Scientists at the U.K.’s University of Newcastle suggest that a bacterial membrane protein that acts as a type of membrane vacuum cleaner could represent a promising new target for rendering harmful Gram-negative strains susceptible to existing as well as new antibiotics. Blocking the protein, which is part of the Mla (maintenance of outer membrane lipid asymmetry) system, would effectively disrupt the composition of one of the two layers of the bacterium’s outer membrane, which normally acts as a barrier to toxic chemicals, such as antibiotics.
“If a way could be developed to inhibit the Mla system via a drug, this would then make bacteria much more sensitive to a number of antibiotics (including ones currently on the market), because the outer membrane would now be leaky,” commented lead researcher Bert van den Berg, Ph.D., professor of membrane protein structural biology at Newcastle University, speaking with GEN. “Such a drug would not only be an antibiotic in itself, but would also potentiate other antibiotics (the latter might be the most important feature).”
The researchers report on studies investigating the structure and function of the Mla protein, in Nature Microbiology, in a paper entitled “Structural Basis for Maintenance of Bacterial Outer Membrane Lipid Asymmetry.”
Gram-negative bacteria are surrounded by both an inner membrane (IM) and an outer membrane (OM). While the IM is formed by a symmetric phospholipid (PL) bilayer, the OM is structured as an asymmetric bilayer, comprising an inner leaflet of PLs and an outer leaflet that is composed of lipopolysaccharides (LPSs). The LPS layer effectively forms a sugar coating on the surface of Gram-negative bacteria, which resists hydrophobic molecules and blocks entry by harmful compounds, such as antibiotics. This makes the delivery of drugs into Gram-negative bacteria particularly problematic.
Some PLs are spontaneously inserted into the outer leaflet of the OM, however. They don’t mix well with the LPS and form islands of PLs, which make patches of symmetrical bilayer, through which toxic molecules, including some types of antibiotic, can penetrate. To avoid this happening, the PLs need to be removed from the outer leaflet. “OM asymmetry, or more precisely the LPS in the outer leaflet, is the basis for the high intrinsic resistance of Gram-negative bacteria toward antibiotics and other toxic compounds,” van den Berg explained to GEN. “This is due to the very special properties of LPS that are unique to Gram-negative bacteria. The asymmetry is disturbed when PLs enter the outer leaflet (which happens spontaneously), and this makes the membrane leaky for many compounds. This, in turn, is bad for the bacterium.”
One of the mechanisms that cells use to remove the PLs is the Mla system, which was first described in 2009. “There are two other systems that remove phospholipids from the OM,” van den Berg continued, “but these generate reaction products that still require removal and thus are indirect. The Mla system is the only system known to directly maintain OM asymmetry.”
The Newcastle team focused on the MlaA protein, which is the OM-sited component of the six-protein Mla system. The researchers carried out X-ray crystallography to elucidate the 3D structure of MlaA, combined with computation and functional assays to identify important residues in the protein.
In silico predictions have indicated that MlaA is a lipoprotein, but the work at Newcastle has now shown this to be incorrect. “Unexpectedly, MlaA is an alpha-helical integral membrane protein (not a humble lipoprotein as predicted), mostly located in the inner leaflet of the OM but partly inserted into the outer leaflet,” writes lead co-research and lead author, Javier Abellon-Ruiz, in an accompanying blog.
“The most surprising insight was that MlaA is mostly embedded in the OM, which was not at all predicted,” van den Berg further commented to GEN. “MlaA has also a structure (alpha-helical) that is common in other membrane proteins but very rare in OM proteins.”
“But what was even more surprising was the presence of an amphiphilic pore in MlaA,” Abellon-Ruiz noted. Effectively, the Mla protein is structured like a donut, sited in the inner leaflet of the OM, which acts like a one-way channel, allowing removal of PLs from the outer OM leaflet, but preventing inner-leaflet PLs from entering the pore. “It removes phospholipids from the outer leaflet of the OM, somewhat akin to a vacuum cleaner,” van den Berg stated. “[This insight] highlights the importance of asymmetry and suggests that it is worth investigating whether the Mla system could be targeted by new drugs.” What isn't clear, however, is how easy or difficult it would be for the bacterium to develop resistance, he acknowledges.
The Newcastle team is now carrying out mechanistic studies to further investigate the Mla system. “We will also try to define what kind of compounds benefit from MlaA removal (which types of molecules can now enter the cell more easily?).… If these studies are promising we could think about trying to see if we can find an inhibitor of the Mla system,” GEN was told. “…such studies will most likely be carried out in collaboration with the pharma industry.”