Structural basis for the lipid asymmetry of the Gram-negative bacterial outer membrane

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Gram-negative bacteria are characterised by having a cell envelope consisting of two membranes, an inner (or cytoplasmic) membrane (IM) and an outer membrane (OM). The two membranes are very different: the two leaflets of the inner membrane both contain phospholipids (PL) and can therefore be considered symmetric. By contrast, the OM is an asymmetric bilayer with an inner leaflet of PL and an outer, surface-exposed leaflet composed almost exclusively of lipopolysaccharide (LPS). The properties and structure of LPS are very different from those of PL, and the main consequence of the presence of LPS is that Gram-negative bacteria are surrounded by a polar layer that forms a very effective barrier for neutral and hydrophobic molecules. Given that the majority of drugs are moderately hydrophobic, the unique, asymmetric structure of the OM is a major factor why Gram-negative bacteria have a high intrinsic resistance towards antibiotics and other harmful compounds. Thus, from the bacterium's point of view, the asymmetry of the OM is very important. However, due to reasons that are not yet clear but likely are a consequence of normal cell growth, PL can accumulate in the outer leaflet of the OM. Those PL form "islands" amid a sea of LPS that increase the permeability of noxious compounds. Thus, PL need to be removed from the outer leaflet to restore the OM permeability barrier.The Mla (maintenance of lipid asymmetry) system is widespread in Gram-negative bacteria and is likely the most important system for maintaining OM lipid asymmetry. It consists of six proteins that are thought to form a "reverse" transport system for PL from the OM to the IM. The protein MlaA is the OM component of the Mla system and is thought to selectively extract PL from the outer OM leaflet. How this happens is unclear, mainly due to a lack of structural information on MlaA. We have determined the first structures of MlaA proteins (by X-ray crystallography) to understand how MlaA functions. These preliminary data are very interesting in that they show that MlaA has a unique structure, but more importantly they provide clear and testable clues regarding function. In this proposal we will use the structures as starting points to determine how MlaA extracts PL from the OM outer leaflet and subsequently transfers them to a binding protein (MlaC) in the periplasmic space. We will use an interdisciplinary approach with functional assays, biophysical methods, computational approaches and X-ray crystallography. Our project will elucidate a fundamental and important process in Gram-negative bacteria and will inform on the prospect of targeting the Mla system of Gram-negative pathogens as a means to decrease virulence and to potentiate various antibiotics.