November 16, 2007

Of Destroying Angels and Death Caps: Understanding the World's Most Lethal Fungi

A quick review of Hallen et al., Gene family encoding the major toxins of lethal Amanita mushrooms (forthcoming in the Proceedings of the National Academy of Sciences).

Blogging on Peer-Reviewed ResearchOne of the questions people who aren't mycologists ask me most frequently is why I would be interested in spending my life studying fungi. With so many interesting things in the world to work on understanding, why bother with mushrooms? My response is always that I find them fascinating because they've come up with so many novel ways of approaching the basic problems of life. From the way they digest nutrients to the ways they reproduce, fungi constantly amaze me.

It's because of this that I find the genus Amanita to be among the most interesting. If I had the time to focus on a single genus without having to exclude everything else, I might well settle on this one. Every time I turn around, it seems, there's something new to be found out about them. I've been told by my colleagues who study peroxidases (compounds that fungi use to digest cellulose and, sometimes, lignin) that we don't find what one would expect in this particular genus. I've been looking at chitinases myself, and the chitinase gene sequenced from A. muscaria is also a strange one, with an activation domain that looks much more like one expected from an animal than a fungus. In evolutionary terms, Amanita is a young genus, and a degree of divergence from its relatives isn't unexpected, but this bunch of fungi seems happy to go to perplexing extremes in distinguishing itself.

And that's where the findings noted in a recent paper on the mechanics of the one clade of Amanita's (section Phalloideae) α-amanitin production enters the picture. Thanks to the persistent efforts of Heather Hallen, Jonathan Walton and their team at Michigan State University, we now know of yet another way in which some of the most deadly poisonous mushrooms on the planet have separated themselves from their kin — they've come up with a way of producing their toxins that's more efficient than the way everyone else is doing it. Kudos to you, section Phalloideae.

A little background is in order. Section Phalloideae is a group of 30 or so amanitas that includes such killers as A. phalloides (death cap), A. ocreata, A. virosa, and A. verna (destroying angels) and A. bisporigera. While they produce a number of toxins, the most lethal of these are the amanitins. α-amanitin is the most deadly of the lot when ingested orally; a lethal dose of the stuff for a human being consists of around 0.1 mg per kilogram of body mass. This means that a single mushroom contains enough toxin to kill an average-sized human being and then some. The toxin works by inhibiting RNA polymerase II, binding to a particular subunit of that crucial enzyme that's distinct in mammals. Essentially, ingesting the amanitin stops a mammal's body from transcribing DNA to RNA. No RNA, no translation to protein. That is, simply put, a death sentence. The effects tend to be most pronounced in the liver and digestive tract, so a liver transplant might save a victim's life, but for the most part those who ingest amanitin are the walking dead. Nasty stuff, that.

α-amanitin is a cyclic polypeptide. Other fungi produce similar compounds, too, and they do so by first translating a bit of their genome to a protein called a synthetase, specifically a nonribosomal polypeptide synthetase (NRPS). The synthetase triggers peptide synthesis elsewhere, as the name suggests. In the clever Phalloideae clade, however, this intermediate step has been done away with entirely. They don't make any NRPSs at all. Instead, the backbone of amanitins and other cyclic peptides are produced right on the ribosome as the end result of translation of genes. This allows for a quicker and more energy-efficient production of these compounds that are seen in any other fungus known. There is some similarity noted in the paper, as well as a few important difference, between this novel pathway in Amanita spp. and animals such as cone snails and salamanders, though the exact nature of the peptides produced differs chemically between animal and amanita (e.g., cyclization in animals occurs through forming disulfide bridges across the peptide backbone, whereas additional peptide bonds are formed in the fungi). Nonetheless, finding this mechanistic jump from a typically fungal way of doing things to one that looks like how we animals do it is rather interesting, no?

The fact that the Phalloideae do things in such a novel manner created a major challenge for the researchers who have now produced these results. Hallen, as I've been told by colleagues, spent eight years looking for the genes involved in producing their toxins precisely because the usual genetic markers one would expect (for the production of NRPSs, for instance) aren't there to be found. It took a shotgun sequencing and analysis of the genome to finally track down a family of genes that encode the products including α-amanitin, such as AMA1 and PHA1, the latter encoding yet another toxic peptide, phallacidin. Each of the sequences encodes for a proprotein which is cleaved at a proline to yield a product. In the case of the amanitins, the proprotein is 35 amino acids long and the products are peptides eight amino acids in length that become cyclized, yielding the stuff that makes the mushroom a killer to any mammal foolish enough to try eating it. Again, this all happens without the intermediate requirement of a synthetase. The venom's primary peptide backbone comes right off the ribosome ready for action.

That this mechanism has been discovered in the Phalloideae make them an excellent candidate for future application as a tool for synthesizing other small cyclic peptides for biotechnology. Someday, the amanitas may be harnessed for making drugs, for instance. Now that we understand how the system works, we may see a day, hypothetically, when tweaking of the AMA1 gene results in the production of a compound useful for preventing the proliferation of unwanted tumor cells or to shut off the production of unwanted virons in some unfortunate host. Centuries ago, proto-scientists dreamed of transforming lead into gold. Thanks to research like this, there could come a day when natural-born killers like Amanita bisporigera are turned into life-saving therapeutic factories. Most of could certainly use a little extra gold, but how many more of us might need a biotechnical achievement like this some day, and how many could use it right now? The possibilities are tremendous.

As of my writing this, the paper has still not appeared on the PNAS Early Edition website. If you're interested in learning more, however, keep checking in on the site; it will get there eventually, I'm told. Here's the citation, including the DOI even though clicking it right now will give you a "file not found." Keep on trying.

Hallen HE, Luo H, Scott-Craig, JS and Walton, JD. 2007. Gene family encoding the major toxins of lethal Amanita mushrooms. Proceedings of the National Academy of Sciences. doi: 10.1073/pnas.0707340104

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