The study, published August 25 in PLOS Biology, traces the evolutionary history of peptides—short fragments of proteins—back to the earliest placental mammals. This broad group includes humans and nearly all mammals alive today.

Laboratory experiments showed that some peptides reconstructed from extinct ancestors were more effective against drug-resistant bacteria than certain peptides found in modern species.

Matt Barber, the study’s senior author and an evolutionary biologist at the University of California College of Arts and Sciences, said these long-lost biological defenses could provide a starting point for developing treatments to complement or replace antibiotics that no longer work.

“For those who study pathogens, it is always back in our minds that antibiotics were one of the most important advances in medicine of the 20th century,” Professor Barber said. “But bacteria are now, and have been for a long time, continuing to evolve resistance to them.”

He added that researchers are interested in whether enhanced antimicrobial peptides could eventually be revived or modified for use as therapeutic agents.

How ancient antimicrobial defenses evolved

About 160 million years ago, near the end of the Jurassic period, the common ancestor of all placental mammals emerged. Around the same time, lactoferrin—the protein at the center of Barber’s research—also appeared.

Lactoferrin is an immune protein found in almost all body fluids except blood, including breast milk, tears, saliva, nasal mucus and intestinal mucus. One of its primary functions is to keep iron away from pathogens. Because bacteria depend on iron, lactoferrin makes it harder for microorganisms to access this essential resource by binding tightly to it.

Lactoferrin also has a direct defense against pathogens. It contains antimicrobial peptides that can damage bacterial membranes and create holes that cause bacterial cells to burst.

“Antimicrobial peptides are an important part of the body’s first line of defense,” said Titus Schill, lead author of the paper and a doctoral student in Barber’s lab. “They can target a wide range of pathogens, and because of their potency, scientists have attempted to synthesize a variety of them for therapeutic use.”

Relatives of lactoferrin do not share the same ability to kill bacteria. This suggests that the trait developed sometime after lactoferrin first appeared during mammalian evolution.

To determine when this antimicrobial function emerged and how it changed, the research team traced lactoferrin’s evolutionary history and reconstructed versions of the protein belonging to extinct ancestors.

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

“The theory of evolution is essentially a billion-year-old scientific experiment, right?” he said.

“We’re looking at the results of what works and what doesn’t. Examining how traits are naturally produced and selected through evolution can provide information that can help design new antimicrobial tools.”

Reconstructing proteins from 160 million years ago

To recreate the extinct antimicrobial peptide, Schill compared lactoferrin gene sequences from living species such as humans and cows. The researchers then mapped the evolutionary relationships among those sequences and used statistical methods to estimate the most likely gene sequence carried by a common ancestor.

This process, known as ancestral sequence reconstruction, allowed the team to trace the protein’s history back approximately 160 million years. The method was pioneered by Joseph Thornton, a former university scientist whose old laboratory space is now occupied by Barber’s group.

Schill synthesized the predicted genes and used cells to produce the reconstructed ancient proteins. The team then tested them against several pathogens associated with human disease, including Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli and Streptococcus.

The oldest revived antimicrobial peptides were able to disrupt bacterial membranes. However, the bacteria appeared to repair the damage and survive exposure to the peptides.

Reconstructed versions from more recent mammalian ancestors, dating back millions of years, gradually became more effective. In some cases, they performed better than corresponding antimicrobial peptides found in modern humans.

A single mutation dramatically increased antimicrobial activity

The researchers found that much of the increase in potency resulted from surprisingly small structural differences. A single mutation in the amino acid chain—the building blocks of a protein—was enough to greatly improve the antimicrobial peptide’s effectiveness.

“What was surprising and unexpected was that small changes in these areas could have such a large impact,” Barber said.

Clinical trials have tested derivatives of human lactoferrin peptides for infectious diseases. However, the researchers found several examples in which only a small number of evolutionary changes were needed to increase activity beyond that of the human forms.

Ancient peptides are not ready to replace antibiotics

Barber and Schill caution that revived antimicrobial peptides are unlikely to become new drugs anytime soon. Unlike traditional antibiotics, these peptides are structurally less stable and are rapidly degraded in the body.

Even so, reconstructing their evolutionary history could reveal useful strategies for designing future treatments. Understanding how antimicrobial peptides changed over millions of years may help scientists develop approaches that make it harder for pathogens to evolve resistance.

“Like antibiotics, pathogens will be able to evolve against antimicrobial peptides,” Barber said. “But if we can understand and predict how these molecules become resistant, we hope to find better ways to target them and develop combination therapies that better circumvent resistance.”

This study was funded by the National Institutes of Health.