November 13, 2007

How Destroying Angels Make Their Poison

Word comes this morning of a possible solution to the mystery of how the deadliest of Amanitas manufacture their poison, alpha-amanitin. It's the most lethal of mushroom toxins and the reason that I frequently advise readers to avoid consuming any possible Amanita mushroom. We know how the poison works; it inhibits gene transcription. Your genetic machinery stops making proteins, including the enzymes necessary to sustain life, your cells starts dying off and then you do as well.

Still, we didn't know quite how the mushrooms manage to make this virulent compound. Most mushrooms produce toxins by transcribing a gene, translating mRNA to an enzyme, and finally using the enzyme to activate toxin production. The poison is then ready to take its toll on unsuspecting fungivores. In the case of deadly amanitas such as A. phalloides, A. virosa, and A. verna, however, researchers have been able to find the gene for producing alpha-amanitin.

Using recently developed, powerful 454 sequencing technology and a shotgun sequence approach, it has now been demonstrated that the Amanitas have evolved an unprecedented trick; they've done away with the intermediate enzyme entirely! Alpha-amanitin is itself a gene product. We go from DNA to RNA to toxin directly. This hasn't been seen in any other fungus; it's a novel pathway and so an exciting find.

While the paper has not yet been published by PNAS quite yet (I've been trying to get a copy all morning!), there has been a press anouncement:

Genetic technology reveals how poisonous mushrooms cook up toxins

Heather Hallen spent eight years looking for poison in all the wrong places.

Alpha-amanitin is the poison of the death cap mushroom, Amanita phalloides. The Michigan State University plant biology research associate was looking for a big gene that makes a big enzyme that produces alpha-amanitin, since that’s how other fungi produce similar compounds. But after years of defeat, she and her team called in the big guns – new technology that sequences DNA about as fast as a death cap mushroom can kill.

The results: The discovery of remarkably small genes that produce the toxin – a unique pathway previously unknown in fungi...

“We think we have a factory that spits out lots of little sequences to make chemicals in Amanita mushrooms,” said Jonathan Walton, MSU plant biology professor who leads Hallen’s team. “Our work indicates that these mushrooms have evolved a mechanism to make dozens or even hundreds of new, previously unknown chemicals, besides the toxins that we know about.”

Of the thousands of species of mushrooms, only about 30 produce alpha-amanitin. Most of them look much like their edible cousins. But poisonous mushrooms are powerful in folklore and in history. In 54 A.D., Emperor Tiberius Claudius was fed a death cap mushroom by his wife Agrippina to put her son Nero on the throne of Rome...

Walton’s lab works to understand the biochemical pathways by which natural products are synthesized in fungi. Fungal natural products that benefit human health include penicillin and the immunosuppressant drug cyclosporin. Studying their biosynthesis could lead to the discovery and development of new medicines.

To find the elusive gene for alpha-amanitin, they used what they term “brute force” – a new machine at MSU that can sequence immense quantities of DNA quickly. The 454 LifeSciences pyrosequencer generates 100 Mb DNA sequence in one overnight run - twice the size of a fungal genome. Traditional sequencing methods require months to yield the same quantities. What they found was a gene that encodes the toxin directly – with no need to first synthesize an enzyme that in turn would make the toxin.

“The RNA goes in, and out comes the backbone of the toxin,” Hallen said. After its initial synthesis, the toxin is then modified in several ways by the mushroom to make it exceptionally poisonous...
The paper itself should be available on the Proceedings of the National Academy of Sciences website within the next day or two. As soon as I can get hold of it, I'll most certainly be sharing a review here.

454 sequencing technology is itself a powerful new tool that allows us to blast tiny and heterogeneous samples of DNA and get back completed sequences very quickly and efficiently. It can shave literally months off the turnaround time for getting these sequences using more traditional means, and it has recently allowed us to learn about the genomes and genetic mechanics of everything from mammoths to mushrooms.

Anyhow, I'm looking forward to reading Walton and Hallen's paper, and if anybody should happen to bump into them over at Michigan State, please extend my sincere congratulations on solving the puzzle. Good stuff, no doubt!

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