Molecular and structural insights into Chironex fleckeri venom

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

Rapid and permanent cytotoxic effects of venom from Chiropsella bronzie and Malo maxima on human skeletal and cardiac muscle cells

Jellyfish envenomation is a global public health risk; Cubozoans (box jellyfish) are a prevalent jellyfish class with some species causing potent and potentially fatal envenomation in tropical Australian waters. Previous studies have explored the mechanism of action of venom from the lethal Cubozoan Chironex fleckeri and from Carukia barnesi (which causes “Irukandji syndrome”), but mechanistic knowledge to develop effective treatment is still limited. This study performed an in-vitro cytotoxic examination of the venoms of Chiropsella bronzie and Malo maxima, two understudied species that are closely related to Chironex fleckeri and Carukia barnesi respectively. Venom was applied to human skeletal muscle cells and human cardiomyocytes while monitoring with the xCELLigence system. Chiropsella bronzie caused rapid cytotoxicity at concentrations as low as 58.8 μg/mL. Malo maxima venom caused a notable increase in cell index, a measure of cell viability, followed by cytotoxicity after 24-h venom exposure at ≥11.2 μg/mL on skeletal muscle cells. In contrast, the cardiomyocytes mostly showed significant increased cell index at the higher M. maxima concentrations tested. These findings show that these venoms can exert cytotoxic effects and Malo maxima venom mainly caused a sustained increase in cell index across both human cell lines, suggesting a different mode of action to Chiropsella bronzie. As these venoms show different real-world envenomation symptoms, the different cellular toxicity profiles provide a first step towards developing improved understanding of mechanistic pathways and novel envenomation treatment

Structural characterisation of predicted helical regions in the Chironex fleckeri CfTX-1 toxin

The Australian jellyfish Chironex fleckeri, belongs to a family of cubozoan jellyfish known for their potent venoms. CfTX-1 and -2 are two highly abundant toxins in the venom, but there is no structural data available for these proteins. Structural information on toxins is integral to the understanding of the mechanism of these toxins and the development of an effective treatment. Two regions of CfTX-1 have been predicted to have helical structures that are involved with the mechanism of action. Here we have synthesized peptides corresponding to these regions and analyzed their structures using NMR spectroscopy. The peptide corresponding to the predicted N-terminal amphiphilic helix appears unstructured in aqueous solution. This lack of structure concurs with structural disorder predicted for this region of the protein using the Protein DisOrder prediction System PrDOS. Conversely, a peptide corresponding to a predicted transmembrane region is very hydrophobic, insoluble in aqueous solution and predicted to be structured by PrDOS. In the presence of SDS-micelles both peptides have well-defined helical structures showing that a membrane mimicking environment stabilizes the structures of both peptides and supports the prediction of the transmembrane region in CfTX-1. This is the first study to experimentally analyze the structure of regions of a C. fleckeri protein

Detecting and confirming residual hotspots of lymphatic filariasis transmission in American Samoa 8 years after stopping Mass Drug Administration

The Global Programme to Eliminate Lymphatic Filariasis (LF) aims to eliminate the disease as a public health problem by 2020 by conducting mass drug administrations (MDA) and controlling morbidity. Once elimination targets have been reached, surveillance is critical for ensuring that programmatic gains are sustained and challenges include timely identification of residual areas of transmission. WHO guidelines encourage cost-efficient surveillance, such as integration with other population-based surveys. In American Samoa, where LF is caused by Wuchereria bancrofti, and Aedes polynesiensis is the main vector, the LF elimination program has made significant progress. Seven rounds of MDA (albendazole and diethycarbamazine) were completed from 2000 to 2006, and Transmission Assessment Surveys were passed in 2010/2011 and 2015. However, a seroprevalence study using an adult serum bank collected in 2010 detected two potential residual foci of transmission, with Og4C3 antigen (Ag) prevalence of 30.8% and 15.6%. We conducted a follow up study in 2014 to verify if transmission was truly occurring by comparing seroprevalence between residents of suspected hotspots and residents of other villages. In adults from non-hotspot villages (N=602), seroprevalence of Ag (ICT or Og4C3), Bm14 antibody (Ab) and Wb 123 Ab were 1.2% (95% CI 0.6-2.6%), 9.6% (95% CI 7.5-12.3%) and 10.5% (95% CI 7.6-14.3%), respectively. Comparatively, adult residents of Fagali'i (N=38) had significantly higher seroprevalence of Ag (26.9%, 95% CI 17.3-39.4%), Bm14 Ab (43.4%, 95% CI 32.4-55.0%), and Wb123 Ab 55.2% (95% CI 39.6-69.8%). Adult residents of Ili'ili/Vaitogi/Futiga (N=113) also had higher prevalence of Ag and Ab, but differences were not statistically significant. The presence of transmission was demonstrated by 1.1% Ag prevalence (95% CI 0.2% to 3.1%) in 283 children aged 7-13 years who lived in one of the suspected hotspost, including a 9 year old child. Our results provide field evidence that integrating LF surveillance with other surveys is effective and feasible for identifying potential hostpots, and conducting surveillance at worksites provides an efficient method of sampling large populations of adults

Detecting and confirming residual hotspots of lymphatic filariasis transmission in American Samoa 8 years after stopping mass drug administration

The Global Programme to Eliminate Lymphatic Filariasis (LF) aims to eliminate the disease as a public health problem by 2020 by conducting mass drug administration (MDA) and controlling morbidity. Once elimination targets have been reached, surveillance is critical for ensuring that programmatic gains are sustained, and challenges include timely identification of residual areas of transmission. WHO guidelines encourage cost-efficient surveillance, such as integration with other population-based surveys. In American Samoa, where LF is caused by Wuchereria bancrofti, and Aedes polynesiensis is the main vector, the LF elimination program has made significant progress. Seven rounds of MDA (albendazole and diethycarbamazine) were completed from 2000 to 2006, and Transmission Assessment Surveys were passed in 2010/2011 and 2015. However, a seroprevalence study using an adult serum bank collected in 2010 detected two potential residual foci of transmission, with Og4C3 antigen (Ag) prevalence of 30.8% and 15.6%. We conducted a follow up study in 2014 to verify if transmission was truly occurring by comparing seroprevalence between residents of suspected hotspots and residents of other villages. In adults from non-hotspot villages (N = 602), seroprevalence of Ag (ICT or Og4C3), Bm14 antibody (Ab) and Wb123 Ab were 1.2% (95% CI 0.6–2.6%), 9.6% (95% CI 7.5%-12.3%), and 10.5% (95% CI 7.6–14.3%), respectively. Comparatively, adult residents of Fagali’i (N = 38) had significantly higher seroprevalence of Ag (26.9%, 95% CI 17.3–39.4%), Bm14 Ab (43.4%, 95% CI 32.4–55.0%), and Wb123 Ab 55.2% (95% CI 39.6–69.8%). Adult residents of Ili’ili/Vaitogi/Futiga (N = 113) also had higher prevalence of Ag and Ab, but differences were not statistically significant. The presence of transmission was demonstrated by 1.1% Ag prevalence (95% CI 0.2% to 3.1%) in 283 children aged 7–13 years who lived in one of the suspected hotspots; and microfilaraemia in four individuals, all of whom lived in the suspected hotspots, including a 9 year old child. Our results provide field evidence that integrating LF surveillance with other surveys is effective and feasible for identifying potential hotspots, and conducting surveillance at worksites provides an efficient method of sampling large populations of adults.The project was funded by grants from the Australian Institute of Tropical Health and Medicine (www.aithm.jcu.edu.au, #13122014) and the Faculty of Medicine and Biomedical Sciences at the University of Queensland (www.uq.edu.au, #2127835). CLL was supported by an Australian National Health and Medical Research Council (www.nhmrc.gov.au) Fellowship (1109035

Structural Characterisation of Predicted Helical Regions in the Chironex fleckeri CfTX-1 Toxin

The Australian jellyfish Chironex fleckeri, belongs to a family of cubozoan jellyfish known for their potent venoms. CfTX-1 and -2 are two highly abundant toxins in the venom, but there is no structural data available for these proteins. Structural information on toxins is integral to the understanding of the mechanism of these toxins and the development of an effective treatment. Two regions of CfTX-1 have been predicted to have helical structures that are involved with the mechanism of action. Here we have synthesized peptides corresponding to these regions and analyzed their structures using NMR spectroscopy. The peptide corresponding to the predicted N-terminal amphiphilic helix appears unstructured in aqueous solution. This lack of structure concurs with structural disorder predicted for this region of the protein using the Protein DisOrder prediction System PrDOS. Conversely, a peptide corresponding to a predicted transmembrane region is very hydrophobic, insoluble in aqueous solution and predicted to be structured by PrDOS. In the presence of SDS-micelles both peptides have well-defined helical structures showing that a membrane mimicking environment stabilizes the structures of both peptides and supports the prediction of the transmembrane region in CfTX-1. This is the first study to experimentally analyze the structure of regions of a C. fleckeri protein

An in vitro comparison of venom recovery methods and results on the box jellyfish, Chironex fleckeri

The emergence of novel venom extraction techniques over the last half-century has greatly facilitated advances in the field of cnidarian research. A new recovery protocol utilizing ethanol as the primary stimulant in nematocyst discharge was recently published, however in vitro examination of the venom on organic models was not performed. This present study reports an original comparison of the chemically-induced discharge technique in vitro with a commonly used saltwater extraction method. Size-exclusion chromatography revealed distinct differences in venom profiles between the two methods: the saltwater recovery method FPLC profile and SDS-PAGE gel were similar to previously published results, whereas the ethanol-induced method was not. SDS-PAGE gel revealed distinct 40-55 kDa bands of previously identified cardiotoxic proteins recovered from the saltwater method, whereas the ethanol-induced method yielded degraded venom protein bands. A concentration-response curve generated through xCELLigence Real-Time Cell Analysis (RTCA) revealed a dramatic decrease in human cardiomyocyte activity when venom recovered via saltwater discharge was applied to these cells. With the exception of one sample, all ethanol-induced recovered venom failed to prompt a concentration-dependent decrease in cell survival when applied to human cardiomyocytes, resulting in a significant difference in IC50 concentrations between the compared venom samples. The data presented here facilitates an improved understanding of the parameters and analyses that are essential when developing and utilizing novel techniques for future cnidarian venom extraction research and supports the conclusion that recovery of venom from the tentacles of the box jellyfish Chironex fleckeri by ethanol is not an effective, efficient, or comprehensive extraction method compared to the published method of saltwater degradation of tentacles and bead mill extraction

Dose and time dependence of box jellyfish antivenom

Background\ud \ud The effectiveness of the currently available box jellyfish (Chironex fleckeri) antivenom has been subject of debate for many years. To assess whether the box jellyfish antivenom has the ability to attenuate venom-induced damage at cellular level, the present study analyzed the dose and time dependence of the antivenom in a cell-based assay.\ud \ud Methods\ud \ud Different doses of antivenom were added to venom and subsequently administered to cells and the cell index was measured using xCelligence Technology (ACEA Biosciences). Similarly, antivenom and venom were incubated over different time periods and cell survival measured as stated above. For both experiments, the cell index was plotted as a measure of cell survival against the dose or incubation time and significance was determined with the use of a one-way ANOVA with a LSD post hoc test.\ud \ud Results\ud \ud Increasing concentrations of antivenom significantly augmented cell survival, with a concentration of approximately five times the currently recommended dose for human envenomation, causing the first significant increase in cell survival compared venom alone. Further, cell survival improved with increasing incubation time of venom and antivenom prior to addition to the cells, indicating that box jellyfish antivenom requires approximately 70 minutes to neutralize C. fleckeri venom.\ud \ud Conclusion\ud \ud The presented results suggest that the currently recommended dose of antivenom requires adjustment, and more importantly, a human trial to test the effects of higher concentrations is also necessary. Further, antivenom has delayed neutralizing effects (i.e. after 70 minutes) which underlines the eminence of immediate and prolonged cardiopulmonary resuscitation in victims suffering from a C. fleckeri venom-induced cardiovascular collapse