NEW YORK (GenomeWeb) – An effort by Australian researchers has yielded a new de novo assembly of the box jellyfish Chironex fleckeri transcriptome, as well as a comprehensive analysis of proteins in the cniderian's venom including several toxins predicted by the transcriptomic data.
In their study published this week in BMC Genomics, the investigators wrote that the results, which offer an expanded list of the proteins associated with the very severe and sometimes life-threatening symptoms of the box jellyfish sting, could help guide future comparative studies, additional protein discovery, and potentially the development of more effective sting treatments.
Stings by C. fleckeri produce excruciating pain and an intense burning sensation, and can also cause dermonecrosis, dyspnea, transient hypertesnion, hypotension, and in severe cases, cardiovascular arrest and death. Most human encounters are relatively mild, though extremely painful, but the animals have also caused more than 60 deaths since first reported in the 1980s.
In their BMC study, the researchers, from the Australian Institute of Marine Science and the QIMR Berghofer Medical Research Institute, set out to try to identify a comprehensive set of toxins responsible for the severe effects of C. fleckeri stings using Illumina sequencing and tandem mass spectrometry.
Jason Mulvenna, the study's senior author, told GenomeWeb in an email that combining a transcriptome assembly with proteomics was key to the study's success.
"A transcriptome of the tentacle [can't] actually tell us what was in the venom [because] protein toxins look a lot like non-toxin proteins and this complicates the identification," Mulvenna wrote.
"Conversely you can't do much with proteomics data unless you have sequences to search spectra against. By combining the two methods we could generate sequence data for all tentacle proteins using transcriptomics and then use proteomic analysis of extracted venom to filter out the toxins from the transcriptome," he explained.
Initially, the group purified total RNA from whole C. fleckeri tentacle tissue, generating more than 43,000,000 paired reads on an Illumina HiSeq 2000. They then de novo assembled these reads into 34,438 transcripts.
After further analysis, the team identified 20,548 transcripts mapping to 20,562 predicted protein sequences which they then recorded for later exploration by MS/MS.
The researchers also compared their assembled transcripts to the UniProt animal toxin database to attempt to identify sequences associated specifically with C. fleckeri toxins. They initially found 455 high-scoring hits, and then further narrowed this group to 179 after removing those with stronger links to putative non-toxin proteins.
The final 179 likely toxin transcripts represented 10 different venom protein families, the authors wrote, including metalloproteinases, major constituents of spider venom, which were the most highly represented, as well as other proteases and protease inhibitors, lectins, lipases, CRISP venom proteins, and two families of snake venom molecules.
The transcriptome analysis also picked up several known members of a toxin family specific to cniderians — including 15 previously characterized C. fleckeri venom toxin isoforms — as well as three novel proteins of the same family, and nine others represented as partial transcriptomic sequences.
To confirm which of these putative toxin proteins were also detectable in C. fleckeri venom, the team followed their transcriptome assembly with LC-MS/MS analysis of protein preparations derived from the animals' nematocysts.
The team used AB Sciex’s TripleTOF 5600 MS/MS system to analyze total protein concentration of two nematocyst preparations from C. fleckeri tissue, one containing mainly mastigophores, which are nematocysts believed to hold the lethal venom components, as well as a second mixture enriched for isorhizas, which are thought to have a non-penetrative role in ensnaring the jellyfish's prey, and trirhopaloids, which have an undetermined role in envenomation, according to the authors.
The researchers then searched any proteins identified using MS/MS against the predicted set from their transcriptome assembly. They also reanalyzed existing MS/MS data from previous studies of C. fleckeri.
Across all these analyses, the investigators identified 263 proteins, and characterized them into eight functional groups. The set included 26 of the putative toxins generated by the transcriptome sequences, the majority of which were members of the cniderian-specific toxin family.
The MS/MS results also included 13 other putative toxins also represented in the transcriptome from other families.
According to the authors, because no C. fleckeri reference exists and they had to do a de novo assembly, it's likely that further sequencing will be required to fully characterize the species transcriptome.
Additionally, the authors wrote that it is possible that their MS/MS analysis missed low-abundance toxin proteins that still play a role in the symptoms of a sting. Accordingly, the team has additional work underway to try to identify these low-abundance molecules by improving methods of venom purification to decrease the proportion of structural proteins present, and also improving the fractionation of toxins before MS/MS.
Moving forward, Mulvenna said that the team is now working on linking the protein toxins they identified to the human response to envomation to identify targets for inhibition of the sting's effects.
"Now that we know the proteins that are in the venom we are using things like protein arrays to identify which human proteins a lot of these toxins are interacting with. Once we know this we can start finding inhibitors for the toxins with the most harmful activities. We also suspect some immune involvement in the symptoms caused by the jellyfish and are looking at mediators of an immune response," he explained.
Additionally, he said the investigators are now working on the genome of C. fleckeri, which could help refine their knowledge of its transcriptome and also help identify some low-abundance toxins.
Finally, the team is also doing some comparative venomics focused on the Irukandji jellyfish, another group of cubozoan jellyfish with medical importance in Australia.
"These jellyfish cause somewhat different symptoms and by comparing the venom of C. fleckeri to these species we hope to get some idea of how the two groups cause the symptoms that they do," Mulvenna said.