Outer membrane protein biogenesis
Gram-negative bacteria contain a double membrane system that acts to protect them from the environment while permitting the selective uptake of nutrients and removal of toxic substances. The outer of these two membranes makes direct contact with the environment and is composed of a lipid inner leaflet, a lipopolysaccharide outer leaflet and harbours two classes of protein, beta-barrel proteins (more commonly known as OMPs for ‘outer membrane protein’) and peripheral lipoproteins (proteins attached to the membrane via a lipid anchor). This membrane make direct contact with the environment and hence is at the frontline of microbial warfare, playing pivotal roles in microbial pathogenesis, virulence and multidrug resistance, mediating many of the lethal processes responsible for infection and disease progression.
The emergence of bacteria that are resistant to available antibiotics represents an enormous and growing global threat requiring new targets and strategies to combat infection. Multidrug resistance is most serious for Gram-negative bacteria, with essentially few antibiotics under development or likely to be available for clinical use in the near future.
Gram-negative bacteria are generally more resistant than Gram-positive bacteria to antibiotics, detergents, and other toxic chemicals because of the presence of an additional membrane surrounding the cell, the outer membrane. This membrane contains a sophisticated asymmetry of lipids with Lipopolysaccharide in the outer leaflet and phospholipid in the inner leaflet. It makes for a highly effective permeability barrier, both by acting as a barrier to hydrophilic molecules but also by acting to slow the penetration of small hydrophobic molecules, explaining their increased resistance to hydrophobic antibiotics and detergents. Whilst the proteins present within the outer membrane are the prime instruments of microbial warfare and play key roles in microbial pathogenesis, virulence and multidrug resistance, mediating many of the lethal processes responsible for infection and disease progression. Outer membrane proteins (OMPs) are also essential for cellular homeostasis allowing excretion of toxic substances, such as antibiotics, and uptake of nutrients.
The research in the laboratory of Dr Tim Knowles is focused on elucidating the mechanisms involved in the fundamental processes of outer membrane biogenesis in Gram-negative bacteria and has several important objectives: (1) to provide fundamental information about how Gram-negative bacteria form. (2) To provide new opportunities to attenuate bacteria in the pursuit of anti-infective strategies. Current antibiotics predominantly target peptidoglycan synthesis and have been very effective in the past. Targeting OM biogenesis offers the potential for a whole new class of antimicrobials urgently required to stay ahead of bacterial resistance.
Projects within this research area include.
Phospholipid transport to the outer membrane
Recently three protein pathways, the Mla, PqiABC and YebST(LetAB) pathways, have been identified that have components in the inner membrane, periplasm and outer membrane and all bind phospholipid suggesting they may be involved in phospholipid transport. How these pathways transport phospholipid and the molecular mechanisms involved in transport still remain to be elucidated however. To answer these questions we are using the latest structural biology techniques including cryo-electron microscopy, X-ray crystallography, nuclear magnetic resonance, neutron reflectometry as well as developing our own novel in house biophysical tools to study these fascinating pathways. Using this approach potential druggable pockets will be identified and allow the identification of compounds that will not only abolish virulence but also impede the restoration of a damaged outer membrane and therefore increase the effectiveness of already available antibiotics.
The Bam complex
A single OM complex, the β-Barrel assembly machine (Bam) complex, has been recognized as essential for the efficient insertion of almost all OMPs into the outer membrane. It is ubiquitous throughout Gram-negative bacteria, however we are only just beginning to understand how it functions. The structure of the complex has been identified but how the components function as part of the complex and how this complex can insert the myriad OMPs targeted to the outer membrane is still to be determined. We are using a multidisciplinary approach, working in both the fields of biophysics and molecular biology to probe the structure of this complex and how it functions. This understanding is critical as the design of compounds that inhibit this process would impede OMP biogenesis and therefore essential physiological, pathogenic and drug resistance functions.
Novel amethods for studying membrane proteins
Our group focuses on two areas:
Membrane protein solubilisation
Working with membrane proteins is technically demanding. The current best technology is the use of detergents which by their very nature often destabilise proteins, inhibit function and decrease sample longevity. Consequently Knowles is developing new technologies to solubilize membrane proteins in the absence of detergents. To date Knowles has developed a styrene maleic acid based system for the solubilisation of membrane proteins, known as SMALP technology, which offers increased stability, longevity and functionality to detergent based systems.
Membrane protein function
Knowles is working with colleagues at the ISIS Neutron and Muon source, Rutherford Appleton laboratory, UK, to develop sensor based systems directly utilising membrane proteins within their phospholipid bilayers atop the sensor surface. These allow a “true to nature” approach to studying membrane protein function including drug screening, receptor ligand interactions and viral-membrane interactions amongst other things.