Tag Archives: Membrane

New compounds fight drug-resistant bacteria by turning their membranes into prison cells

Two new antibiotic compounds join the fight against drug-resistant bacteria.

Staphylococcus aureus seen under the electron microscope.
Image credits Mogana Das Murtey, Patchamuthu Ramasamy.

The compounds have been named corbomycin and complestatin, and are part of the glycopeptide family of antibiotics produced by soil bacteria (the Actinomycetes family in particular). The unique way in which they attack bacteria makes them very promising candidates against drug-resistant infections, the study reports.

Don’t tear down this wall

The study reports that laboratory studies on mice showed that these two substances interact with bacteria in a completely different way from anything we’ve seen before.

“Bacteria have a wall around the outside of their cells that gives them shape and is a source of strength,” said study first author Beth Culp, a PhD candidate in biochemistry and biomedical sciences at McMaster.

“Antibiotics like penicillin kill bacteria by preventing building of the wall, but the antibiotics that we found actually work by doing the opposite — they prevent the wall from being broken down. This is critical for cell to divide.”

Both corbomycin and complestatin have proven themselves effective in combating Methicillin-resistant Staphylococcus aureus (MRSA), a family of bacteria that is highly resistant to antibiotics and is responsible for many serious, potentially life-threatening infections today.

Glycopeptides inhibit the growth of cell membranes by blocking the synthesis of peptidoglycan, which is a vital building block. It may not sound like much of a hassle but this effectively prevents bacteria from multiplying, as they need to generate extra membrane before dividing. These two compounds essentially ensures the bacteria are “trapped in a prison, and can’t expand or grow.”

For the study, the team started with a list of known glycopeptides — a chemical class that includes some of the most powerful and dangerous antibiotics humanity has ever wielded — and the microbial genes that encode their synthesis. They hoped that compounds encoded in different genes would also engage bacteria in different ways. This step set them on the trail of corbomycin and complestatin.

Looking at the family tree of known members of the glycopeptides, researchers studied the genes of those lacking known resistance mechanisms, with the idea they may be antibiotics demonstrating a different way to attack bacteria. Further testing in collaboration with Yves Brun and his team from the Université de Montréal carried out with cell imaging equipment, revealed how they acted on bacterial membranes.

“This approach can be applied to other antibiotics and help us discover new ones with different mechanisms of action,” Culp explains. “We found one completely new antibiotic in this study, but since then, we’ve found a few others in the same family that have this same new mechanism.”

One of the most exciting findings of the study is that the compounds show efficiency even against Enterococcus strains resistant to vancomycin and S. aureus strains that show an intermediate resistance to vancomycin. Vancomycin is used as a last-line-of-defense antibiotic against gram-positive infections that do not respond to any other treatment.

The paper “Evolution-guided discovery of antibiotics that inhibit peptidoglycan remodelling” has been published in the journal Nature.

New, free app modifies antibiotics to work against drug-resistant infections

A new web tool could help us find novel antibiotics that work against Gram-negative bacteria (which tend to gain antibiotic resistance). The app works by offering instructions on converting drugs that kill other bacteria into compounds that work against Gram-negative strains.

Image credits Sheep purple / Flickr.

Gram-negative bacteria have an extra, outer membrane, that renders most antibiotics useless. It helps the bacteria to survive out in nature where many organisms (like fungi) naturally produce antibiotics. This would be fine except for the fact that some Gram-negative bacteria like to cause nasty infections in humans — which don’t respond to treatment and put patients at risk. In order to prove that their tool works, the team used it to modify a drug and successfully tested it against three different Gram-negative bacterial strains.

Computer, design a drug

“It’s really hard to find new antibiotics for Gram-negative pathogens, because these bacteria have an extra membrane, an outer membrane, that’s very good at keeping antibiotics out,” said University of Illinois chemistry professor Paul Hergenrother, who led the research.

Hergenrother explains that no new antibiotics against Gram-negative bacteria have been approved by the Food and Drug Administration in 50 years, leaving us virtually exposed to the pathogens. His team has been hard at work finding a solution for several years now. His team “discovered the molecular features that allowed an antibiotic compound to surpass this barrier” a few years ago, he said, adding that this tool is the implementation of those findings.

The team’s app/web tool is called eNTRyway, and evaluates the potential of known drug compounds to pierce the outer membrane of Gram-negative bacteria. It also estimates whether the drug can perform this at high enough levels to accumulate inside the bacterial cells in functional doses. Even better, this app can also point out how to modify existing drugs for the task of tackling Gram-negative pathogens.

The team used eNTRyway to identify a drug that’s currently in use against Gram-positive infections that, with a little bit of tweaking, could potentially hurt Gram-negative strains. The team then synthesized the drug (by adding an amine group to the original one) and tested it on Gram-negative infections in mice. It proved effective against several different strains, the team reports, successfully accumulating behind the outer membrane of these pathogens.

The whole process took only a few weeks, Hergenrother said. The team hopes that their app will greatly speed up the development of such drugs in the future.

“We can use this tool to rapidly identify compounds that accumulate in Gram-negative bacteria,” he said.

“Keep in mind that before this, over 100 derivatives of this same compound had been made. We found them in patents and papers,” he said. “And none of these other derivatives had notable Gram-negative activity.”

The team went on to identify over 60 antibiotics that could be converted to fight Gram-negative bacteria using a variety of chemical pathways. For example, one of their newly-developed drugs (christened Debio-1452-NH3) disturbs fatty acid synthesis in bacterial cells, but not in mammalian ones.

The paper “Implementation of permeation rules leads to a FabI inhibitor with activity against Gram-negative pathogens” has been published in the journal Nature Microbiology.

Cell image.

When faced with high gravity, cells get “thicker skin” by strengthening their membranes

When gravity increases, cells thicken their skins, new research shows.

Cell image.

Image credits Emma M. Woodcock et al., (2019), Biophysical Journal.

Researchers from the European Space Agency (ESA) and Imperial College, London report that as gravity increases, cells develop more viscous membranes to hold their shape — similar to developing a “thicker skin”. This study is the first to document the changes in cell membrane viscosity that occur at higher and extreme gravities.

Thick, thicker, thickest

“Studying how cells adapt to extreme gravity helps us understand how cells can resist environmental stress in general—from viral attacks and chemical imbalance to extreme cold and extreme heat,” says Dr. Nick Brooks, co-author of the study and Senior Lecturer in the Department of Chemistry at Imperial.

“Another really exciting thing was showing we could do it. Actually being able to conduct detailed microscopy experiments inside of a centrifuge which is spinning at 15g is not very easy.”

Using the European Space Agency’s (ESA) Large Diameter Centrifuge in the Netherlands, the team pitted living cells against a gravitational force 15 times stronger than Earth’s (i.e. 15g). As gravity increased, so did cell membrane viscosity, the team reports. These results may not sound like much, but the authors note that membrane viscosity is an important parameter for cells, as it controls how quickly some key reactions occur — and, indeed, if they can take place at all.

The findings may help us develop better treatments for diseases like diabetes and Alzheimer’s that can change the viscosity within cells. They also simply help us better understand how cells go about their routine lives.

Cells development over time.

Image credits Emma M. Woodcock et al., (2019), Biophysical Journal.

The study was led by Imperial Ph.D. graduate Emma Woodcock as part of the Spin Your Thesis! competition that gives students access to the ESA’s centrifuge — a four-armed, eight-meter in diameter spinning beast. Her study brought together Imperial chemists, physicists, and ESA scientists. At full speed, the centrifuge can spin containers at the ends of its arms at 101km/hour, roughly 60mph. Simulated gravity inside these containers can reach up to 20g. The most powerful simulated gravitational force you likely experienced was on a rollercoaster ride; to give you some context, the most powerful rollercoasters today only touch the 5g mark for a few seconds at a time.

The team made several impressive advancements for this study. They successfully secured a camera and microscope inside the containers and managed to keep them properly focused throughout the experiment. Dr. Marina Kuimova, one of the paper’s co-authors and an Associate Professor in the Department of Chemistry at Imperial also developed molecules that would light up as viscosity in the cellular membranes increased. These molecules have a spinning axis that rotates faster in low viscosity but becomes progressively stalled as the membrane stiffens. When not spinning, it emits bright fluorescent light — and the light it emits is reliable and stable enough that the team could measure it to see how fast the molecule was spinning. This method doesn’t chemically interfere with the cell in any way, making it very promising for future research of this kind.

“This technique opens a whole new way of looking at how cells work,” Dr. Kuimova explains. “We usually monitor smaller changes in viscosity under normal physiological conditions, but it was exciting to apply our technique to conditions when the cells were pushed to the extreme.”

This study worked with general mouse cells and specialized human cells of the endothelium — those that line blood vessels. Both types of cells had very fast responses in stiffness to gravity levels. The team says this suggests it is mostly a physical response caused by components in the cells’ membranes, rather than an active shift in which the cell senses changes in pressure and starts to produce a mechanical change in its membrane as a result.

The human cells saw the greatest and fastest changes, the team reports, which isn’t that surprising; endothelial cells are equipped to deal with pressure changes caused by sharp corners in vessels, plaque build-up, or shifts in heart rate and blood pressure. This effect might illustrate what cells are subjected to with arterial diseases (though that happens on a larger scale), the team writes.

In the future, the team wants to look at how the cell signals to its membrane to increase viscosity. They also want to adapt their method to study the viscosity of different parts of the cell, how this is implicated in diseases and how cell membranes control other things.

The paper “Measuring Intracellular Viscosity in Conditions of Hypergravity” has been published in the Biophysical Journal.

Blood.

Cell-membrane-coated nanobots successfully clear out 66% of bacteria and toxins in blood samples

Medical nanobots are one step closer, as researchers developed simple nanorobots that can be propelled through blood to clear out bacteria and toxins.

Blood.

Image credits Mate Marschalko / Flickr.

A team of engineers from the University of California San Diego has developed a class of ultrasound-powered robots that can scrub blood clean of bacteria and the toxins they produce. While still simple, the proof-of-concept nanobots could pave the way towards safe and rapid methods of decontaminating biological fluids — even in the bodies of living patients.

Bling medicine

The team builds their nanorobots out of gold nanowires coated with platelet and red blood cell membranes. This hybrid membrane is what gives the nanites the ability to clear out biological contaminants. The platelet membrane binds to pathogens such as the antibiotic-resistant strain of Staphylococcus aureus, MRSA, while the red blood cell membranes can absorb and neutralize toxins produced by bacteria.

The gold nanobody is what lets the researchers move the bots around. The metal responds to ultrasound, giving the team the means to power them through the bloodstream without the use of engines or fuel. The bots need to be mobile in order to more efficiently mix with a fluid sample, speeding up the process of detoxification.

The nanobots were created using processes pioneered by the teams of Joseph Wang and Liangfang Zhang, professors in the Department of NanoEngineering at the UC San Diego Jacobs School of Engineering. Wang’s team designed and built the nanobots and the means of ultrasound-powered propulsion, while Zhang’s team developed the process used to coat these in natural cell membranes.

“By integrating natural cell coatings onto synthetic nanomachines, we can impart new capabilities on tiny robots such as removal of pathogens and toxins from the body and from other matrices,” said Wang.

“This is a proof-of-concept platform for diverse therapeutic and biodetoxification applications.”

Furthermore, the natural membranes prevent the nanobots from being ‘biofouled’ — a process by which proteins cake onto the surface of a foreign body, which would prevent the nanobots from functioning. The hybrid membranes were created from natural membranes, separated in one piece from platelets and red blood cells. These were then blasted with high-frequency sound waves, causing them to fuse together.

Nanobot.

The nanobot binding to and isolating a pathogen.
Image credits Fernández de Ávila et al., 2018, Science Robotics.

The robots’ bodies were constructed by then applying these membranes to gold nanowires through chemical means.

The finished devices are roughly 25 times smaller than the width of a hair, the team writes. Ultrasound waves can propel them up to 35 micrometers per second in blood. They were successful in cleaning blood samples contaminated with MRSA and associated toxins — after 5 minutes of being injected, the levels of bacteria and toxins were three times lower in treated samples than untreated samples.

If you’re like me and dream all starry-eyed about the day we’ll treat ourselves with nanobots, this research might make you feel quite happy inside. However, this work is at a very early stage. It’s also focused on something different — the team notes that, while their current nanobots can be used to treat MRSA in blood samples, they aim to have a device that can detoxify all kinds of biological fluids.

We still have a ways to go until then. For the near future, the team hopes to test their devices in live animal models, and to devise a way of creating the robot bodies out of biodegradable materials instead of gold.

The paper “Hybrid biomembrane–functionalized nanorobots for concurrent removal of pathogenic bacteria and toxins” has been published in the journal Science Robotics.

Membrane model.

Researchers took a nanoscale snap of a living cell membrane for the first time in history

Researchers have snapped the most detailed ever image of a cell membrane, all the way down to the nanoscale. The image could finally settle a long-standing debate in biology on how it functions, and introduce a powerful new tool to biologists’ toolkit.

Membrane model.

Image credits John Katsaras et al, PLOS ONE (2017).

Every living cell’s membrane is put together from a thin sandwich of lipids, which are fat molecules, interspersed with other bits of organic materials such as proteins and carbohydrates. It’s a pretty nifty system — the fatty bilayer, for example, keeps all the watery stuff inside the cell from mixing with the watery stuff outside the cell. Proteins act as pumps and decide what goes through the membrane and when, or serve as landing areas for signaling molecules so the cell can talk with its pals. Some carbohydrates act as ID tags. Then there’s one other bit whose function — as far as can be summarized if you keep tabs on cytological debates, which I’m sure most of you do — seems to be solely to sow discord and disagreement into the ranks of biologists.

These tiny bits are known as lipid rafts and, although there’s a pretty solid body of documentation as to what they are and what they do, haven’t really caught with all cellular biologists. The short of it is that they act as independent, more compact domains than the rest of the membrane, making it behave a little wobbly, and their movements allow the cell to activate or inactivate proteins along its membrane.

So, a team led by John Katsaras, Senior Biological Systems Scientist at Oak Ridge National Laboratory’s Neutron Sciences Directorate, decided to take a picture and find out.

“It became a debate,” Katsaras said. “Some people believed they exist, while others believed they didn’t. There was a lot of circumstantial evidence that could support either side.”

The way they went about it could fundamentally change how living nanoscale structures are studied in the future.

Looking at the really small

When biologists want to take a peek at the going-ons inside a cell, they normally use fluorescent compounds designed to attach to a particular molecule and tag it, making it visible under the optical microscope. But since we don’t really know what lipid rafts do (so we don’t know where to add the fluorescent tags), and because they’re probably too tiny to spot under the microscope, this doesn’t really work in their case.

Good luck spotting anything.
Image credits Tobi Luxe.

An electron microscope could probably make them out with ease, but the thing is that to find out how these rafts behave you need to observe a living cell. Since cells are made so tiny, atoms are basically brick-sized compared to them. Electrons, then, are bullet- or pellet-sized. To a living cell, an electron microscope is basically a death-spewing chaingun. So that won’t work either.

In the end, the team decided to use a mix of genetic and chemical labeling techniques to add a hydrogen isotope to the membranes of living Bacillus subtilis cells. Then, they used a method called neutron scattering to chart the arrangement of different molecules in the bacterium’s cell membrane. Neutron scattering was picked because it’s less energetic than electron microscopy, meaning the particles aren’t (necessarily) deadly to the bacteria.

So why are the isotopes there? Well, although less energetic, neutrons are way heavier than electrons. So it’s not exactly deadly, but the particles are powerful enough to affect the cell and interfere with its membrane’s internal processes. Furthermore, while it could spot the rafts, neutron scattering couldn’t tell it apart from the rest of the membrane, so the team needed to tag them with something that stands out.

Bag and tag

Since 99.98% of all hydrogen atoms currently in existence only have a single proton for a nucleus, the isotopes the team used, which have an extra neutron attached to the nucleus and are known as deuterium, is pretty conspicuous. And while they chemically function the same (since neutrons don’t affect the atom’s valence/electrical balance), physically they do differ enough to scatter neutrons in a different way — so they were both easy to spot and unlikely to occur naturally.

The team genetically edited a new strain of B. subtilis with a slightly different ratio of hydrogen to deuterium in its membrane compared to wild strains. If there were no rafts, they should see a uniform distribution of these altered fat molecules throughout the membrane.

Instead, their imaging showed areas with pronounced differences in lipid arrangement, which matched the proposed size of the lipid rafts — very strong evidence for their existence. Even better, the technique they developed for the study could fundamentally change how biologists peer into the workings of living cells.

“The people who study these things tend to use particular types of probes,” says Katsaras.

“They didn’t use neutron scattering because it wasn’t in the biologist’s wheelhouse. Our novel experimental approach opens up new areas of research.”

These differentiated areas aren’t visible in the team’s model, but it does an exemplary job of showing how a cell’s outer layers are structured — watery cytoplasm covered with the lipid layer the team was investigating in the middle, and the outer cell wall at the top.

The full paper “The in vivo structure of biological membranes and evidence for lipid domains” has been published in the journal PLOS ONE.