Structural and functional characterisation of the Pseudomonas aeruginosa lipid-modifying translocase MprF

Thesis

The multiple peptide resistance factor (MprF) is a bifunctional membrane protein found in the cytosolic membrane of many Gram-positive and Gram-negative bacteria, including pathogens such as Pseudomonas aeruginosa and Staphylococcus aureus. MprF modifies inner leaflet lipid headgroups through aminoacylation, and translocates modified lipid to the outer leaflet, resulting in a reduction in anionic character of the bacterial surface. The altered surface charge provides resistance to cationic antimicrobial peptides (CAMPs) and commercial positively charged antimicrobials. MprF presents a novel target to combat antimicrobial resistance, but structural information is limited, especially for MprF from common clinical pathogens. Moreover, the substrate specificity and energisation of lipid transport by MprF are yet to be characterised.

In this thesis, I structurally and functionally characterised the P. aeruginosa MprF (PaMprF). I determined the structure of PaMprF by cryo-electron microscopy (cryo-EM) to 3.28 Å using a nanobody to increase particle size and improve sample behaviour. Comparison of PaMprF to a known MprF structure from Rhizobium tropici (RtMprF) reveals significant structural differences and suggests novel routes of lipid transport. Furthermore, I established an in vitro lipid transport assay to probe the mechanism of PaMprF-mediated lipid translocation. With this assay - the first application of a direct in vitro lipid transport assay to the MprF family - I demonstrated that PaMprF does not require an external energy source and can transport a wide range of different lipid species. My data indicate that PaMprF is a lipid scramblase, which is contrary to previous suggestions. By combining the lipid transport assay with site-directed mutagenesis, I investigated possible lipid transport pathways through PaMprF, resulting in novel hypotheses for its mechanism of action.

Authors: Hankins, MTK

• DOI: 10.5287/ora-er7bmm567
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English