Research reveals hidden antibiotics in non-immune proteins

The main actors in the immune system’s cast of proteins are antibodies, which neutralize or identify foreign substances such as viruses and bacteria, and cytokines, which regulate communication and responses between cells. However, this intricate defense mechanism against infections — the leading cause of death in human history until the discovery of antibiotics — has a previously unknown secondary player that offers a fresh perspective on the body’s protective shield. Research conducted by the Machine Biology Group at the University of Pennsylvania, led by Spanish scientist César de la Fuente, has uncovered a new category of antimicrobial agents known as encrypted peptides. These peptides are hidden within molecules with various functions throughout the body, including in the eyes. The researchers’ findings were published on Tuesday in Trends in Biotechnology by Cell Press.

With this work, De la Fuente’s team has begun to decode one of the mysteries of the human proteome, the term for the complete set of proteins in an individual. In these molecules — which perform specific functions in all systems, such as the nervous, cardiovascular or digestive systems — they found chains of amino acids (peptides) whose role was previously unknown.

“They [the encrypted peptides] are hidden in proteins that we had never thought could play a role in the immune system,” explains De la Fuente. After two years of work, the team discovered that 98% of the peptides analyzed and sequenced from different parts of the body, including the eyes, are found in proteins not previously related to the body’s defense against pathogens.

The researcher likens these encrypted peptides to what was once termed junk DNA — genetic sequences thought to be without function, but which subsequent studies have revealed to have roles which had previously gone undetected.

The encrypted peptides are components of proteins that perform regular functions within the body’s different systems. “But we have discovered that the amino acid chains have an extra use and play an antimicrobial role and modulate the immune response,” the researcher explains. This concept is referred to as the “cross-communication hypothesis,” which posits that proteins from systems outside the immune system interact with immune components to enhance the body’s defense mechanisms.

The team’s approach suggests that most of the encrypted peptides act as a first line of defense against bacterial invasions. Their primary antimicrobial action involves disrupting the pathogen’s membrane, thereby weakening it and compromising its protective barrier. The second line of defense involves modulating or activating the immune response — essentially calling for reinforcements to aid in the pathogen’s elimination.

Of the synthesized peptides, eight —collagenin-3, collagenin-4, zipperin-1, zipperin-2, and immunosins 2, 3, 12, and 13 — exhibited remarkable anti-infective activity in preclinical mouse models, and were able to reduce bacterial infections in the skin and thigh by up to four orders of magnitude. In terms of their immunomodulatory properties, these peptides activated key inflammatory mediators involved in the immune response to infections, such as interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-α), and monocyte chemoattractant protein-1 (MCP-1). “In culture plates, 90% showed antimicrobial properties,” adds De la Fuente.

One of the most intriguing systems analyzed in the search for encrypted peptides is the ocular system. Human eyes are unique in that they cannot allow a normal inflammatory response, as this could impair vision. This phenomenon is known as immune privilege.

De la Fuente’s team examined the proteins in the eye to determine whether encrypted peptides also play a role in this “privilege.” “It is an interesting environment for us, and the findings complete the answer to the classic question of how the eye is protected,” the researcher explains.

The discovery of the anti-infectious function of encrypted peptides has two significant implications for future research. First, it reveals a complementary system to the known mechanisms for combating microbes. Second, it opens the possibility of leveraging these newly identified sequences to develop antibiotics aimed at bacteria that have developed resistance, which can lead to serious health consequences, including death.

“These previously unconsidered molecules [peptides] could play a crucial role in the immune system’s response to infections. This could not only transform our understanding of immunity, but also offer new opportunities to tackle infections that are resistant to drugs,” says the researcher.

Antimicrobial resistance (AMR) “represents a crucial global health threat that is associated with high morbidity and mortality, prolonged hospital admission, and increased health-care costs,” according to a review published in The Lancet Microbe, and in which De la Fuente participated along with 11 other researchers.

A study conducted by the Global Burden of Disease identifies six of the most-concerning pathogens from the long list of drug-resistant bacteria: Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae, Streptococcus pneumoniae, Acinetobacter baumannii, and Pseudomonas aeruginosa. Recently, food poisoning caused by a variant of Escherichia coli (O157) among customers of a McDonald’s restaurant in the United States resulted in one death and around 50 additional cases, 10 of which required hospitalization.

“To address this threat, it is essential to develop innovative antimicrobial strategies, such as drug repositioning in combination with the few clinically relevant antibiotics,” says Younes Smani, lead investigator of the Bacterial Infections group at the Andalusian Center for Developmental Biology (CABD), who was not involved in the University of Pennsylvania study.

Smani led a study published in Frontiers in Pharmacology that identifies 27 thiophene derivatives based on tamoxifen, a drug used in cancer treatments, and its compound, raloxifene. Among these derivatives, three compounds demonstrated significant antibiotic potential against multiresistant strains of bacteria, including two of the most dangerous pathogens: Acinetobacter baumannii and Escherichia coli.

Another promising area of research involves phages, which are viruses capable of targeting and killing bacteria. A series of studies examining AMR cases treated with phage therapy yielded mixed results. Out of 20 patients, most of whom had infections related to cystic fibrosis, 11 showed a positive response to the therapy. However, in only five individuals was the infection completely eliminated, while another six experienced a partial response. The remaining patients either did not respond or their outcomes were inconclusive.

In parallel, the Cooperative Research Center for Biomaterials in Spain has established a new research group focused on bottom-up cell biology and bioengineering. This group aims to explore the molecular processes involved in bacterial cell biology, specifically how bacteria form and reorganize their cell walls, divide, and communicate with one another or their host organisms. The goal is to understand these biological mechanisms in order to devise novel strategies to combat antibiotic resistance.

The research team aims to use reverse engineering, a process that team leader Natalia Baranova describes as “reconstructing cellular processes from the ground up, with a bottom-up perspective.” She explains: “We disassemble molecular components and reconstruct them in a similar way to how we would with cars or bridges. In this way, we can discover how nature has selected these specific components as critical, that is, we aim to understand the relationship between molecular composition and ultimate biological function.”

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