Box jellyfish toxins: investigating novel protein structure and function

A collaborative project undertaken at the Australian Institute of Marine Science, Monash University and James Cook University, and supervised by Diane Brinkman, Australian Institute of Marine Science

Several species of box jellyfish, including Chironex fleckeri (Figure 1), produce venoms that are harmful and potentially life-threatening to humans.  Their venoms contain a complex mixture of toxic proteins which are stored and discharged by small, highly pressurised stinging capsules called nematocysts (Figure 2).

Victims of box jellyfish stings suffer a broad range of species-specific effects, suggesting that box jellyfish venoms vary in toxin composition, biological activity and potency.  C. fleckeri is Australia's most dangerous jellyfish, yet prior to this research project, only two of a number of toxins contained in its venom had been isolated and fully sequenced.  However, knowledge of their amino acid sequences is fundamental to the definitive identification of the venom proteins and prediction of their structures and biological/ecological functions.

Applying a combination of molecular, biochemical, proteomic and bioinformatic approaches, our project focused on the isolation and identification of C. fleckeri venom proteins and investigation of their structural and functional characteristics. Using tandem mass spectrometry we profiled the proteome of C. fleckeri venom and found that the most abundant proteins contained in C. fleckeri nematocysts belong to a small family of pore-forming cnidarian toxins (Brinkman et al. 2012). Several representatives of the toxin family were identified including two previously sequenced C. fleckeri toxins, CfTX-1 and CfTX-2. Further biochemical studies led to the isolation and sequencing of two additional toxins, CfTX-A and CfTX-B, and a third putative toxin, CfTX-Bt, that are also related to the toxin family. Phylogenetic analyses of amino acid sequences of the toxin family inferred that the toxins have diversified into at least two structurally and functionally distinct subfamilies (Type I and II) during evolution. Immunological studies showed that antibodies raised against the Type I and II toxins are not cross-reactive, presumably due to a lack of mutual epitopes brought about by their structural differences. Comparative biological assays demonstrated that CfTX-1 and -2 (Type I toxins) caused rapid cardiovascular collapse in anaesthetised rats (25µg/kg) while CfTX-A and -B (Type II toxins) produced only minor effects at the same dose. In contrast, CfTX-A and -B were more potently haemolytic (>30 times) than CfTX-1 and -2. Together, our data suggests that the Type I toxins have a higher specificity for vertebrate cardiac cells than Type II toxins and therefore are more likely to be the primary toxins involved in human envenoming. Furthermore, only Type II toxins have been identified as the major components of other cubozoan venoms, which may explain why C. fleckeri is exceptionally more dangerous to humans than other box jellyfish species (Brinkman et al., in press).

3D molecular modelling of the C. fleckeri toxins (Figure 3) predicted that the cubozoan toxins share high structural similarity with pore-forming Cry toxins produced by the bacterium Bacillus thuringiensis. Structural homology between the C. fleckeri toxins and Cry toxins suggests the toxins may have a similar pore-forming mechanism of action, while structural diversification within the toxin family may modulate target specificity. Expansion of the cnidarian toxin family has therefore provided new insights into the evolutionary diversification of box jellyfish toxins from a structural and functional perspective (Brinkman et al., in press).

Publications/Conferences

1) Brinkman, D.L., Konstantakopoulos, N., McInerney, B.V., Mulvenna, J., Seymour, J.E., Isbister, G.K., Hodgson, W.C. (in press) Chironex fleckeri (box jellyfish) venom proteins: expansion of a cnidarian toxin family that elicits variable cytolytic and cardiovascular effects. Journal of Biological Chemistry. doi:10.1074/jbc.M113.534149.

2) Brinkman, D.L., Aziz, A., Loukas, A., Potriquet, J., Seymour, J., Mulvenna, J. (2012) Venom proteome of the box jellyfish Chironex fleckeri. PLOS ONE e47866.

3) Brinkman, D.L., Mulvenna, J., Konstantakopoulos, N., Hodgson, W.C., Isbister, G.K., Seymour, J.E., Burnell, J.N. (2012) Molecular diversity of box jellyfish toxins. Toxicon 60, 148-149. 17th World Congress of the International Society on Toxinology, Honolulu, Hawaii, July 08-13, 2012.

4) Brinkman D.L., Konstantakopoulos N., McInerney B., Seymour J.E., Ávila-Soria G., Isbister G.K. and Hodgson W.C. (2011) Toxins of Australia’s most venomous jellyfish, Chironex fleckeri. Symposium presentation, ComBio2011.  Combined annual meetings of the ASBMB, ASPS and ANZSCDB, Cairns, Australia, September 26-29, 2011.

 

Figure 1. The large, multi-tentacled box jellyfish, Chironex fleckeri (Order Chirodropida). Adult specimens can grow to 20-30 cm in bell height and weigh up to 6 kg. Photograph Jamie Seymour.

Figure 2. Nematocysts isolated from the tentacles of C. fleckeri (magnification 400x)Box jellyfish produce a variety of nematocysts that differ in shape, size and function.  The internal structures of undischarged nematocysts are visible using light microscopy.  When specific types of nematocysts discharge, a harpoon-like tube rapidly ejects from the tip of each nematocyst and injects venom into the jellyfish’s victim or prey.  Photograph Diane Brinkman.

Figure 3. Predicted 3D molecular models of C. fleckeri toxins, CfTX-1 and CfTX-A (modelled using I-TASSER and visualised using Jmol)