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The work, described in a paper published in PLOS Biology on Aug. 25, traced the evolutionary history of peptides, short fragments of proteins, back to the earliest placental mammals. This broad group includes humans and nearly all mammals living today. Laboratory experiments showed that some reconstructed peptides from extinct ancestors were more effective against drug resistant bacteria than some versions found in modern species.

These long lost biological defenses could give researchers useful starting points for developing treatments that complement or replace antibiotics that no longer work, according to Matt Barber, senior author of the study and an evolutionary biologist at the UO College of Arts and Sciences.

"For anybody who studies pathogenic bacteria, it's always in the back of our minds that antibiotics are one of the most important breakthroughs in medicine in the 20th century," Barber said. "But bacteria are, and have been for a long time, evolving resistance to them."

He continued: "We're definitely interested in whether by resurrecting or engineering some enhanced antimicrobial peptides, we could use these as therapeutics down the road."

Tracing an Ancient Antimicrobial Defense

About 160 million years ago, near the end of the Jurassic Period, the common ancestor of all placental mammals, whose young develop in the womb, appeared. Around that same time, lactoferrin, the protein at the center of Barber's research, also emerged.

Lactoferrin is an immune protein present in almost every body fluid except blood, including breast milk, tears, saliva, snot, and intestinal mucus. One of its primary jobs is to keep iron away from pathogens. Bacteria inside the body depend on iron, but lactoferrin binds tightly to the element, making it much harder for microbes to access.

Lactoferrin also carries its own direct defense against pathogens. It contains an antimicrobial peptide capable of damaging bacterial membranes, creating holes that can rupture the cells.

"Antimicrobial peptides are a key part of the body's first line of defense," said Titas Sil, lead author of the paper and a doctoral student in Barber's lab. "They can target a broad range of pathogens, and due to their potency, scientists have been trying to synthesize a variety for therapeutic uses."

Close relatives of lactoferrin do not share this bacteria killing ability. That suggests the trait appeared sometime after lactoferrin emerged during mammalian evolution. To determine when the ability developed and how it changed over time, the team retraced lactoferrin's evolutionary history and reconstructed versions belonging to its extinct ancestors.

For Barber, that evolutionary record offers a natural archive of biological experiments that could help researchers develop new medical tools.

"Evolution is essentially a billions-year-old science experiment, right?" he said.

"We're seeing the results of what worked and what didn't work. Looking at how traits are naturally produced and selected through evolution, you can get information that could be useful for designing new antimicrobial tools."

Rebuilding Proteins From 160 Million Years Ago

To recreate the extinct antimicrobial peptide, Sil began by comparing lactoferrin gene sequences from living species such as humans and cows. She then mapped the evolutionary relationships among those sequences and used statistical methods to estimate the most likely genetic sequences carried by their common ancestors, extending the reconstruction back roughly 160 million years.

The advanced method is known as ancestral sequence reconstruction, which was pioneered by Joseph Thornton, a former UO scientist whose old laboratory space is now occupied by Barber's group.

Sil synthesized the predicted genes and used cells to produce the reconstructed ancient proteins. She then tested them against several pathogens linked to human disease, including Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli, and Streptococcus.

The oldest resurrected antimicrobial peptides were able to disrupt bacterial membranes, but the bacteria apparently repaired the damage and survived exposure to the peptides. Versions reconstructed from more recent mammalian ancestors, dating back a few million years, became progressively more effective. In some cases, they performed better than the corresponding modern human peptides.

One Mutation Produced a Major Change

Researchers traced much of that increased potency to a remarkably small structural difference. A single mutation in the chain of amino acids, the protein "building blocks," was enough to make the antimicrobial peptide substantially more effective.

"What was surprising and unexpected was how small changes in these domains could have such large effects," Barber said. "There have been clinical trials using derivatives of human lactoferrin peptides to treat infections. But there were several instances where (Sil) showed that you don't need a lot of changes for evolution to enhance the activity of these peptides beyond the human versions."

Barber and Sil caution that resurrected antimicrobial peptides are unlikely to become new medicines anytime soon. Unlike conventional antibiotics, these peptides are less structurally stable and are rapidly broken down inside the body.

Even so, reconstructing their evolutionary history could reveal useful strategies for designing future treatments. By learning how antimicrobial peptides changed over millions of years, researchers may be able to create new approaches that make it harder for pathogens to develop resistance.

"Similar to antibiotics, pathogens are going to be able to evolve against antimicrobial peptides," he said. "But if we understand and can anticipate how they become resistant to these molecules, we can hopefully find better ways to target them or develop combination treatments that better avoid resistance."

This research was funded by the National Institutes of Health.