University College London (UCL) researchers have generated what they claim are the sharpest images ever to show the complex architecture of the protective outer membrane (OM) of some types of potentially deadly pathogenic bacteria, which acts to shield these microorganisms from antibiotic drugs.
The study, carried out in collaboration with scientists at National Physical Laboratory, King’s College London, University of Oxford and Princeton University, reveals that this proactive outer membrane of Gram-negative bacteria comprises dense networks of protein building blocks, alternated by patches that have no proteins, but which are enriched with glycolipids that keep the outer membrane tight. The finding that these protective membranes demonstrate weaker, as well as stronger spots could help scientists to develop more effective drugs against antimicrobial resistant strains.
“The outer membrane is a formidable barrier against antibiotics and is an important factor in making infectious bacteria resistant to medical treatment,” explained Bart Hoogenboom, PhD, London Centre for Nanotechnology at UCL and UCL Physics & Astronomy. “However, it remains relatively unclear how this barrier is put together, which is why we chose to study it in such detail. By studying live bacteria from the molecular to cellular scale, we can see how membrane proteins form a network that spans the entire surface of the bacteria, leaving small gaps for patches that contain no protein. This suggests that the barrier may not be equally hard to breach or stretch all over the bacterium, but may have stronger and weaker spots that can also be targeted by antibiotics.”
Hoogenboom is co-corresponding author of the team’s published paper in Proceedings of the National Academy of Sciences (PNAS), which is titled, “Phase separation in the outer membrane of Escherichia coli.”
Gram-negative bacteria such as Escherichia coli are surrounded by an outer membrane that protects cells against the immune systems of plants and animals, contributes to the mechanical stability of the cell, and excludes many classes of antibiotics, which then contributes to antimicrobial resistance, the authors explained. The tough outer membrane of Gram-negative bacteria prevents certain drugs and antibiotics from penetrating the cell, and is part of the reason why antimicrobial resistance of such bacteria (including A. baumannii, P. aeruginosa, and enterobacteriaceae such as Salmonella and E. coli) is now considered a greater threat than that of Gram-positive bacteria such as resistant S. aureus.
This OM is comprised of an asymmetric bilayer of phospholipids in the inner leaflet, lipopolysaccharides (LPS) in the outer leaflet, and many outer-membrane proteins (OMPs), the investigators further commented, but how these membrane components are arranged isn’t well understood. “The OM is also populated with many β-barrel outer-membrane proteins (OMPs), some of which have been shown to cluster into supramolecular assemblies,” they stated. “However, it remains unknown how abundant OMPs are organized across the entire bacterial surface and how this relates to the lipids in the membrane.’
To better understand the architecture of the OM, the scientists used atomic force microscopy to image the complete surface of E. coli strains. The technique effectively involves running a tiny needle over the living E. coli bacteria, thus “feeling” their overall shape. Since the tip of the needle is only a few nanometres wide, this makes it possible to visualise molecular structures at the bacterial surface.
The resulting images showed that the whole outer membrane of the bacteria is crammed with microscopic holes formed by proteins that allow the entry of nutrients while preventing the entry of toxins. Although the outer membrane was known to contain many proteins, this crowded and immobile nature had been unexpected.
Surprisingly, the images also revealed many patches that did not appear to contain proteins. These patches do contain a glycolipid normally found on the surface of Gram-negative bacteria. “Applying such large-scale, high-resolution imaging on engineered E. coli strains and complementing it by specific labeling of abundant OMPs, we identify large-scale and near-static protein-rich networks interspersed with nanoscale domains that are enriched in LPS,” the team noted. “Key components of the protein-rich networks are abundant trimeric porins such as OmpF, in addition to (the monomeric) OmpA, which forms noncovalent interactions to the underlying cell wall. By contrast, no significant protein content is detected in the LPS-rich domains, which are also found to grow and merge with other patches during cell elongation.”
The team also saw that a different type of pimple-like patch formed when parts of the membrane were flipped inside out due to mutations. In this case, the appearance of these defects correlated with enhanced sensitivity to bacitracin, an antibiotic usually only effective against Gram-positive, but not against Gram-negative bacteria.
Georgina Benn, PhD, who carried out the microscopy on the bacteria in the Hoogenboom lab at UCL, explained further, “The textbook picture of the bacterial outer membrane shows proteins distributed over the membrane in a disordered manner, well-mixed with other building blocks of the membrane. Our images demonstrate that that is not the case, but that lipid patches are segregated from protein-rich networks just like oil separating from water, in some cases forming chinks in the armour of the bacteria. This new way of looking at the outer membrane means that we can now start exploring if and how such order matters for membrane function, integrity and resistance to antibiotics.”
As the authors further noted, “Taken together, these results represent the highest resolution microscopy data of live cells reported to date and define the supramolecular architecture of the E. coli OM … Importantly, they provide a framework within which to understand associations between different OMPs, LPS, and phospholipids in the OM.”
The team speculate that their findings may help explain ways by which bacteria can maintain a tightly packed, protective barrier while still allowing rapid growth: the bacterium E. coli doubles in size and then divides in 20 minutes under favourable conditions. The investigators suggest that the glycolipid patches may allow for more stretch of the membrane than the protein networks, making it easier for the membrane to adapt to the growing size of the bacterium.
“We conclude that the OM is a mosaic of phase-separated LPS-rich and OMP-rich regions, the maintenance of which is essential to the integrity of the membrane and hence to the lifestyle of a gram-negative bacterium,” they wrote. “…this framework also provides a perspective to assess how bacterial sensitivity to immune effectors and antimicrobials may depend on local as well as global properties of the OM.”