Biophysics of Antimicrobal Peptides

University of Strasbourg professor, Burkhard Bechinger, on discovering new antibiotics, the interaction of peptides with bacterial membranes and promising work all over the world

videos | February 17, 2015

You might have heard about the occurrence of multi-resistance infections, that are now more and more widely spread, particularly in hospitals, but also outside. So there are young people, all of a sudden dying of having caught an infection, and there’s no way to treat these people. So this is a serious problem for our society and, of course, for the health of all humans. And so this multi-resistance is more and more abundant, and at the same time there are less and less new pharmaceuticals to treat these bacterial infections or fungal infections as well.

So, we are concerned about these problems, as well as many other people. And what people have done is that they have looked in the nature – what kind of antibiotics are there? And what they have found is peptides, first in frogs and insects, but now also in humans. They are called, for example, ‘defensins’ – to defend, of course. And they are very important for our health, and we try to understand how these peptides work, and maybe find a better solution, compared to the common antibiotics that are used now, which would develop less of a resistance.

So, we are not a laboratory that’s concerned with the microbiology and the medicine behind that. But what we try to do is we try to understand how these peptides, these compounds work. And we know they work on membranes, basically the exterior of all our cells that keeps the cells together and protects them from the environment. And that’s our specialty – the biophysics of these biomembranes.

And so we started to study these peptides very early on. They were discovered already in the 1960s, but people did not recognize the antibiotic effect, or they might have recognized, but they didn’t recognize the utility. There are very early publications that very few people know nowadays that describe these peptides. But only in the late 1980s there were, basically, two teams that developed or that recognized these peptides are antibiotics, and they found them either in insects or in frogs. So these two teams worked in different systems.

These peptides are relatively short, and they have something in common — it’s that they are very heavily charged. They have some hydrophobic amino acids that like to interact with the oily phase, but they have a lot of positive charges that like to interact, of course, with negative surfaces, for example. And they are, let’s say, from 20 to 30 amino acids long — not very long for a peptide.

And people thought: “that’s good, and they might interact with membranes” and did tests and they, indeed, found a change in structure. They form helices, separating nicely the hydrophobic parts underneath and the charged residues on the top. They could see that these helices make holes in membranes and increase the permeability. “All of this is very interesting” — they thought, of course. They looked at literature and found membrane channels, where all these helices would be lined, a sort of transmembrane, going through the membrane, interacting with one another and forming these holes.

So, that was the idea, but there was very little data on that. So, about 1990 we started to look at them with biophysical methods, particularly solid-state NMR, for that purpose. It was sort of a period that we developed this technique, and it was very interesting.

So, what we found — and that was very surprising to us, because we didn’t expect that at all — was that these peptides actually don’t go through the membrane, but they lay on the surface. Of course, you may wonder how they can make holes then, because we have these peptides, but there is so much more of the membrane. And why would water flow through and ions go through then? — and that were very puzzling questions. And so we started to look at this problem in more detail using a variety of techniques, like CD — circular dichroism spectroscopy, infrared spectroscopy, but mainly solid-state NMR spectroscopy. And what we did for that purpose was, actually, we made a supported bilayers of these membranes. We deposited membranes on little surfaces, and included these peptides. And that’s how we could see, for example, that they lie parallel to the surface and not transmembrane.

But we can also look at the lipids, of course, and what we figured out was that it’s not just the peptide alone, but they have to sort of very closely include the lipids that surround them. So the lipids are actually not solid, they are a very soft material like a gel, it’s oily. If you have these lipids, these membranes in your sample, you can smear it out like a gel. So it’s not liquid, it’s not solid, but it’s soft enough to adapt. So you have this peptide here, and the lipids can sort of form themselves around and adapt.

But now look at this — you have that peptide here that sinks in halfway into the membrane, but not fully. That means, you have a lot of space underneath, and of course, this space doesn’t stay empty, and you have the lipids here, left and right. And, of course, the lipids try to move in from both sides. And that causes the membrane, actually, to start bending. But of course, because there is another part of the membrane, it cannot bend forever. But it’s strained, the membrane isn’t in equilibrium anymore. There is strain now on this membrane, and if there is only one peptide, it’s fine, but if there are more and more of these peptides, the disruptive little molecules that try to disturb the membrane, packing, then it might rupture. And that’s what we think is happening.

But it’s against what people initially imagined, so it took a bit of time to convince ourselves that’s the way it works. Now we are pretty convinced that the interaction with peptides and the lipids, the ensemble of the membrane has to be considered.

And that’s actually good, because if you think about resistance, how does resistance occur in most bacteria? You give a patient an sort of an antibiotic, so you expose these pathogens to the antibiotic. And of course, these antibiotics can then also include mutations, and change a receptor molecule. And some of these receptors that have been mutated all of a sudden don’t interact with the antibiotic anymore. Usually it’s enough to change just one amino acid in a receptor to make these bacteria resistant. But you cannot do that to a membrane, it’s a very complex system. You cannot just change one thing and everything changes.

The interaction of this peptide system membrane is based on such varied channel mechanisms that the induction of resistance is very hard for the bacteria. It’s still possible, but it’s much harder. So this bears great promise, now that we understand a bit better how the things work, that we copied nature in a way to see how things work and maybe do something that is easier to prepare and apply to a patient, that we can find something that isn’t prone to resistance as fast and something very different from what’s around now.

So how can we use this knowledge now that these peptides lie flat on the surface and don’t go transmembrane? Of course, the idea is now to develop small molecules that have also this charged part and a hydrophobic part that sinks in, and the charged part is kept on the surface. And then the lipids are pushed apart and there is curvature strain.

So there are several groups that think about this concept and develop new molecules. There is a group in Japan, for example, that has designed very small compounds, like the size of usual medications that we take nowadays. But there are also other groups that just develop polymers that are basically long chains of molecules that are repetitive, and some parts of these polymers are positively charged and then there’s the hydrophobic interaction. And this might, for example, be used to coat surfaces that have to be sterile, because they also have antimicrobial activity.

Then there are other groups don’t use peptides in a regular sense, because a usual peptide can be digested in the stomach.But they replace the links between the amino acids that make up these peptides, and these links are stable also in the stomach. And so all these are applications that have very promising future, I think, because they can be used to fight this problem of multi-resistance.

And I think that’s sort of the future of this. Of course, we want some treatment, for now we always find one antibiotic, only in few cases, we are really desperate and don’t find anything.
But it’s getting more and more serious, so at some point we will need something completely new. And now that we understand these peptides, this gives us a chance to develop something that can replace these conventional antibiotics.

Of course, the peptides themselves might not be very useful as a medication, because if you eat a peptide it’s like eating meat or a protein, they will be digested. But now people started to develop small molecules on the same principles – to have the head with the positive charge and to have something hydrophobic underneath. So it will sink into the membranes, push the lipids apart, and cause curvature. And these things probably work in the same way as these peptides do. And they can be synthesized, they can be given orally and so on.

We are very early on and this is a dream right now. There are several start-up companies that work on that. We don’t do that, we do basic science, doing biophysics on these systems. But what we and others, of course, have found on these peptides will help the development of these new compounds, and I think that will be the future.

Professor, head of NMR and membrane biophysics team of the CNRS and University of Strasbourg
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