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

Secondary Supervisor(s): Professor Andy Lovering

University of Registration: University of Birmingham

BBSRC Research Themes: Understanding the Rules of Life (Microbiology, Structural Biology)

Project Outline

The rise of antibiotic-resistant bacteria presents a serious and escalating global threat, emphasizing the urgent need for new strategies and targets to combat infections. This is particularly concerning in Gram-negative bacteria, where multidrug resistance is a major issue. With few antibiotics in development and limited prospects for new treatments, finding alternatives is crucial.

Gram-negative bacteria are generally more resistant than Gram-positive bacteria, largely due to an additional outer membrane. This outer membrane has a complex lipid structure, with lipopolysaccharides (LPS) on the outer layer and glycerophospholipids (GPLs) on the inner layer. This arrangement forms a highly effective barrier that blocks hydrophilic molecules and slows the penetration of hydrophobic ones, contributing to the bacteria's heightened resistance to antibiotics and detergents. The outer membrane also contains proteins essential for microbial pathogenesis, virulence, and drug resistance, which drive processes involved in infection and disease progression. These outer membrane proteins (OMPs) are vital for maintaining cellular homeostasis, expelling toxic substances like antibiotics and facilitating nutrient uptake.

Understanding outer membrane biogenesis is critical for uncovering new antibiotic targets. A key area of interest is the transport mechanisms responsible for delivering GPLs to the outer membrane. While it has been known for decades that Gram-negative bacteria synthesize and transport GPLs bidirectionally between the inner and outer membranes, the exact mechanisms have remained elusive. Recent studies have implicated several proteins in these processes. Among these are proteins containing Mammalian Cell Entry (MCE) domains, initially identified in mycobacteria but widely present across bacterial species. In E. coli, MlaD, part of the maintenance of outer membrane lipid asymmetry (Mla) pathway, removes mislocalized GPLs from the outer membrane and returns them to the inner membrane, helping maintain membrane integrity. Two other MCE proteins, PqiB and LetB, have also emerged as key candidates for GPL transport between membranes.

The Pqi operon in E. coli contains three genes: pqiA, pqiB, and pqiC. pqiA encodes an integral inner membrane protein, pqiB encodes a periplasmic protein anchored in the IM, and pqiC encodes an outer membrane-associated lipoprotein. PqiB forms a homo-hexamer that co-purifies with GPLs. Together they form a hydrophobic tunnel spanning the periplasm that may facilitate transport. PqiA shows significant homology with LetA, part of a complex with LetB. Like PqiB, LetB contains MCE domains and spans the periplasm, suggesting a similar transport mechanism.

Despite advances in identifying these proteins, the exact mechanisms by which they transport GPLs—whether they exhibit directionality or selectivity, or what drives transport—remain unknown. Moreover, their precise biological roles in the cell are still not fully understood. To address these questions, advanced structural biology techniques, including cryo-electron microscopy and neutron reflectometry, will be used, along with biophysical assays to measure transport and cell-based assays to assess their cellular significance.

Elucidating the mechanisms of outer membrane biogenesis in Gram-negative bacteria - The AsmA family of proteins

Secondary Supervisor(s): Professor Andrew Lovering

University of Registration: University of Birmingham

BBSRC Research Themes: Understanding the Rules of Life (Microbiology, Structural Biology)

Project Outline

The rise of antibiotic-resistant bacteria presents a serious and escalating global threat, emphasizing the urgent need for new strategies and targets to combat infections. This is particularly concerning in Gram-negative bacteria, where multidrug resistance is a major issue. With few antibiotics in development and limited prospects for new treatments, finding alternatives is crucial.

Gram-negative bacteria are generally more resistant than Gram-positive bacteria, largely due to an additional outer membrane. This outer membrane has a complex lipid structure, with lipopolysaccharides (LPS) on the outer layer and glycerophospholipids (GPLs) on the inner layer. This arrangement forms a highly effective barrier that blocks hydrophilic molecules and slows the penetration of hydrophobic ones, contributing to the bacteria's heightened resistance to antibiotics and detergents. The outer membrane also contains proteins essential for microbial pathogenesis, virulence, and drug resistance, which drive processes involved in infection and disease progression. These outer membrane proteins (OMPs) are vital for maintaining cellular homeostasis, expelling toxic substances like antibiotics and facilitating nutrient uptake.

Understanding outer membrane biogenesis is critical for uncovering new antibiotic targets. A key area of interest is the transport mechanisms responsible for delivering GPLs to the outer membrane. While it has been known for decades that Gram-negative bacteria synthesize and transport GPLs bidirectionally between the inner and outer membranes, the exact mechanisms have remained elusive.

A recently described class of lipid transporter, first identified in eukaryotes, has been found to facilitate bulk inter-organelle lipid transport at contact sites by forming bridge-like structures with a hydrophobic groove that allows lipids to pass through. These transporters, characterized by repeating β-groove (RBG) domains in their predicted structure, have been classified as the RBG protein superfamily.

RBG proteins have subsequently been found to be conserved across didermic bacteria. In E. coli, a family of six proteins has been identified showing homology to each other and are referred to as AsmA-like, named after one of their members. They are anchored to the inner membrane via an N-terminal alpha helix and feature a large periplasmic domain containing an RBG domain. This family is now considered likely to be the long-sought after bulk transporters responsible for transferring GPLs from the inner membrane to the outer membrane.

Three members of the AsmA-like protein family—TamB, YhdP, and YdbH—have been shown to be functionally redundant but not equivalent, as their loss results in different phenotypes. However, evidence suggests they all directly facilitate the anterograde transport of GPLs between the IM and OM. The roles of the remaining AsmA proteins (AsmA, YicH, and YhjG) are still unclear, although AsmA has been linked to outer membrane protein assembly.

Despite their apparent importance, the precise molecular functions and mechanisms of action for each AsmA-family protein remain elusive. This project aims to unravel these roles, which could provide insights into bacterial pathogenesis and open new pathways for antibiotic development. The project will be interdisciplinary, incorporating advanced structural biology techniques such as cryo-electron microscopy, X-ray crystallography, and neutron reflectometry, alongside biophysical assay development, molecular dynamic simulations, and cell biology approaches.

Developing an in vitro mimetic of the gram-negative bacterial membrane

Secondary Supervisor(s): Dr Luke Clifton

University of Registration: University of Birmingham

BBSRC Research Themes: Understanding the Rules of Life (Microbiology, Structural Biology)

Project Outline

This project focuses on developing a pioneering in vitro system to study bacterial envelope biogenesis. This system will incorporate many of the facets of the in vivo membrane whilst providing unprecedented scope for studying membrane structure, transport processes, host pathogen interactions and drug screening.

Gram-negative bacteria represent 9 of the 12 priority pathogens identified by the WHO, highlighting the urgent need for new antibiotics to address the growing threat of antimicrobial resistance. One of the most promising targets for antimicrobials development is the bacterial cell envelope. In Gram-negative bacteria, this envelope is a complex and essential structure, composed of two membranes sandwiching a peptidoglycan layer, which together maintain the cell's integrity and shape. The biological processes within this envelope often span both membranes, including multidrug efflux (e.g., AcrB-TolC), secretion (e.g., T3SS), nutrient import (TonB), and membrane biogenesis (Lpt), making them challenging to study. Until now, accurately replicating the Gram-negative envelope in vitro has been impossible, with experimental approaches typically focusing on simpler, single-membrane systems in isolation.

This project focuses on developing a surface based double bilayer mimetic to provide a novel way to study envelope biology.

We have identified a multiprotein complex in E. coli known as PqiABC, which consists of the integral inner membrane protein PqiA, the inner membrane-anchored periplasmic protein PqiB, and the outer membrane lipoprotein PqiC. Together, these proteins form a trans-envelope tunnel that spans and anchors to both the inner and outer membranes. This complex can be reconstituted from its individual components and tethered to a surface. These properties enable the stepwise reconstruction of the double membrane architecture on a planar surface using PqiABC as a scaffold, allowing precise control over its formation.

By introducing a known non-functional variant, PqiABC will provide the ideal scaffold to study multiple trans-membrane biogenesis processes. Given our proposed deposition process, we believe additional components can be readily incorporated at each stage, allowing for the study of other systems

This proposal therefore focuses on developing this system and incorporating and testing multiple trans-envelope systems, for example the Mla pathway, a well-established retrograde phospholipid transport system that includes the outer membrane component MlaA, the periplasmic lipid chaperone MlaC, and the inner membrane ATPase MlaFEDB. Incorporation and activity will be assessed through a combination of quartz crystal microbalance and neutron reflectometry based approaches.

If success we will next assess the limitations of the system, attempting to incorporate more complex intermembrane machineries, for example the entire outer membrane protein biogenesis pathway. This strategy aims to enable real-time monitoring of all stages of outer membrane protein biogenesis. From transport across the inner membrane and periplasmic shuttling via chaperones, to the folding of integral outer membrane proteins at the outer membrane.

There is a critical need to understand outer membrane biogenesis as it is essential for various biological functions, including cellular homeostasis, pathogenesis, and virulence. Outer membrane proteins (OMPs) are central to these processes, yet their transport, folding, and insertion into the outer membrane remain poorly understood. Developing an in vitro OMP biogenesis platform would provide a unique tool to investigate and manipulate OMP biogenesis, offering novel insights into how these proteins are targeted and folded. In the long term, this could lead to new approaches for antimicrobial development.

Targeting the Mla Pathway: A Strategy to Combat Multidrug-Resistant Gram-Negative Bacteria

Secondary Supervisor(s): Professor Liam Cox

University of Registration: University of Birmingham

BBSRC Research Themes: Integrated Understanding of Health (Pharmaceuticals)

Project Outline

The emergence of antibiotic-resistant bacteria poses a serious and growing global threat, emphasizing the urgent need for new targets and strategies to combat infections. Multidrug resistance is particularly concerning in Gram-negative bacteria, with few antibiotics currently in development and limited prospects for new clinical treatments in the near future.

Gram-negative bacteria tend to be more resistant to antibiotics, detergents, and other harmful chemicals than Gram-positive bacteria, primarily due to the presence of an additional outer membrane (OM) surrounding the cell. This outer membrane has a complex lipid asymmetry, with lipopolysaccharides (LPS) in the outer layer and phospholipids in the inner layer, creating a highly effective barrier. This barrier blocks hydrophilic molecules and significantly slows the penetration of hydrophobic molecules, contributing to the bacteria's increased resistance to antibiotics and detergents.

The outer membrane also contains proteins that play essential roles in microbial pathogenesis, virulence, and multidrug resistance, driving many of the processes responsible for infection and disease progression. These outer membrane proteins (OMPs) are crucial for cellular homeostasis, facilitating the excretion of toxic substances like antibiotics and aiding in nutrient uptake.

Understanding the biogenesis of the outer membrane is vital, both as a key biological process and as a potential pathway for developing new antibiotic targets. One such pathway, the Maintenance of outer membrane Lipid Asymmetry (Mla) system (Wotherspoon et al. 2024), is found in all Gram-negative bacteria and contributes to virulence, vesicle formation, and the preservation of the outer membrane’s barrier function. The Mla pathway maintains lipid asymmetry by removing misplaced lipids from the outer leaflet of the outer membrane and returning them to the inner membrane through three protein complexes: the MlaA-OmpC complex in the outer membrane, the periplasmic phospholipid shuttle protein MlaC, and the inner membrane ABC transporter complex MlaFEDB (Malinverni and Silhavy 2009, Hughes et al. 2019).

Leveraging our expertise in structural biology and our focus on antimicrobial resistance, we will concentrate on developing inhibitors for the Mla pathway. Using a combination of computational methods and high-throughput screening, we will identify potential hits. Biophysical and structural biology techniques will help determine binding modes, while synthetic chemistry will be employed to modify and enhance the properties of promising compounds. These candidates will then undergo various in vivo screening studies to assess their efficacy.

References

Hughes, G.W., Hall, S.C.L., Laxton, C.S., Sridhar, P., Mahadi, A.H., Hatton, C., Piggot, T.J., Wotherspoon, P.J., Leney, A.C., Ward, D.G., Jamshad, M., Spana, V., Cadby, I.T., Harding, C., Isom, G.L., Bryant, J.A., Parr, R.J., Yakub, Y., Jeeves, M., Huber, D., Henderson, I.R., Clifton, L.A., Lovering, A.L. and Knowles, T.J. (2019). "Evidence for phospholipid export from the bacterial inner membrane by the Mla ABC transport system." Nat Microbiol 4(10): 1692-1705.

Malinverni, J.C. and Silhavy, T.J. (2009). "An ABC transport system that maintains lipid asymmetry in the gram-negative outer membrane." Proc Natl Acad Sci U S A 106(19): 8009-8014.

Wotherspoon, P., Johnston, H., Hardy, D.J., Holyfield, R., Bui, S., Ratkeviciute, G., Sridhar, P., Colburn, J., Wilson, C.B., Colyer, A., Cooper, B.F., Bryant, J.A., Hughes, G.W., Stansfeld, P.J., Bergeron, J.R.C. and Knowles, T.J. (2024). "Structure of the MlaC-MlaD complex reveals molecular basis of periplasmic phospholipid transport." Nat Commun 15(1): 6394.