Neutrons reveal surprises on the action of natural antibiotics
Antimicrobial peptides (AMPs) are natural antibiotics very effective against resistant bacteria. Despite their interesting properties, AMPs remain hard to use. A study now published marks a remarkable step further in understanding how AMPs work. Taking full advantage of neutron and X-ray scattering, researchers obtained results that are both important and surprising.
1. Natural antibiotics
Antimicrobial peptides (AMPs) are a class of natural antibiotics omnipresent throughout evolution. They are used by bacteria to fight other bacteria and they are part of the human immune system. AMPS are very effective against antibiotic-resistant bacteria, and have several – still not very well known - modes of action. Despite their interesting properties, AMPs remain hard to use, as they possess some toxicity and tend to suffer degradation in the body.
“To be able to use AMP antibiotics in a hospital, we need to reduce the negative effects. If we understand how they actually work, we can have a clearer strategy to reduce these effects,” explains Reidar Lund, professor at the University of Oslo (Norway), coordinator of the study now published.
2. Natural antibiotics and cell membrane patches
Like many drugs, AMPs act on the cell membrane. The basic structure of membranes is a double layer of lipids. This double layer tends to form differentiated patches, or domains, that don’t easily mix with the rest of the membrane. While much is still do be understood, they are believed to be essential for the function of the cell. Perturbation of the patch structure in human cells may be related to neurodegenerative diseases and cancer.
The new study addresses the question of how the membrane domain structure is affected by AMPs. The obtained results are far reaching:
“We moved one step further in understanding how AMPs work,”says Lund, adding:“It turns out that the vision of AMPs destroying bacteria by making holes in the membrane was too simplistic. The effect is much deeper, there is a reorganisation of the membrane surface.”
AMPs thus have severe effects in the organisation of lipids in the membrane, and thus on the domain structure, and thus on how the membrane works. But what was actually the observation and why is it surprising?
“We discovered that patches grow when AMPs are added, and this is somehow surprising,” reveals Vladimir Rosenov Koynarev, PhD student at the University of Oslo and first author of the article,“We would rather expect them to shrink or even to disappear – when one adds something amphiphilic (with a water-loving part and a water-hating part, like AMPs) the patches and the rest of the membrane usually become more likely to mix”.
In other words, the line tension (related to the energetic cost of mixing) decreases. In the present case, the line tension increases, and researchers relate this to the observed, concentration-dependent thinning of the membrane in the patches. These results show that the concentration at which AMPs are effective is much lower than the concentration at which the membrane is destroyed.
Schematic representation of a cell mebrane (lipid double layer with patches) and of the effect of AMPs.
3. Natural antibiotics, cell membrane patches and scattering techniques
But what is there about membrane domains that makes them at the same time controversial and hypothesised to play a major role in cell functions?
“It’s rather recent knowledge and research,” explains Koynarev, “One tricky thing about domains is that you can’t see them with optical techniques. The membrane surface looks exactly the same, the thickness may change but only by a few Angstroms.” Indeed, neutron and X-ray scattering techniques, together with the use of artificial membranes models, are playing a crucial role in giving insight into membrane domains. |
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In this study, researchers used small angle neutron and X-ray scattering techniques (SANS/SAXS) to look inside artificial membranes at the nanometre scale (1-100 ns). Neutron data were collected at the ILL, using instrument D22, and at the PSI (Switzerland). X-ray data were collected at the European Synchrotron ESRF also in Grenoble. “Neutrons allowed to see the domain distribution, size and growth, while X-rays allowed to see that the thickness of the membrane changes,” explains Lioner Porcar, ILL scientist responsible for D22, adding: “with neutrons we are looking at the plane of the membrane, it’s another scale, and we can see overall effects.”
“Neutrons and contrast techniques were key in this study,” stresses Lund: hydrogen atoms in the sample molecules can be replaced by deuterium atoms in selected locations. Taking advantage of the very different neutron scattering properties of the two isotopes, deuterated molecules can be tracked. In this study, samples were prepared in such a way that the all signals averaged to zero for the initial membranes. When patches changed due do AMPs, a clear signal appeared.
On the complementarity of the different data sets, Koynarev explains that “usually to analyse the data we make computer simulations based on models, which we compare to the measurement results, seeing which model better describes reality. The more different sets of data in different conditions the model is able to describe, the more confident we are that the model is right”.
The article’s peer review process is described as “long but fruitful” by Lund: “The main critique to the initial draft was that we did not actually know the distribution of the AMPs in the first place. Was it really uniform? Did they have a preference for rafts, or the opposite?” From the PSI results it was impossible to tell. To be able to answer this question and publish the article, the researchers obtained fast access to D22 at the ILL through the so-called EASY proposal mechanism. The high flux available quickly provided convincing evidence that AMPs inserted evenly in all membrane regions.
4. What next?
Besides novelty and surprise, the results bring many questions to be addressed in future experiments. Koynarev gives a couple of examples: “We explain that patches grow due to the line tension increase, which in turn we explain with the membrane shrinking. But why does it shrink? Domains grow, but what is their composition?” Reidar Lund and Lionel Porcar hope to continue experiments in D22 soon, as much is still to be explored.
Bio-inspired materials, antimicrobial resistance, and next generation drug carriers have all been recognised by EU and UN as priority research areas in the UN 2030 Agenda for Sustainable Development. The recently approved EU-funded doctoral network “CLIMB - Complex lipid membranes for science and technology”, in which both the University of Oslo and the ILL participate, will be an important test ground for scattering techniques in these fields. It aims to advance the current knowledge and to contribute to knowledge transfer between academia and the companies involved.
ILL Instrument: D22
Reference: Vladimir Rosenov Koynarev, Kari Kristine Almåsvold Borgos, Joachim Kohlbrecher, Lionel Porcar, Josefine Eilsø Nielsen, and Reidar Lund, Antimicrobial Peptides Increase Line Tension in Raft-Forming Lipid Membranes. Journal of the American Chemical Society 2024 146 (30), 20891-20903.
DOI: https://doi.org/10.1021/jacs.4c05377
ILL Contact: Lionel Porcar