Bacteria may soon replace viruses as our primary pandemic concern. Among potential candidates, Gram-negative bacteria lead the pack, thanks to their unique biological barrier that shields them from antibiotics. In our shrinking antibiotic arsenal, polymyxin B stands out as a last-resort drug, as it can breach the bacterial outer membrane, although not without side effects. In this study, published in ACS Omega, the researchers examined this process at the molecular scale. Their work was also featured as a cover image for the journal issue.
The work was carried out as part of Nicoló Paracini's PhD research, which was part-funded by ISIS as part of the Facility Development Studentship scheme. Along with his supervisors, Jeremy Lakey from Newcastle University and Luke Clifton from ISIS, he used neutron reflectometry on the OffSpec beamline at ISIS to investigate how the antibiotic polymyxin B interacts with a model bacterial membrane.
Nature's armour: the bacterial outer membrane
Bacteria fall into two main categories: Gram-positive and Gram-negative. A key distinction is that Gram-negative bacteria have an additional impermeable membrane. This outer membrane differs from typical phospholipid bilayers and consists instead of phospholipids on its inner layer and lipopolysaccharides (LPS) on its outer surface. These LPS molecules form an impermeable barrier for many antibiotics, but polymyxin B can breach it by destabilising the LPS interactions in the membrane. This leads to the irreparable damage and cell death that's needed to beat the infection.
Probing membranes with neutrons
Bacterial cell membranes consist of lipid bilayers approximately five nanometres thick, which makes detailed atomic-scale investigation extremely challenging. Neutron reflectometry offers a solution by measuring the structure of these ultra-thin layers at the molecular level. Due to quantum mechanics, neutrons behave as waves and create interference patterns when reflecting off thin films - similar to the colours seen in soap bubbles, but at the nanometre scale. Cold neutrons, with wavelengths comparable to X-rays (in the angstrom range), perfectly suit studying biological membranes. Unlike X-rays, neutrons carry only a few meV of energy rather than several keV, preventing membrane damage during measurement.
The work brought together expertise from different neutron facilities. The experiments were carried out at ISIS, but the data were analysed at a later stage by Nicoló while he was at the Institut Laue–Langevin (ILL), using software developed at the Australian Centre for Neutron Scattering (ANSTO).
Temperature-dependent antibiotic penetration
Their results revealed that polymyxin B only damages bacterial membranes at 37°C but had no effect at room temperature (20°C). This dramatic all-or-nothing response demonstrates how the temperature-induced phase transition of LPS molecules, from gel to fluid state, enables antibiotic penetration.
The group were also able to make the most of the sensitivity of neutrons to different isotopes of hydrogen. They used deuterated LPS and lipids to precisely map the antibiotic's distribution across the membrane and found that it accumulates in the LPS hydrophobic region.
While the fight against antibiotic resistance continues, understanding the physical mechanisms of antimicrobial action helps us develop more effective strategies against bacterial resistance. This knowledge proves crucial for preserving our remaining antibiotic options and developing new ones.
“This research shows the power of neutron reflectometry in providing detailed information on biomolecular interactions, in this case resolving precisely how this important last resort antibiotic interacts with pathogen membranes at the molecular level," explains ISIS scientist Luke Clifton.
Since completing his PhD, Nicoló has been working in the reflectometry group at the ILL and has recently moved to the European Spallation Source (ESS). “The time I spent at ISIS during my PhD has been extremely formative on the scientific and human level. Facilities that work with such a wide breadth of science like ISIS, the ILL, and soon the ESS, rely on many great people from wildly different backgrounds. I've been lucky enough to work in all three facilities and it has been a truly enriching experience."
The full paper can be found at DOI: 10.1021/acsomega.4c07648
