Multidrug resistance (MDR) has been a significant problem for decades and limits the use of available chemotherapeutic drugs in treatments of bacterial infections and cancers. One of the key reasons for the development of MDR is the expression of polyspecific drug efflux pumps in cells. MsbA is an ATP-binding cassette (ABC) protein in Gram-negative bacteria that can mediate the efflux of a wider range of antibiotics but also mediates the flopping of phospholipids and Lipid-A core across the plasma membrane. The MsbA protein has a similar fold as the mammalian multidrug resistance P-glycoproteins (P-gp, ABCB1) and might share functional features and substrate transport mechanisms with ABCB1. Furthermore, MsbA’s Lipid-A transport in Gram-negative bacteria is vital for the biogenesis and maintenance of the outer membrane, which, in turn, is essential for the survival of the cell. Therefore, this protein is a potential target for novel antibacterial agents against pathogenic Gram-negative bacteria. The structural and functional properties of MsbA have been studied over the past two decades. Crystal structures of MsbA have revealed different transporter conformations. Recent cryo-EM structures provide molecular insights into the ability of MsbA to transport Lipid-A. However, due to limitations in the available biochemical assays, the detailed mechanisms and the energetics requirements of lipid transport by MsbA remain unclear. In this PhD project, I developed an in-vitro lipid transport assay for phospholipids with unmodified, long-acyl chains as well as for the hexa-acylated Lipid-A. To track these lipids during the transport reaction, they contain a biotin tag on the lipid headgroup. This novel lipid transport assay improves previous methods using water-soluble phospholipid analogues labelled with a fluorescent nitrobenzoxydiazole moiety on a short acyl chain. Using my method, I directly demonstrate the transport of phospholipid, and Lipid-A by MsbA for the very first time. This thesis will then focus on exploring the energetics requirements of lipid transport by MsbA. A previous study suggested that the ATP-dependent transport of ethidium, chloramphenicol and erythromycin by MsbA is stimulated by the chemical proton gradient. When testing the energetic requirements of phospholipid transport by MsbA, I found that the proton gradient is also important for achieving significant rates of ATP-dependent phosphatidylethanolamine transport. In contrast, Lipid-A transport is active with the input of ATP only. Although chemical proton gradient does not stimulate Lipid-A transport, the membrane potential does enhance the transport reaction. Finally, this thesis will focus on the transport pathways for drugs and lipids in MsbA. Mutagenesis work was carried out to prove that the central binding cavity is shared in the binding of small molecules, phosphatidylethanolamine, and Lipid-A. Due to the differences in chemical structures of the substrates, they interact with different amino acid residues in the same binding cavity. In conclusion, this thesis describes the use of a novel transport assay in the exploration of the pathways and energetic requirements of lipid transport by MsbA. By comparing the results from these functional studies with data on drug transport as well as the available structural information for this transporter, a more comprehensive model for the mechanisms of lipid and drug transport by this important ABC exporter is proposed. The substantive part of the work described in this thesis is published. Specifically, the major results related to MsbA described in Chapter 3 and 4 are published on Communications Biology (1), and the major results related to the complexity of Hoechst 33342 described in Chapter 4 are published on Scientific Reports (2).

1. D. Guo et al., Energetics of lipid transport by the ABC transporter MsbA is lipid dependent. Communications Biology 4, 1379 (2021).
2. B. M. Swain et al., Complexities of a protonatable substrate in measurements of Hoechst 33342 transport by multidrug transporter LmrP. Scientific Reports 10, 20026 (2020).