One of the deadliest venoms in the world can be found in the flimsy tentacles of this box jellyfish. When you get right down to it, box jellyfish are little more than goo. The majority of their volume is mesoglea, a non-living, jello-like substance, which is sandwiched between two thin tissue layers. They have no teeth to bite with, no claws to scratch with — none of the weaponry we generally think of when we imagine a ruthless predator. Yet these boneless, brainless boxies are among the deadliest animals on Earth. The box jellyfish Chironex fleckeri can kill a full grown man in less than five minutes, and the venom it wields in its tentacles contains of some of the most rapid, potent toxins in the world. Exactly what those toxins are, though, has remained somewhat of a mystery. Scientists have been trying to determine the composition of box jelly venom for decades, but have only uncovered some of its potent constituents. And while there's still more to learn, last week, a research team from Queensland, Australia published the most extensive analysis of Chironex venom proteins to date, revealing some of the diverse arsenal that these gelatinous killers are equipped with. Box jellies, like other members of the phylum Cnidaria, are armed with stinging cells along their tentacles. In each is a structure called a nematocyst which contains the venom and a harpoon-like structure on a biological thread. When nematocysts are triggered, their harpoon shoot out at speeds that can exceed 40 miles per hour, creating as much penetrative force as some bullets. Victims of box jellies can be hit with millions of these tiny stinging cells in a matter of seconds, causing large, painful welts. In severe cases, the venom causes systemic effects, including acute cardiovascular collapse and death in a matter of minutes or more delayed but potentially deadly symptoms — a condition known as Irukandji Syndrome. "Despite the economic and medical impact that this jellyfish has on Australia (and similar species world-wide) we know very little about what is exactly in the venom," explained Jason Mulvenna, Team Head of Infectious Disease and Cancer at QIMR Berghofer Medical Research Institute and coauthor of the paper published in BMC Genomics. The team's goal was to perform the most in-depth analysis of Chironex venom to date, producing both a proteome (a library of the proteins present) and a transcriptome (a library of which genes are expressed). This combination of approaches has become more and more popular in recent years as technological advances have opened the doors to faster and easier genetic sequencing as well as more precise protein determination. While they could have used either the genetic or proteomic approaches to look at venom proteins, the combination was particularly powerful. Transcriptomes tell you which genes are actively being expressed, but it can be hard to tell which of those genes are actually acting as venom toxins and which are involved in day-to-day cellular maintenance. Similarly, while proteins can be directly sequenced, it can be difficult to make sense of those kinds of data without genetic information, and there is no published genome for Chironex. So using both approaches was key to the team's success. From the genetic side, they constructed a tentacle transcriptome using next generation sequencing. This is done by separating out all of the messenger RNA sequences (or 'transcripts') — the first step on the pathway from gene to protein. They then chopped these into small pieces and sequenced them. Much like recreating a book from 10-word sentence fragments, the team was able to use special computer programs to align the little pieces, eventually creating a library of the expressed genes. Then it was time for the protein side. To construct a 'proteome', the team had to isolate venom from the jelly tentacles. Unlike snakes or spiders, jellyfish cannot be 'milked' — so the team had to separate nematocysts from fresh tentacles, make them "sting", and then separate the venom excreted from the capsule itself. That venom product could then be separated into individual components and identified using gel electrophoresis, liquid chromatography, and mass spectrometry. The tentacles yielded over 20,000 predicted protein sequences that the researchers compared with known proteins in the UniProt database to identify potential toxins. They ended up with 179 likely toxins from ten different protein families. Their tandem proteomic analyses specifically identified 13 of these that were in venom in relatively high abundance: seven proteases, four of which were metalloproteinases, an alpha-macroglobulin domain containing protein, two peroxiredoxin toxins, two CRISP proteins and a turripeptide-like protease inhibitor. Another study from earlier this year had similarly constructed a transcriptome, and both research groups found metalloproteases and protease inhibitors. The team also found new variations of the known cnidarian pore-forming proteins, or porins, that have been detected in a diverse set of species. Four were known from Chironex fleckeri when the scientists began their quest — the team found evidence for fifteen different variants. "We now know that there is a whole bunch of unique toxins, only found in jellyfish, that may explain why the box jellyfish is one of the most venomous creatures known to man," said Mulvenna. "Now we know what is in the jellyfish venom we can do two things; we can start coming up with novel treatments for jellyfish stings that directly target the proteins we identified in the venom; and we can start seeing if these novel toxins are useful to us medically," explained Mulvenna. More targeted therapeutics are a welcome idea, as previous studies have questioned whether box jelly antivenom is effective. A recent study showed that a specific blocker of the porin toxins worked far better than the antivenom in an animal model of envenomation, for example. Now that there are new targets to consider, scientists may be able to create a better treatment regime that stops the venom's most deadly activities. Perhaps ironically, those deadly activities may also be harnessed for good. More and more, scientists are mining venomous animals for novel pharmaceuticals. After all, these animals have had millions of years to hone the actions of their toxins — jellies, for example, have existed for some 600 million years, tweaking their venom along the way. Though the process of drug discovery is long and difficult (and thus any box jelly-derived drugs would be years if not decades away from production), the database of toxins created in this study can now be screened for useful activities like anti-cancer properties. While the combined approach allowed for these novel discoveries, there is still much more to be done. "The upside of using this technique is speed and cost — we can generate a transcriptome quickly and use proteomics to refine and correct it," explained Mulvenna. However, the methods used can overlook potent toxins that are present in lower concentrations, and thus further study is needed to detect and sequence the proteins that are less abundant. In addition, correctly assembling the entire transcriptome without a genome as a guide is a bit like trying to piece together hundreds of pages of text after they've gone through a shredder. Mistakes stand out when the short sequences are mapped back to the constructed full-length ones; in this case 44% of the short sequences didn't match, which Mulvenna says is "a result of errors in the assembly." This suggests that even with the dual approach, there are proteins being produced that we still don't know about. The study design also is only able to detect protein toxins. Many venoms are complex chemical cocktails with diverse toxins. But for now, the team has more than enough to work with. "Now the fun starts as we start working on individual proteins to find out what they do and why they are so potent," Mulvenna said. Citation (Open Access!): Brinkman, Diane L., Xinying Jia, Jeremy Potriquet, Dhirendra Kumar, Debasis Dash, David Kvaskoff, and Jason Mulvenna. “Transcriptome and venom proteome of the box jellyfish Chironex fleckeri.” BMC Genomics 16, no. 1 (2015): 407. DOI: 10.1186/s12864-015-1568-3
Note: on Sunday, I went off on bad science coverage of this paper, saying that I was so disgusted by the awful reporting that I couldn't even write the post I had wanted to about the study itself. This is that post.