Structure and mechanism of the Mla lipid transport system in the multidrug-resistant bacterium A. baumannii

In recent years, we have observed a dramatic increase in "superbugs", bacteria that are resistant to all known antibiotics. This is a particularly important worry in hospital settings, where superbugs cause untreatable infections in patients undergoing routine medical procedures. More than 20,000 people die from superbug infections every year.
In many bacteria, this drug resistance is caused by a thick lipid later (the outer membrane) on their surface. This membrane acts as a physical barrier, preventing many drugs from entering the bacterial cell. The lipid composition of this outer membrane is very specifically regulated, as it is critical for its physical integrity. However, the mechanisms used by bacteria to transport lipids to and from the outer membrane are still poorly understood.
Recently, a set of proteins termed the Mla system (for Maintenance of Lipid Asymmetry) was shown to be essential to maintain an intact outer membrane. The aim of this project is to characterize the molecular basis for lipid transport by this system. Specifically, we will study the Mla system from the bacterium Acinetobacter baumannii, a known multi-drug resistant bacterium responsible for up to 20% of antibiotic-resistant infections world-wide.
We will study how the different Mla proteins interact with each other, and how they recruit and transport lipids. In particular, we will exploit the latest advances in high-resolution electron microscopy to characterize these proteins at the atomic level.
This will allow us to understand how the proteins from the Mla system recognize lipids and transport them to and/or from the outer membrane. Ultimately, this could be exploited for the development of therapeutics that prevent antibiotics resistance.

In many gram-negative bacteria, the outer membrane is responsible for broad-range antibiotics resistance. In particular, in the multi-drug resistant pathogen Acinetobacter baumannii, outer membrane integrity is critical for the lack of effect of many common antibiotics. Therefore, characterizing the molecular basis for outer membrane integrity in this bacterium is of high medical importance.
Recently, a transport system responsible for maintaining the outer membrane lipid composition has been identified in E. coli. This Mla system (for Maintenance of Lipid Asymmetry) is very unusual, as it contains a canonical ABC-type transporter, ATPase and periplasmic binding protein, but also several auxiliary proteins of largely unknown function, not found in other ABC transporters. Several Mla proteins have been shown to bind to phospholipids, but the directionality and mechanism of transport remains elusive.
The aim of this proposal is to characterize the molecular mechanism of phospholipid transport by the Mla system, in the human pathogen A. baumannii. Specifically, we will determine the structure of the core MlaBDEF complex, in the apo, substrate- and ATP-bound states. This will allow to establish the directionality for lipid transport, and reveal the role of the auxiliary proteins MlaB and MlaD. In addition, we will characterize the interplay between the three periplasm-located components, MlaA, MlaC and MlaD, and their interaction with phospholipid molecules. This will allow us to propose a mechanism for lipid transport between membranes in gram-negative bacteria, and could be exploited for the screening of small molecule inhibitors acting as antibiotics adjuvants.

This proposal focuses on the characterization of the Mla lipid transport system in the wide-range antibiotic resistant Acinetobacter baumannii. This system plays a major role in the bacterial cell architecture, and is a potential target for new antibacterial therapeutics. This project will provide a number of benefits to the pharmaceutical industry and the general public in the UK and abroad:

Industry:
As a potential antibiotic adjuvant target, structural characterization of the Mla system could be exploited for the design of novel therapeutics. Through collaborations with the Sheffield Science Gateway, we will develop collaborations with pharmaceutical companies that could lead to new antimicrobial drugs.

National Health Services:
Antibiotic resistance it a global health challenge, and a major priority for the NIH. It also poses a significant economic burden on health services. In particular, multi-drug resistant A. baumannii infections in hospital environments are increasing, and the Mla system directly contributes to this problem. By providing potential new avenues for the development of antibacterial drugs, this project will contribute to helping the NHS in its mission to improve the health and wellbeing of patients and communities.

Sheffield and the North Yorkshire:
One of the major cause for antibiotic resistance is the over-consumption of antibiotics. Raising awareness of this problem among the general public is essential in the fight against excess antibiotics prescription. The outreach activities associated with this project will contribute to raise awareness of this important issue in the communities at Sheffield and the region.

Students and staff at the University of Sheffield:
Membrane proteins are notoriously challenging to study, yet about 50 % or existing drugs target membrane proteins. This project will help increase the current pool of expertise in membrane protein biochemistry at the University of Sheffield. In addition, a number of undergraduate and postgraduate students will benefit from being involved with the project, and acquire cutting-edge structural biology techniques. Finally, the PDRA associated with the project will receive many opportunities for scientific training, and well as for acquiring transferable skills.