An ancient protein found in tears and breast milk carries a powerful secret weapon against deadly bacteria.
The protein, called lactoferrin, emerged 160 million years ago, when an early ancestral gene known as transferrin duplicated. Transferrin binds to and shuttles iron throughout the body. But in the 1990s, scientists discovered that lactoferrin had acquired antibacterial properties, identifying an antimicrobial peptide, or AMP, embedded in human and cow lactoferrin. They named the AMP lactoferricin.
Why did a protein meant to carry iron develop antibacterial properties? Scientists used AI and a novel evolutionary approach to find out — and their discoveries could help guide development of new antibiotic alternatives.
“We’re looking back in time to ancient versions of these AMPs and asking: Do we see a more enhanced or altered version of this peptide that existed millions of years ago that we can resurrect and use as a therapeutic?” said Matthew F. Barber, PhD, associate professor of biology at the University of Oregon in Eugene.
Barber’s team reconstructed the amino acid sequences from before and shortly after lactoferrin’s emergence and used the AI program AlphaFold to predict how the structures evolved. They noticed a highly positive charge on the surface of lactoferrin and its more recent ancestors at the point where the AMP breaks away. The positive charge was absent in transferrin’s ancestors — an indication that lactoferrin evolved to have antibacterial properties after breaking off from transferrin. “It really looked like that was something that wasn’t there 165 million years ago,” Barber said.
Evolutionary Fine-Tuning in Great Apes
In 2014, Barber found evidence suggesting transferrin is very rapidly evolving in primates, driven by an "evolutionary arms race” with bacteria trying to scavenge transferrin’s iron. In a 2016 study, Barber and colleagues showed that lactoferrin is also rapidly evolving in humans and other simian primates, but the adaptation is playing out differently. In transferrin, signs of rapid evolution were found only at binding sites. But in lactoferrin, the signals were spread out, including at several places within the lactoferricin region.
Barber’s latest study, published in August in PLOS Biology, looked at the antibacterial effects across primates. Some variations were inconsequential, but one in particular stood out: At the fifth link in lactoferricin’s molecular chain, human peptides carry glutamine, a chemically neutral amino acid. But in non-human great apes, evolution swapped arginine into that slot. This substitution gave apes a stronger defense against bacteria like Staphylococcus aureus, though it did little to combat Pseudomonas aeruginosa, a common pathogen in healthcare-associated antibiotic-resistant infections.
In the lab, the researchers set out to see if humans could borrow that evolutionary upgrade. They engineered the swap in human lactoferricin — and the peptide’s antimicrobial activity significantly improved. Not only was it more effective against S aureus, but it also suppressed bacterial growth in four different staphylococcus samples over time.
In Barber’s experiments, ancestral lactoferricin made bacteria more permeable to dyes, suggesting an early ability to rupture bacterial membranes. Contemporary lactoferricin variants may have evolved to rupture membranes faster than bacteria can make repairs.
“It’s a neat evolutionary story of invention of function,” said Brian Lazzaro, PhD, a professor of entomology and ecology and evolutionary biology at Cornell University in Ithaca, New York, who was not involved in the research. “It didn’t just come from a random sequence; it was invented in the middle of an existing protein, where it’s cleaved out and then optimized over evolutionary time.”
Future studies could investigate how such simple mutations can transform peptides from weak to very active. “We’re curious how a single amino acid can do that,” Barber said. “Is it just through interacting with the membrane, or is it allowing it to bind something inside the bacterial cell, like a ribosome or some precursor of the cell wall?”
Therapeutic Potential, Roadblocks
As antibiotic resistance escalates, experts are pushing for more antibiotic alternatives. Therapeutics developed from AMPs, including lactoferricin, are among the potential candidates. Lactoferrin-derived peptides are being evaluated as treatment for various infections, inflammatory conditions, and diseases, including COVID-19.
However, peptide-based drugs face persistent challenges, including shelf stability. “Even things like salt concentration can affect their activity,” Barber said. “AMPs are inactivated by, for example, human serum. If you try to IV this into somebody, the AMP may not be effective.”
Researchers are also looking to other organisms, such as frogs and insects, in search of AMPs to develop as therapeutics. Given that even small genetic differences can affect peptides’ antibacterial potency — as with lactoferricin’s effectiveness against Staphylococcus aureus in humans versus other great apes — “we might want to be looking beyond humans for these possibilities,” Barber said.
Going forward, his lab is interested in exploring how microbes become resistant to AMPs. They also hope to expand their research beyond single mutations in individual peptides to genetic variation among AMPs in large datasets.
And their evolutionary approach could spark similar investigations beyond lactoferrin. “There’s no reason why it has to be restricted to this particular protein family,” Lazzaro said.
Barber and Lazzaro reported no relevant financial relationships.
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