Ancient Peptides Resurrected from Lactoferrin’s Evolutionary Past Could Point to New Antibiotics

University of Oregon biologists have resurrected prehistoric proteins up to 160 million years old that carry natural antimicrobial properties. The scientists worked their way up the tree of life of the iron-binding protein lactoferrin, reconstructing peptides dating back to the earliest placental mammals, the diverse lineage that includes humans and nearly all mammals alive today. In laboratory tests, the researchers found that some of the extinct peptides were more potent against drug-resistant bacteria than some of their present-day counterparts. Evolution’s ancient remedies could offer new starting points for scientists designing treatments that supplement or replace antibiotics that no longer work, said research lead Matt Barber, PhD, 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. But bacteria are, and have been for a long time, evolving resistance to them. We’re definitely interested in whether by resurrecting or engineering some enhanced antimicrobial peptides, we could use these as therapeutics down the road.” Barber is senior author of the researchers’ published paper in PLOS Biology, titled “Retracing the origin and evolution of a cryptic antimicrobial peptide within mammalian lactoferrin.” Some 160 million years ago, near the end of the Jurassic period, the ancestor of all placental mammals emerged and so did lactoferrin, an immune protein found in nearly every body fluid (except blood), including breast milk, tears, saliva, and various types of mucus. The main function of lactoferrin is to withhold iron from pathogens. Bacteria in the body need iron to fuel their advances, but lactoferrin acts as a vault, tightly sealing the key resource away. In addition to securing iron from bacterial reach, lactoferrin has evolved built-in tools to fight against pathogens. Most notably, it has an antimicrobial peptide (AMP), lactoferricin, that punches holes in the membranes of bacteria, rupturing the cell. “Antimicrobial peptides are a key part of the body’s first line of defense,” said first author Titas Sil, 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.” None of lactoferrin’s close protein relatives have that bacteria-killing ability, suggesting that the property arose sometime after lactoferrin emerged in the mammalian lineage. To find out when and how it has evolved since, the researchers worked backward through its evolutionary history and resurrected its ancestors. “Here we retrace the origin and evolution of the abundant mammalian protein lactoferrin and it embedded AMP, lactoferricin,” they wrote. A look at the past might give ideas for a healthier future, Barber said. “Evolution is essentially a billions-year-old science experiment, right? 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.” To resurrect the extinct antimicrobial peptide, Sil first compared the gene sequences of lactoferrin in present-day humans and cows. Mapping their evolutionary relationships, she statistically inferred the most likely sequences of their common ancestors, reaching back about 160 million years. That state-of-the-art technique is known as ancestral sequence reconstruction, which was pioneered by Joseph Thornton, a former UO scientist whose previous lab space is now home to Barber’s group. “To retrace the mutations that led to the emergence of a novel antimicrobial function, we reconstructed ancestral lactoferrin and transferrin sequences across diverse mammals,” the researchers noted. “Ancestral sequence reconstruction (ASR) provides a powerful approach to characterize the function of ancient proteins.” After synthesizing the predicted gene and regenerating the ancient protein in cells, Sil then tested their potency against several pathogens associated with human diseases, including Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli, and Streptococcus. The earliest resurrected antimicrobial peptides disturbed the bacterial membranes, but the pathogens were somehow able to repair the damage and tolerate the peptide. But peptides of later mammalian ancestors, about a few million years old, displayed progressively stronger antimicrobial activity, sometimes outperforming the modern, human versions. “Leveraging mammalian lactoferrin and its embedded AMP lactoferricin as a model, we observed that even the earliest lactoferricin ancestor possessed the ability to permeabilize bacterial membranes and alter membrane potential, a property that was further enhanced in later ancestors,” they stated. The collective findings, they added, “… indicate that the lactoferricin domain initially possessed membrane permeabilizing activity, which intensified during evolution to produce potent bactericidal effects.” That difference came down to a small structural change: a single mutation in the amino acid chain, that made the antimicrobial peptide more potent. “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 the development of new drugs with extinct antimicrobial peptides is unlikely to be immediate. Compared to conventional antibiotics, the peptides are structurally less stable and quickly broken down in the body. Even so, tracing their history matters, Barber said. Understanding how antimicrobial peptides evolved in the past is one of the best ways to inspire new treatment designs that pathogens can’t attack. “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.” In their paper the authors concluded, “Together, our study illustrates how novel immune protein functions can arise, evolve, and diversify to strengthen host defense against microbial pathogens.”