Blue Blubber Jellyfish, Catostylus mosaicus. By using a combination of proteomics and transcriptome sequencing, potential toxin families are identified in the oral arms and tentacles included PLA2, lipases, CRiSPs, cystatins and serine protease inhibitors (Kunitz and Kazal peptides). This study reveals venom components of oral arms and tentacles of a blubber jellyfish for the first time and enhances our understanding of the mechanism of its sting.
The fifth chapter describes the pharmacological profile of the hell’s fire anemone (Actinodendron sp.) venom. Due to the lack of transcriptome data available, an activity-guided fractionation approach (chromatography/tandem mass spectrometry/bioassays) is described to purify and characterise novel bioactives (toxins) with interesting pharmacological properties. The potential anemone toxin explained here (α3β2 nAChR blocker) is strong candidate as a lead compound for the development of diagnostic and therapeutic.
Taken together, this thesis unravels the molecular evolutionary history of cnidarian toxin families, details a novel venom extraction technique which may transform cnidarian venom research, and also illustrates how proteomics, transcriptome sequencing and activity-guided fractionation can be used to discover new proteins and peptide toxins that can be bioactive resources for drug discovery and development.