Elucidating the mechanisms of outer membrane biogenesis in Gram-negative bacteria

Project Outline

Projects within the Knowles laboratory focus on the biophysical characterisation of the mechanisms involved in outer membrane biogenesis in Gram-negative bacteria with the overall aim of identifying novel targets for the development of the next generation of antimicrobials.

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 (OM). This membrane contains a sophisticated asymmetry of lipids with LPS 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 OM biogenesis in Gram-negative bacteria and has several important objectives: (1) to provide fundamental information about how Gram-negative bacteria form and therefore by proxy mitochondria and chloroplasts. (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 are available in two research areas.

Focus - Phospholipid transport to the outer membrane

Recently three protein pathways, the Mla, PqiABC and YebST 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. Indeed our recent research in to the Mla pathway (Nature Microbiology) has shown that this pathway can function to transport phospholipids towards the outer membrane and is the first evidence of a phospholipid transport pathway to the outer membrane in Gram-negative bacteria. 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 OM and therefore increase the effectiveness of already available antibiotics.

Focus - 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 OM. It is ubiquitous throughout Gram-negative bacteria, however little is known about how it functions. The structures of the individual components have been identified but little information about how the components function as part of the complex nor how this complex can insert the myriad OMPs targeted to the OM has been 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.

References

Hughes, G, et al. (2019), Evidence for phospholipid export from the bacterial inner membrane by the Mla ABC transport system Nature Microbiology. doi: 10.1038/s41564-019-0481-y [epub ahead of print]

Browning, D.F., et al., Cross-species chimeras reveal BamA POTRA and beta-barrel domains must be fine-tuned for efficient OMP insertion. Mol Microbiol, 2015. 97(4): p. 646-59.

Knowles, T.J., et al., Structure and function of BamE within the outer membrane and the beta-barrel assembly machine. EMBO Rep, 2011. 12(2): p. 123-8.

Techniques

This project requires the utilisation of numerous biophysical and molecular biology techniques to answer the key questions posed. Cutting edge biophysical techniques will be used such as neutron scattering, reflectometry and detergent free SMALP methods, and will require working with collaborators at national facilities such as ISIS, RAL,UK and producing substantial bodies of data that will require extensive computer analysis, fitting and modelling. To complement the biophysical analysis, molecular biology will be required to probe the effects of mutagenesis, gene knockout and suppressor mutation on phenotype and thus it is likely that a systems biology approach will be used to understand the role specific mutations make to the phenotype.

Structure and function of Batten disease proteins

Project Outline

The neuronal ceroid lipofucscinoses are a group of inherited neurodegenerative disorders with primarily childhood onset. The most common form, also known as Batten disease or CLN3 disease, is caused by recessively inherited mutation in the CLN3 gene. This encodes a multispan protein that appear to regulate vesicular trafficking but its exact functions are not well understood.

The CLN3 gene is predicted to encode a multi-spanning transmembrane protein but it does not conform to any known structure: potentially it represents a novel protein fold. If the structure was to be solved, it would indicate likely mechanisms of action and disease-causing mutations identified in Batten disease patients could be mapped to the 3D structure to highlight the key functional regions of the protein. This project aims to solve the structure and elucidate the function of CLN3.

The student will express and purify CLN3 from human cells. CLN3 will be engineered to include affinity tags in predicted loops, based on preliminary studies. The protein will then be purified from cells and characterised by a battery of analytical tools, including analytical ultracentrifugation and circular dichroism, before structural analysis by cryoEM.

Key mutations will be incorporated into the CLN3 gene by site-directed mutagenesis, these mutations, based on structure/known naturally occurring mutations, will provide key information on how the protein functions.

To complement the structural studies, we will employ proximity labelling to identify the protein partners of CLN3 in complexes within the membranes of living cells. This will shed significant light on how CLN3 operates in cells. Crucially, proximity labelling allows protein complexes to be identified in situ while the cells are intact and living. There is no need to lyse the cells with detergents, which is likely to disrupt protein complexes. We have used it successfully for other membrane proteins, including another neuronal ceroid lipofuscinosis protein, CLN7.

In this project, the student will use CRISPR/Cas9 gene editing to incorporate a proximity labelling enzyme into the CLN3 gene at a site unlikely to disrupt the structure in cultured human cells. This will by-pass the need to overexpress the CLN3 gene and prevent excessive labelling and false identification of non-CLN3-interacting proteins. Cell biology and protein biochemistry methods will then be used to isolate the proteins found directly adjacent to CLN3 in the membranes of live cells and mass spectrometry to identify the protein partners.

That mutations in CLN3 that cause Batten disease have been known for more than 25 years yet the function of the CLN3 protein is still not properly understood is astounding. Together, this two-pronged approach will provide key structural understanding of the Cln3 protein, potentially highlighting a novel protein fold, but also help to elucidate the function of this protein within the cell and identifying any partners it may bind to.

Techniques

Cell biology, structural biology, cryoEM, gene editing, protein biochemistry, mass spectrometry.