Diane Brinkman

Biochemical and molecular characterisation of cubozoan protein toxins.

Class Cubozoa includes several species of box jellyfish that are harmful to humans. The venoms of box jellyfish are stored and discharged by nematocysts and contain a variety of bioactive proteins that are cytolytic, cytotoxic, inflammatory or lethal. Although cubozoan venoms generally share similar biological activities, the diverse range and severity of effects caused by different species indicate that their venoms vary in protein composition, activity and potency. To date, few individual venom proteins have been thoroughly characterised, however, accumulating evidence suggests that cubozoan jellyfish produce at least one group of homologous bioactive proteins that are labile, basic, haemolytic and similar in molecular mass (42-46 kDa). The novel box jellyfish toxins are also potentially lethal and the cause of cutaneous pain, inflammation and necrosis, similar to that observed in envenomed humans. Secondary structure analysis and remote protein homology predictions suggest that the box jellyfish toxins may act as alpha-pore-forming toxins. However, more research is required to elucidate their structures and investigate their mechanism(s) of action. The biological, biochemical and molecular characteristics of cubozoan venoms and their bioactive protein components are reviewed, with particular focus on cubozoan cytolysins and the newly emerging family of box jellyfish toxins.

Partial purification of cytolytic venom proteins from the box jellyfish, Chironex fleckeri

Venom proteins from the nematocysts of Chironex fleckeri were fractionated by size-exclusion and cation-exchange chromatography. Using sheep erythrocyte haemolysis as an indicator of cytolytic activity, two major cytolysins, with native molecular masses of approximately 370 and 145kDa, and one minor cytolysin ( approximately 70kDa) were isolated. SDS-PAGE and western blot protein profiles revealed that the 370kDa haemolysin is composed of CfTX-1 and CfTX-2 subunits ( approximately 43 and 45kDa, respectively); the most abundant proteins found in C. fleckeri nematocyst extracts. The 145kDa haemolysin predominately contains two other major proteins ( approximately 39 and 41kDa), which are not antigenic towards commercially available box jellyfish antivenom or rabbit polyclonal antibodies raised against whole C. fleckeri nematocyst extracts or CfTX-1 and -2. The kinetics of CfTX-1 and -2 haemolytic activities are temperature dependent and characterised by a pre-lytic lag phase ( approximately 6-7min) prior to initiation of haemolysis. Significant amino acid sequence homology between the CfTX proteins and other box jellyfish toxins suggest that CfTX-1 and -2 may also be lethal and dermonecrotic. Therefore, further in vivo and in vitro studies are required to investigate the potential roles of CfTX-1 and -2 in the lethal effects of C. fleckeri venom.

Venom Proteome of the Box Jellyfish Chironex fleckeri

The nematocyst is a complex intracellular structure unique to Cnidaria. When triggered to discharge, the nematocyst explosively releases a long spiny, tubule that delivers an often highly venomous mixture of components. The box jellyfish, Chironex fleckeri, produces exceptionally potent and rapid-acting venom and its stings to humans cause severe localized and systemic effects that are potentially life-threatening. In an effort to identify toxins that could be responsible for the serious health effects caused by C. fleckeri and related species, we used a proteomic approach to profile the protein components of C. fleckeri venom. Collectively, 61 proteins were identified, including toxins and proteins important for nematocyte development and nematocyst formation (nematogenesis). The most abundant toxins identified were isoforms of a taxonomically restricted family of potent cnidarian proteins. These toxins are associated with cytolytic, nociceptive, inflammatory, dermonecrotic and lethal properties and expansion of this important protein family goes some way to explaining the destructive and potentially fatal effects of C. fleckeri venom. Venom proteins and their post-translational modifications (PTMs) were further characterized using toxin-specific antibodies and phosphoprotein/glycoprotein-specific stains. Results indicated that glycosylation is a common PTM of the toxin family while a lack of cross-reactivity by toxin-specific antibodies infers there is significant divergence in structure and possibly function among family members. This study provides insight into the depth and diversity of protein toxins produced by harmful box jellyfish and represents the first description of a cubozoan jellyfish venom proteome.

Transcriptome and venom proteome of the box jellyfish Chironex fleckeri

BackgroundThe box jellyfish, Chironex fleckeri, is the largest and most dangerous cubozoan jellyfish to humans. It produces potent and rapid-acting venom and its sting causes severe localized and systemic effects that are potentially life-threatening. In this study, a combined transcriptomic and proteomic approach was used to identify C. fleckeri proteins that elicit toxic effects in envenoming.ResultsMore than 40,000,000 Illumina reads were used to de novo assemble ∼ 34,000 contiguous cDNA sequences and ∼ 20,000 proteins were predicted based on homology searches, protein motifs, gene ontology and biological pathway mapping. More than 170 potential toxin proteins were identified from the transcriptome on the basis of homology to known toxins in publicly available sequence databases. MS/MS analysis of C. fleckeri venom identified over 250 proteins, including a subset of the toxins predicted from analysis of the transcriptome. Potential toxins identified using MS/MS included metalloproteinases, an alpha-macroglobulin domain containing protein, two CRISP proteins and a turripeptide-like protease inhibitor. Nine novel examples of a taxonomically restricted family of potent cnidarian pore-forming toxins were also identified. Members of this toxin family are potently haemolytic and cause pain, inflammation, dermonecrosis, cardiovascular collapse and death in experimental animals, suggesting that these toxins are responsible for many of the symptoms of C. fleckeri envenomation.ConclusionsThis study provides the first overview of a box jellyfish transcriptome which, coupled with venom proteomics data, enhances our current understanding of box jellyfish venom composition and the molecular structure and function of cnidarian toxins. The generated data represent a useful resource to guide future comparative studies, novel protein/peptide discovery and the development of more effective treatments for jellyfish stings in humans. (Length: 300).

Chironex fleckeri (Box Jellyfish) Venom Proteins: Expansion of a Cnidarian Toxin Family that Elicits Variable Cytolytic and Cardiovascular Effects

Background: Box jellyfish produce a unique family of toxic venom proteins. Results: The toxins are structurally similar, yet two subgroups confer different cytolytic activities in red blood cells and cardiovascular effects in rats. Conclusion: Diversification within the toxin family may influence toxin function/specificity. Significance: Characterization of the toxins provides new insight into their potential roles in human envenoming. 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.

Lipids as biomarkers for carbon cycling on the Northwest Shelf of Australia: results from a sediment trap study

Abstract Sediment traps were deployed on the Northwest Shelf (NWS) of Australia in November 1996, to determine fluxes of organic matter and inorganic elements from the photic zone to deeper waters in a transect extending from Exmouth Shelf to Exmouth Plateau. Infiltrex II water samplers collected particulate and dissolved organics from the water column near the trap sites. Surface sediments and sediment cores were also collected over the study region. Lipid biomarkers were used to determine the sources of organic carbon and its cycling processes on the NWS. Dry weight fluxes from the traps ranged from 124 to 616 mg m −2 day −1 and particulate organic carbon (POC) fluxes ranged from 22 to 42 mg m −2 day −1 . The biogenic lipids consisted of biomarkers indicative of marine zooplankton, phytoplankton and bacteria, plus traces of land plant markers. A large contribution of unresolved complex material (UCM), which is indicative of petroleum, was detected at four times the biogenic hydrocarbon flux at shallow stations, and up to seven times the biogenic hydrocarbon flux at the most offshore station. There is essentially no river input, and only trace aeolian-derived material to contribute to primary production on the NWS of Australia. Most of the organic matter produced are rapidly recycled in the water column and the small fraction of lipids that settle to the sediments is already partially degraded and undergoes further rapid degradation in the surface sediments. Natural oil seeps also provide utilisable organic carbon to the system. The production and vertical flux rates of organics determined in this study are comparable to those reported in studies of shallow traps in oceanic areas from long-term studies in the Arabian Sea, and other coastal margins such as the Bay of Biscay (France) and California (USA). In offshore areas, most “living” lipid materials passed through the GFF filters thus invalidating POC estimates based on high volume sampling. To adequately assess living (particulate) carbon, gentle filtration of low volume seawater samples is more accurate, as shown by this solid phase absorption study of individual lipid biomarkers.

Acute ecotoxicology of natural oil and gas condensate to coral reef larvae.

Risks posed by oil spills to coral reefs are difficult to evaluate, partially due to the absence of studies that adequately assess toxicity to relevant coral reef species. Here we experimentally tested the acute toxicity of condensate, representing a fraction of light crude oil, to coral (Acropora tenuis) and sponge (Rhopaloeides odorabile) larvae. The metamorphosis of coral larvae was inhibited at total petroleum aromatic hydrocarbon (TPAH) concentrations of water accommodated fractions (WAF) as low as 103 μg l−1, similar to concentrations detected in seawater following large spills. The sensitivity of coral larvae increased by 40% when co-exposed to UV light that they might encounter in shallow reefal systems. Condensate WAF was more toxic to coral larvae than predicted by summing the toxicity of its main components (benzene, toluene, p-xylene and napthalene). In contrast, the sensitivity of sponge larvae to condensate WAF (>10,000 μg l−1 TPAH) was far less than coral in the presence and absence of UV, but similar to that of other marine invertebrates. While these results highlight the relative sensitivity of coral larvae to oil, further research is needed to better understand and predict the impacts and risks posed by hydrocarbons to tropical reef systems.

Comparative study of the toxic effects of Chrysaora quinquecirrha (Cnidaria: Scyphozoa) and Chironex fleckeri (Cnidaria: Cubozoa) venoms using cell-based assays.

The venoms of jellyfish cause toxic effects in diverse biological systems that can trigger local and systemic reactions. In this study, the cytotoxic and cytolytic effects of Chrysaora quinquecirrha and Chironex fleckeri venoms were assessed and compared using three in vitro assays. Venoms from both species were cytotoxic to fish gill cells and rat cardiomyocytes, and cytolytic in sheep erythrocytes. Both venoms decreased cell viability in a concentration-dependent manner; however, the greatest difference in venom potencies was observed in the fish gill cell line, wherein C. fleckeri was 12.2- (P = 0.0005) and 35.7-fold (P < 0.0001) more potently cytotoxic than C. quinquecirrha venom with 30 min and 120 min cell exposure periods, respectively. Gill cells and rat cardiomyocytes exposed to venoms showed morphological changes characterised by cell shrinkage, clumping and detachment. The cytotoxic effects of venoms may be caused by a group of toxic proteins that have been previously identified in C. fleckeri and other cubozoan jellyfish species. In this study, proteins homologous to CfTX-1 and CfTX-2 toxins from C. fleckeri and CqTX-A toxin from Chironex yamaguchii were identified in C. quinquecirrha venom using tandem mass spectrometry. The presence and relative abundance of these proteins may explain the differences in venom potency between cubozoan and scyphozoan jellyfish and may reflect their importance in the action of venoms.

Phototoxic effects of PAH and UVA exposure on molecular responses and developmental success in coral larvae

Exposure to polycyclic aromatic carbons (PAHs) poses a growing risk to coral reefs due to increasing shipping and petroleum extraction in tropical waters. Damaging effects of specific PAHs can be further enhanced by the presence of ultraviolet radiation, known as phototoxicity. We tested phototoxic effects of the PAHs anthracene and phenanthrene on larvae of the scleractinian coral Acropora tenuis in the presence and absence of UVA (320-400 nm). Activity of superoxide dismutase (SOD) enzyme was reduced by anthracene while phenanthrene and UVA exposure did not have any effect. Gene expression of MnSod remained constant across all treatments. The genes Catalase, Hsp70 and Hsp90 showed increased expression levels in larvae exposed to anthracene, but not phenanthrene. Gene expression of p53 was upregulated in the presence of UVA, but downregulated when exposed to PAHs. The influence on stress-related biochemical pathways and gene expresson in A. tenuis larvae was considerably greater for anthracene than phenanthrene, and UVA-induced phototoxicity was only evident for anthracene. The combined effects of UVA and PAH exposure on larval survival and metamorphosis paralleled the sub-lethal stress responses, clearly highlighting the interaction of UVA on anthracene toxicity and ultimately the corals development.

Effects of coal contamination on early life history processes of a reef-building coral, Acropora tenuis

Successful reproduction and larval dispersal are important for the persistence of marine invertebrate populations, and these early life history processes can be sensitive to marine pollution. Coal is emerging as a contaminant of interest due to the proximity of ports and shipping lanes to coral reefs. To assess the potential hazard of this contaminant, gametes, newly developed embryos, larvae and juveniles of the coral Acropora tenuis were exposed to a range of coal leachate, suspended coal, and coal smothering treatments. Fertilisation was the most sensitive reproductive process tested. Embryo survivorship decreased with increasing suspended coal concentrations and exposure duration, effects on larval settlement varied between treatments, while effects on juvenile survivorship were minimal. Leachate exposures had negligible effects on fertilisation and larval settlement. These results indicate that coral recruitment could be affected by spills that produce plumes of suspended coal particles which interact with gametes and embryos soon after spawning.