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

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.

Gram-negative bacteria have a cell envelope consisting of an inner membrane (IM) and an outer membrane (OM). Whereas the IM is a regular, symmetrical phospholipid bilayer, the OM is asymmetric with an inner leaflet of phospholipids (PL) and an outer leaflet composed almost exclusively of lipopolysaccharide (LPS). LPS molecules contain several phosphate groups that are cross-linked via divalent metal ions, generating a polar layer on the cell surface that is a very effective barrier for neutral and hydrophobic molecules. Thus, the unique, asymmetric structure of the OM is a major factor why Gram-negative bacteria are intrinsically resistant towards antibiotics and other harmful compounds. The extreme asymmetry of the OM is entropically unfavorable, resulting in accumulation of PL in the outer OM leaflet as a consequence of normal cell growth. Those PL cluster together into rafts that increase the permeability of lipophilic compounds. Thus, PL need to be removed from the outer leaflet to maintain the OM permeability barrier. This process is carried out by the Mla (maintenance of lipid asymmetry) system consisting of six different proteins that together form a retrograde transport mechanism for PL. How the Mla system functions is unknown, mainly due to a lack of structural information. We have determined the first structures of the OM component MlaA, which show that it has a unique structure and provide testable clues as to how MlaA selectively extracts PL from the outer OM leaflet. In this proposal we will elucidate the OM-centered events of the Mla pathway by using an interdisciplinary approach incorporating 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.

The potential of pathogenic bacteria to become resistant to all currently used antibiotics and consequently cause a global health crisis is widely accepted. At the same time, new drugs are not being brought to market due to the high risks of failure in drug development that are caused by a lack of knowledge of fundamental bacterial cellular processes. The problem of multidrug resistance is especially acute for Gram-negative bacteria, largely as a consequence of the presence of an additional, outer membrane (OM) on the surface of the cell. The most striking feature of the OM is its asymmetric nature, with an outer leaflet composed almost exclusively of lipopolysaccharide (LPS) and an inner leaflet of phospholipids (PL). The unique architecture is the reason that the OM is a very effective barrier for small molecules, including most antibiotics. Any process that interferes with the asymmetry of the OM (i.e., decreases the surface density of LPS) will compromise the OM permeability barrier and consequently decrease cell viability in the presence of small molecule stressors. Currently, three systems are known to affect OM integrity: the Bam OM biogenesis machine, the LPS transport pathway (Lpt) and the recently discovered Mla (outer membrane lipid asymmetry) pathway. A defect in the Mla pathway results in accumulation of PL in the OM outer leaflet and this leads to increased susceptibility for certain small molecules. The potential impact of this project lies therefore in the promise of the Mla pathway as a viable target for future antimicrobials. Importantly, an inhibitor of the Mla pathway would likely potentiate a variety of other antibiotics as well. Finally, MlaA is an especially attractive target since our structural preliminary data show that this protein is surface exposed, raising the possibility of MlaA targeting without the need of a drug to cross any membrane.

Thus, while this proposal is based on the strength of the PI in fundamental, structure-function studies of OM proteins, we are excited about the potential of the Mla pathway as a future drug target. We will therefore (i) collaborate with an expert in Salmonella biology (Prof. Bumann) to assess the importance of MlaA for virulence, and (ii) initiate a collaboration with Glaxo Smith Kline (US). GSK will define more precisely what types of compounds are potentiated in the absence of MlaA and will initiate small-molecule screens to identify potential inhibitors (see collaboration letter). The PI and GSK are already part of the Innovative Medicines Initiative "Translocation", a consortium of academic labs and big-pharma aiming to understand the fundamentals of small-molecule influx via classical diffusion channels of Gram-negative bacteria. This existing link will facilitate moving the findings from this proposal to the next, impact-driven stage.