Antimicrobial resistant (AMR) micro-organisms cause a major global threat to modern medicine, resulting in over 700,000 deaths annually. Gram-negative, double-membraned bacteria (such as Escherichia coli) are particularly difficult to treat, in part because they have an outer membrane (OM) that acts as a barrier against antibiotics. Therefore, it is critical to understand how bacteria build and maintain their OM, potentially allowing for novel therapeutic approaches that disrupt this barrier.
The Gram-negative bacterial cell envelope consists of an inner membrane (IM) and an OM separated by an aqueous periplasmic space. To build and maintain this OM, bacteria must transport lipids and hydrophobic proteins across the aqueous periplasm. Increasingly, evidence suggests that membrane components are transported via protein pathways that form shuttles or direct bridges between the IM and OM (Ekiert et al., 2017, Isom et al., 2020, Coudray et al., 2020). Understanding how these proteins function provide new insights into how bacteria build the OM, and potentially enable us to design new drugs targeting this membrane.
This PhD project will take place in the newly established research group of Dr Georgia Isom, at the Sir William Dunn School of Pathology. The group focuses on understudied proteins/protein complexes that are implicated in transport of OM components in E. coli (Ekiert et al., 2017, Isom et al., 2020, Coudray et al., 2020). This project will give you the opportunity to learn state-of-the-art techniques in structural biology, biochemistry and genetics, enabling you to answer some of the following important questions:
1) How do proteins that transport OM components function mechanistically? To address this we will use a combination of cryo-EM and X-ray crystallography to solve the structure of proteins/protein complexes.
2) What are the substrates of these proteins? Understanding which molecules the proteins interact with will allow us to determine what exactly they are transporting (e.g. lipids or proteins). To investigate this we will use a combination of biochemical approaches including protein-substrate binding assays, mass spectrometry, and in vivo crosslinking.
3) What are the phenotypes of bacteria that lack these proteins? We will construct gene knockouts in E. coli and assess changes in cell morphology, sensitivity to OM stresses/antibiotics, and OM lipid and protein composition.