Mysterious mechanism in small peptide holds promise for combating antibiotic-resistant bacteria
By John B. Rosalez on May 20, 2022 Molecular Mechanics
Scientists used neutrons to better understand how an antimicrobial peptide known as aurein 1.2 kills antibiotic-resistant bacteria by attacking their membranes. With increasing peptide concentration (yellow), aurein 1.2 induces lipid molecule clusters (white) within the membrane, making it thicker, stiffer, and more prone to degradation under stress. Their findings could lead to new strategies for treating infections where antibiotics have failed. (Image credit: ORNL/Jill Hemman)
Since the advent of penicillin more than 90 years ago, antibiotics have saved countless lives by preventing and treating bacterial infections. However, bacteria are rapidly developing new ways to resist antibiotics, making some of modern medicine's most potent drugs less effective against many life-threatening infections, such as gonorrhea, methicillin-resistant Staphylococcus aureus (MRSA), and Clostridioides difficile (C. among others. Appreciated today the Centers for Disease Control and Prevention (CDC) that nearly 3 million drug-resistant infections occur each year in the United States alone.
This rise in resistance has fueled an aggressive search for new methods to combat bacterial threats. One possible strategy to combat antibiotic resistance is to develop chains of amino acids called peptides that have a penchant for neutralizing bacterial invaders.
A group of peptides known as antimicrobial peptides (AMPs) serve as an innate line of defense against microbial diseases in animals, plants, and other multicellular organisms. Given their ability to rapidly identify and combat a variety of pathogens, these peptides are considered promising therapeutic candidates for treating bacterial infections where antibiotics have failed.
Neutron experiments led by scientists at the Department of Energy's (DOE) Oak Ridge National Laboratory (ORNL) have provided new details on how AMPs block bacterial infections. By studying a potent AMP called Aurein 1.2, the team pieced together the molecular mechanics behind the peptide's ability to cause significant damage to bacterial cells in small amounts. Their findings, published in BBA Advances, could help pharmaceutical experts design drugs that target antibiotic-resistant bacteria more efficiently and effectively.
"AMPs attack bacterial cells by targeting their membranes using electrostatic and hydrophobic forces," explains Shuo Qian, the neutron scattering scientist at ORNL who led this research. "The basic structure of a cell membrane is a double layer of lipid molecules. Many of the lipid molecules that make up the surface of bacterial cell membranes are negatively charged, which is very attractive to positively charged AMPs. Once bound to an invading bacterial cell, the hydrophobic peptides often embed themselves in the membrane to avoid contact with water."
When AMPs are present in sufficient numbers, they dig deep holes in bacterial membranes. This action destroys the membrane structure, leaving the invading bacterial cells weakened. The downside is that developing therapeutics with high concentrations of peptides could be more costly and cause side effects in patients. Drugs that effectively kill antibiotic-resistant bacteria without requiring a high peptide count would be more beneficial to patient health at a better price. Here, aurein 1.2 provided insights into how to solve the problem.
Consisting of just 13 amino acids, Aurein 1.2 is one of the shortest AMPs found in nature. Originally discovered in Australian tree frogs, synthetically produced versions of this peptide have been shown to be effective against various bacteria. However, the molecular mechanism behind its pathogen-fighting abilities has remained largely unknown.
Neutrons can non-destructively probe the chemical elements that make up biological systems, such as peptides and membranes, making neutron scattering an ideal technique for gathering detailed information about these systems.
Previous neutron experiments led by Qian at ORNL, published in Scientific Reports and in Langmuir, provided some early clues to the peptide's mysterious mechanism. These studies showed that Aurein 1.2 does not poke holes in bacterial membranes like other AMPs, but interferes with the electrostatic balance of the membranes. At low concentrations, the peptide's positive charge causes lipid molecules to slow their lateral movement and form clusters with other similarly charged lipid molecules within the membrane. This behavior had not previously been observed in other AMP interactions.
In his latest study, Qian teamed up with Piotr Zolnierczuk, a neutron scattering scientist at ORNL, to better understand how the peptide-induced lipid clusters disrupt bacterial membrane behavior. They used two neutron scattering instruments at ORNL's Spallation Neutron Source (SNS).
Using the EQ-SANS instrument to measure peptide penetration within the membrane, Qian found that aurein 1.2 remained embedded near the membrane surface even at high concentrations. Zolnierczuk then performed neutron spin-echo spectrometer experiments to investigate the influence of aurein 1.2 on membrane flexibility and compressibility.
"Membrane fluctuations occur on the order of tens or hundreds of nanoseconds, and neutron spin echo spectroscopy is the only technique capable of measuring dynamic behavior on this length and time scale," Zolnierczuk said. "By analyzing neutron spin echo data through mathematical models, we can then derive information about a membrane's mechanical properties, such as how soft or stiff the membrane becomes."
The experiments showed that the membrane becomes thinner and more flexible when exposed to low peptide concentrations. At slightly higher concentrations, the peptides cause the membrane to become thicker and stiffer. The results suggest that aurein-1.2 peptides can thicken and stiffen bacterial membranes through their lipid clustering mechanism.
"Under normal conditions, the lipid molecules that make up membranes are constantly moving to accommodate biological activities. It's a very fluid, inhomogeneous environment in a delicate balance. However, we found that when aurein 1.2 causes lipid molecules to slow down and form unexpected clumps, the membranes end up becoming stiffer than usual," Qian said.
In this state, the bacterial membrane is less able to recover from stress, its structural integrity is weakened, and it is less able to perform normal functions. This combination of side effects makes the bacterial cell more likely to collapse under stress. It's similar to how freezing temperatures slow down water molecules, turning flowing water into stiff, brittle ice.
The data generated from this research will help scientists better understand the intricacies of AMP-based defenses against bacteria and could inform future therapeutic strategies to fight infection in a world of increasing antibiotic resistance.
Looking ahead, this type of research will be greatly enhanced by the Second Target Station (STS) currently under construction at ORNL. The STS will provide transformative new capabilities for discovery science and enable breakthroughs in many areas of materials research and development.
One of the first STS instruments, CENTAUR, will provide unprecedented capabilities to study materials across a wide range of length scales with high resolution, measure smaller samples, and study evolving structures in various scientific fields, including biological membranes and others, in a time-resolved manner forms of soft matter.
The research was supported by the DOE Office of Science. This work also used resources from the Center for Structural Molecular Biology at ORNL. The deuterium oxide used in this research was provided by the US Department of Energy Isotope Program administered by the Office of Isotope R&D and Production.
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