Eye-eating amoeba resists treatment-understanding its biochemistry could help

In water, soil and air all around us lives a free-living, single-celled organism called Acanthamoeba. Most of the time, Acanthamoeba keeps to itself. On rare occasions, though, the amoeba infects humans and can cause severe infections of the eyes, skin and even the brain. What's more, doctors can struggle to diagnose and treat the eye infection caused by the organism, called Acanthamoeba keratitis.

"It's a big challenge. The lack of readily available diagnostics is a problem, and then even after you finally get to the diagnosis, we don't have good drugs," says Dr. Jon Stefely, now a metabolism investigator at the Morgridge Institute for Research and assistant professor of biomolecular chemistry at the University of Wisconsin School of Medicine and Public Health. "Our current drugs are untargeted and toxic, and we just need more options for treating these infections."

Stefely is the first author of two new studies about Acanthamoeba in the journals Cell and Cell Press Blue—part of a set of nine papers published concurrently by the MitoCarta Tree of Life Consortium. The broader project aims to create an inventory of mitochondrial proteins across all branches of the tree of life and, in doing so, learn more about evolutionary history and the last common ancestor of all animals, plants, fungi and protists, which lived about 2 billion years ago.

The MitoCarta project was conceived by Dr. Vamsi Mootha, a molecular biologist at the Broad Institute, Massachusetts General Hospital, Harvard Medical School and the Howard Hughes Medical Institute. Stefely completed work on his two first-author papers while a postdoctoral researcher in Mootha's lab. Previously, the MitoCarta project had cataloged a mammalian mitochondrial proteome inventory, noting all the proteins involved in the mitochondria of mammalian cells.

The new set of papers adds proteome inventories to divergent branches of the tree of life by intentionally selecting pathogenic organisms, like Acanthamoeba, from across the tree. This strategy has the added benefit of potentially leading to new drugs to treat those pathogens.

Acanthamoeba keratitis is in particular need of new treatments because, in harsh environments like a human cornea, the amoeba builds a thick, double-layered protective cell wall somewhat like those found in trees and other plants. In this cyst form, Acanthamoeba is hardy and resistant to currently available drugs. The team hoped that by understanding the genes and proteins involved in mitochondrial function, they could find targets for highly specialized drugs that aren't toxic to humans.

"What's been proven historically," says Stefely, "is that if you have a target protein in a biochemical pathway that's completely unique to the microbe, it's a better target than something that has a homolog in humans. A drug that targets a unique microbial protein or pathway is less likely to also harm the infected person."

Building a genetic catalog for Acanthamoeba

But before they could begin to understand the proteome of Acanthamoeba's mitochondria, the team had to understand the organism's basic nuclear genetics. Even though mitochondria have their own tiny DNA compared with the cell's nucleus, most mitochondrial proteins are derived from the nuclear genome.

The genome in the cell's nucleus had been sequenced, but an accurate annotation of where the genes lay in the genome was still lacking—the prior annotation was only about 52% accurate. Once annotation of the overall genome was improved, the researchers could move on to targeting mitochondrial proteins. But even that first step—annotating an organism's genome—is a challenging endeavor.

"If you imagine a page of words in a book, it would be like all the words were squished together and in a language that you don't know," says Stefely. "You would have to ask, 'Where are the words? How do I separate one word from the next?' It's hard to tease apart."

In their first paper, Stefely and the team used a technique called long-read RNA sequencing on Acanthamoeba cells. RNA is part of the dynamic cellular process of translating DNA into proteins, and reading RNA sequences lets researchers see what parts of the genome are being transcribed. The long-read technique can collect transcripts of entire genes, rather than capturing shorter snippets that must be pieced together later.

With long-read RNA sequencing and other empirical techniques, the researchers increased the accuracy of Acanthamoeba genome annotation to 98%, detailing the precise locations of almost 16,000 genes. Of those, about a third were unique to the amoeba compared with the human genome and to baker's or brewer's yeast, an organism commonly used by biologists. They also found 20 families of proteins involved in building cyst walls that were absent in humans. Both these genes and proteins could be targets for future drug development.

Researchers nominate drug targets in the mitochondria

With the annotated genome in hand, Stefely and the team turned their attention to Acanthamoeba's mitochondria in their second paper. In progressively purified samples of Acanthamoeba mitochondria, they used mass spectrometry to track proteins that became more abundant. If a protein's abundance increased as the proportion of mitochondria in the samples rose, they determined it was a mitochondria-localized protein.

"There are roughly 300 proteins in Acanthamoeba mitochondria that are unique when compared to human and yeast mitochondria," says Stefely.

They also found that Acanthamoeba mitochondria have the unusual ability to switch functions between oxygen-rich and oxygen-deprived conditions. This trait might help the amoebae survive and thrive in places like a human cornea or deep in a lake. Acanthamoeba also turned out to be a model organism for studying the last common ancestor of all animals, plants, fungi and protists, retaining many proteins and pathways predicted to have been present in this ancient ancestor.

Going forward, Stefely plans to investigate the individual proteins and molecular pathways the team has identified as unique to Acanthamoeba, which will help determine which are most likely to make good drug targets.

"It's going to take a lot of focused work on individual proteins and pathways, but we'll start chipping away at this exciting project," says Stefely. "A long-term goal is to annotate functions for all of those targets, but we'll take them one small set at a time, and there is a lot of potential for new discoveries."

That work will be aided by close collaborations with other Morgridge investigators who specialize in mass spectrometry, RNA sequencing, cell metabolism, structural biology and biomedical imaging.

Stefely will also spend five weeks a year at UW Health University Hospital as part of his appointment in the School of Medicine and Public Health. This experience working directly with patients is part of his personal motivation to discover new treatments for protozoan infections that occur in Wisconsin, like Acanthamoeba keratitis.

"I've seen many examples where patients are really suffering from these infections, and sometimes we just don't have a good treatment," he says. "Seeing those challenges in diagnosis and treatment in the hospital motivates the work that we do in the lab. We try to bring those challenges and questions back to our basic science research team here at Morgridge. We have a very exciting opportunity to both discover new fundamental mechanisms of biology and at the same time help lay the foundation for new therapies."