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2 Supported by a National Health and Medical Research Council Clinical Career Development Fellowship. * This research project was funded by a grant from the Herman Slade Foundation and facilitated by access to the Australian Proteome Analysis Facility supported under the Australian Government's National Collaborative Research Infrastructure Strategy (NCRIS).
The box jellyfish Chironex fleckeri produces extremely potent and rapid-acting venom that is harmful to humans and lethal to prey. Here, we describe the characterization of two C. fleckeri venom proteins, CfTX-A (∼40 kDa) and CfTX-B (∼42 kDa), which were isolated from C. fleckeri venom using size exclusion chromatography and cation exchange chromatography. Full-length cDNA sequences encoding CfTX-A and -B and a third putative toxin, CfTX-Bt, were subsequently retrieved from a C. fleckeri tentacle cDNA library. Bioinformatic analyses revealed that the new toxins belong to a small family of potent cnidarian pore-forming toxins that includes two other C. fleckeri toxins, CfTX-1 and CfTX-2. Phylogenetic inferences from amino acid sequences of the toxin family grouped CfTX-A, -B, and -Bt in a separate clade from CfTX-1 and -2, suggesting that the C. fleckeri toxins have diversified structurally and functionally during evolution. Comparative bioactivity assays revealed that CfTX-1/2 (25 μg kg−1) caused profound effects on the cardiovascular system of anesthetized rats, whereas CfTX-A/B elicited only minor effects at the same dose. Conversely, the hemolytic activity of CfTX-A/B (HU50 = 5 ng ml−1) was at least 30 times greater than that of CfTX-1/2. Structural homology between the cubozoan toxins and insecticidal three-domain Cry toxins (δ-endotoxins) suggests that the toxins have a similar pore-forming mechanism of action involving α-helices of the N-terminal domain, whereas structural diversification among toxin members may modulate target specificity. Expansion of the cnidarian toxin family therefore provides new insights into the evolutionary diversification of box jellyfish toxins from a structural and functional perspective.
Chironex fleckeri (Cnidaria: Cubozoa) is a large, venomous, Australasian box jellyfish that preys on fish and crustaceans but also inflicts painful and potentially fatal stings to humans. Contact with the jellyfish tentacles triggers the explosive discharge of nematocysts (i.e. stinging capsules) that inject extremely potent and rapidly acting venom into the victim or prey. The effects of C. fleckeri envenoming can involve severe localized and systemic effects, including cutaneous pain, inflammation and necrosis, hypertension followed by hypotension, cardiovascular collapse, and cardiac arrest (
). These highly abundant venom proteins belong to a family of taxonomically restricted cnidarian toxins (42–46 kDa) that includes CqTX-A, CrTX-A, and CaTX-A from box jellyfish species Chironex yamaguchii (
)), respectively, as well as other representatives from Cubozoa, Scyphozoa, and Hydrozoa. In cubozoans, the toxin family is associated with potent hemolytic activity and pore formation in mammalian erythrocytes as well as nociception, inflammation, dermonecrosis, cardiovascular collapse, and lethality in rats (
). Although hemolysis has not been reported in human envenoming, the in vivo effects in rats suggest that these toxins may be the primary cause of similar effects in humans.
A recent proteomic study confirmed the presence of CfTX-1 and -2 in C. fleckeri venom and also identified a large number of potential homologues of CqTX-A, CrTX-A, and CaTX-A using tandem mass spectrometry and de novo sequencing (
). Although clearly related to CfTX-1 and -2, these new homologues do not cross-react with CfTX-1 and -2 antibodies and are thus likely to be structurally and functionally different from the characterized toxins. In this study, we describe the purification and molecular characterization of two CfTX-like toxins from C. fleckeri venom that are closely related in sequence to CaTX-A as well as a third, putative toxin that is also homologous to CaTX-A. Through computational analyses and bioactivity assays, we examine the structural and functional characteristics of the new toxins, explore the molecular diversity of the expanded toxin family, and discuss the implications for the biological role of these toxins in box jellyfish stings.
Expansion of the cnidarian toxin family to include CfTX-A, CfTX-B, and CfTX-Bt has provided further insight into the molecular, structural, and functional diversity of the major toxins produced by box jellyfish. Phylogeny-based predictions suggest that the cubozoan toxins have diversified into at least two toxin groups (Type I and II), with the newly described CfTX-A, -B, and Bt grouped among the Type II toxins (Fig. 9). The Type I and Type II toxins are all predicted to contain signal peptides and, with the exception of CfTX-Bt, form dual-domain mature proteins. However, unlike their Type I counterparts, Type II toxins contain a short propart (5–7 residues) ending with the classical dibasic proteolytic cleavage site (RR/KR) between the signal peptide and mature protein. N-terminal proparts are a common feature in precursor proteins and fulfill a variety of important roles during protein biosynthesis and activation. The propart can facilitate controlled and efficient transport within the secretory pathway, promote correct folding, direct posttranslational modifications, and, in protoxins, prevent unwanted toxicity to the host cell prior to propart cleavage and toxin activation (
). The purpose of the propart found exclusively in the Type II toxins remains unclear, but its presence may modulate toxin folding, posttranslational modifications, or intracellular trafficking that in turn leads to diversified function/specificity or variations in toxin localization between the two toxin groups. Alternatively, the Type II toxins may be more toxic to cnidarians (invertebrates) than Type I toxins, and thus the propart may provide additional protection to the host organism during toxin production.
Another difference between the toxin groups is that mature Type II toxins are typically shorter in sequence length and consequently lower in theoretical molecular mass than Type I toxins (Fig. 15). This variation in molecular mass is consistent with experimental observations for CfTX-1 and -2 (Type I) and CfTX-A and -B (Type II) separated by reducing SDS-PAGE (Fig. 1b). It is also noted that the theoretical molecular masses of all mature CfTX-like proteins, including those of other cubozoans, are consistently higher than the apparent molecular mass estimated by SDS-PAGE (
). Notwithstanding the inherent inaccuracies of SDS-PAGE-based estimations, the discrepancies in molecular mass may result from additional posttranslational proteolytic processing during toxin maturation/activation.
Despite only moderate amino acid sequence similarities between the Type I and II toxins, both toxin types are predicted to form similar secondary and tertiary structures. In this study, high structural homology was predicted between the N-terminal domains of the CfTX-A, -B, and -Bt and 3d-Cry toxins, which suggests that their domains share a similar functional role. B. thuringiensis 3d-Cry toxins are a family of proteins that exhibit specific biocidal activities against insect larvae from the orders Lepidoptera (butterflies and moths), Diptera (flies and mosquitoes), and Coleoptera (beetles) (
). The N-terminal domain (Domain I) forms a seven- or eight-helix bundle in which a central hydrophobic helix is surrounded by outer helices. The central and C-terminal domains (Domains II and III) contain mostly β-sheets. The molecular mechanism underlying 3d-Cry toxin activity is complex and is proposed to involve several steps, including protoxin proteolytic processing, multireceptor toxin binding, oligomerization, and pore formation, which subsequently leads to midgut cell lysis and larval death (
). Numerous studies have implicated Domain I in toxin oligomerization, membrane insertion, and pore formation, whereas Domains II and III are implicated in receptor recognition, binding, and toxin specificity (reviewed in Ref.
). Given that the N-terminal domains of CfTX-like and 3d-Cry toxins are structurally analogous, it is therefore plausible that the CfTX-like toxins are involved in oligomerization and pore formation in cardiocytes, erythrocytes, and other susceptible cells. The formation of relatively large ring-shaped pores (12-nm inner diameter/25-nm outer diameter) in human erythrocyte cell membranes following exposure to purified CfTX isoforms (
), the CfTX proteins have a propensity to oligomerize into high molecular mass quaternary structures, implying that although each toxin is individually secreted, they assemble as larger heterogeneous Type I (CfTX-1/2) or Type II (CfTX-A/B) holotoxins.
The functional role(s) of the C-terminal domain in CfTX-like toxins is less clear, but due to its structural similarity (albeit weaker) to Domain II of 3d-Cry toxins, it may be involved in receptor binding and/or toxin specificity. Earlier experimental studies found that purified CaTX-A bound to specific carbohydrates (
), which implicates these carbohydrates as potential sugar moieties in toxin-binding receptors. However, further functional studies are still necessary to establish which domain(s) are involved in receptor binding and whether the carbohydrate-binding affinity of CfTX-like toxins influences target specificity. The discovery of the truncated isoform CfTX-Bt, in which the C-terminal domain is missing, also raises questions about the functional relevance of the C-terminal domain.
Although the cubozoan toxins share a conserved structural scaffold, evolutionary diversification of toxin family members into two broad groups infers that the toxins vary in function and/or specificity. This hypothesis is supported by our data indicating that Type I and II toxin-specific antibodies are not cross-reactive (Fig. 2), presumably due to the absence of mutual epitopes, and that the cardiovascular and cytolytic activities associated with Type I and II toxins are different. Purified CfTX-A and -B (CEX Peak 2) caused relatively minor in vivo cardiovascular effects in anesthetized rats (25 μg kg−1, intravenously), whereas CfTX-1 and -2 (SEC Peak 2) caused cardiovascular collapse within 1 min at the same dose. Fractions from the other SEC peaks caused less potent cardiovascular effects than SEC Peak 2, which could be attributed to CfTX-1 and -2 contamination, as detected by Western blot analysis, and/or the presence of other unidentified cardioactive toxins. Together, these findings suggest 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. The variability of the in vivo cardiovascular effects is also consistent with previous studies on the in vitro effects of C. fleckeri fractionated venom on human cardiac myocytes (
), where only SEC Peak 2 fractions (purportedly containing CfTX-1 and -2) caused rapid cell detachment and death. To date, only Type II toxins have been identified in the venoms of A. moseri, C. rastonii, and C. yamaguchii, which may also explain why C. fleckeri is exceptionally more dangerous to humans than other box jellyfish species. Nonetheless, it is also feasible that Type I toxins are expressed in the other species but at much lower levels.
In contrast to the rat studies, in vitro hemolysis assays demonstrated that Type II toxins CfTX-A and -B elicit more potent hemolytic activity (HU50 = 5 ng ml−1) than Type I toxins CfTX-1 and -2 (HU50 = 161 ng ml−1). Variability in hemolytic activity is also apparent when comparing toxin family members of other box jellyfish. In studies of A. moseri, C. rastonii, and C. yamaguchii, HU50 values for purified CaTX-A and CrTX-A (Type II toxins) were at least 10-fold lower than purified CqTX-A (Type I toxin) (
). However, as mentioned previously, hemolysis has never been reported as a clinical feature of human envenoming, which suggests that the cubozoan toxins preferentially target other cell types in vivo. Although the rat studies suggest that Type I toxins elicit more potent cardiovascular effects in vertebrates than Type II toxins, some experimental evidence suggests that the Type II toxins elicit more potent effects in invertebrates. For example, in earlier studies on crustaceans, researchers found that the LD50 values of Type II toxins CaTX-A and CrTX-A (5–25 μg kg−1) were lower than the Type I toxin CqTX-A (80 μg kg−1) (
). Comparative studies on the invertebrate toxicity of purified CfTX-1/2 and CfTX-A/B have yet to be published, but they would undoubtedly provide important information on the target specificity of the two toxin types. Similarly, as more toxin sequences and structural/functional data are acquired for other venomous cnidarians, our understanding of the molecular diversity and actions of this unique toxin family can be further refined.
We thank Avril Underwood (James Cook University), Andrew Hart (Monash University), Georgina Giannikopoulos (Australian Proteome Analysis Facility), and Annabel Good (IMVS) for assistance with jellyfish collection, rat bioassays, Edman sequencing, and antibody production, respectively.
Prospective study of Chironex fleckeri and other box jellyfish stings in the “Top End” of Australia's Northern Territory.