Characterising macrophage plasma membrane signatures to detect and target intracellular Mycobacterium tuberculosis

This studentship is supported by the Aston Institute for Membrane Excellence. AIME is a unique, interdisciplinary, intersectoral research and training hub for translational membrane science. AIME is a globally unique, cross-disciplinary institute to develop novel membranes for use in applications as varied as drug discovery and water purification. The team behind AIME believes that the full potential of membranes will only be realised by a research team spanning biology, physics and chemistry that can investigate membranes holistically. No other institute has the platform, potential or promise for major breakthroughs in this area. The vision is for AIME to become a ‘one-stop shop’ for interdisciplinary, translational membrane research through access to its facilities and expertise, ideally located in the heart of the UK.

Intracellular pathogens are difficult to detect and treat as they hide within, and reprogramme our own cells, creating a replicative niche by changing the protein and lipid composition of its host. One of the most successful, and deadliest pathogens is Mycobacterium tuberculosis (Mtb), which causes Tuberculosis (TB) disease, infecting 1.4 billion people and killing over 1.2 million people a year.

One way to detect these hidden intracellular pathogens is to characterise unique changes to the protein and lipid composition of the host cell’s plasma membrane (PM). To date, no study has fully elucidated the PM changes in protein and lipid signatures of human macrophages during Mtb challenge, or how these changes modulate bacterial infection. Preliminary work has detected an upregulation of a PM lipid receptor for oxidised low-density lipids (oxLDL) on macrophages infected with Mtb. With intracellular oxLDL associated with lysosomal dysfunction and increased Mtb survival(1) this and other biomarkers will represent targetable proteins to modulate intracellular infections.

This study will lead to the discovery of accessible host biomarkers for detection, and crucially, therapeutic targeting of intracellular infections.

Objectives and Methods

This project will isolate plasma membrane proteins and lipids of macrophages infected with Mtb for proteomic and lipidomic analysis. Characterising the infected host’s PM signatures will allow better detection of these infected cells, and therefore, the intracellular bacteria through fluorescent microscopy or flow cytometry.

Protein abundance is a powerful metric for detecting biomarkers, but to generate functional information and therapeutic targets, understanding protein conformational changes is vital. For this, the project will utilise styrene maleic acid lipid-particles (SMALPs). SMALPs preserve membrane embedded proteins in their native environment compared to detergent based extraction methods, and have been demonstrated previously for both host, and bacterial membrane proteins (2,3). Previous work has utilised model cell lines, this project will optimise and apply these methods to primary human macrophages infected with a BSL-2 strain of Mtb for host-pathogen interactions.

Whether targeting these PM biomarkers is beneficial for the host or pathogen will be tested using therapeutics against key protein and lipid hits. Any changes in survival to the host or bacteria will be quantified by time-lapse microscopy of host-pathogen co-cultures. Additionally, Mtb is known to improve its intracellular survival by modulating its metabolism after drug treatment or during host-specific stresses. To understand this metabolic rewiring, the project will employ RNA extraction methods to assess metabolic activity of intracellular Mtb.

Overall, this project will lead to new PM signatures of infected host cells, improving detection and therapeutic targeting.

1. Vrieling et al., (2019). Oxidized low-density lipoprotein (oxLDL) supports Mycobacterium tuberculosis survival in macrophages by inducing lysosomal dysfunction. PLoS Pathogen. doi: 10.1371/journal.ppat.1007724

2. Routledge et al., (2020). Ligand-induced conformational changes in a SMALP-encapsulated GPCR. Biochimica et Biophysica Acta (BBA) – Biomembranes. doi: 10.1016/j.bbamem.2020.183235

3. Teo et al., (2019). Analysis of sMALp co-extracted phospholipids shows distinct membrane environments for three classes of bacterial membrane protein. Scientific Reports. doi: 10.1038/s41598-018-37962-0

Person Specification

The successful applicant should have been awarded, or expect to achieve, a Masters degree in a relevant subject with a 60% or higher weighted average, and/or a First or Upper Second Class Honours degree (or an equivalent qualification from an overseas institution) in a relevant subject. Some tissue culture experience using mammalian or bacterial cells, and fluorescent techniques (microscopy or flow cytometry) would be ideal. This project would suit someone with a keen interest in interdisciplinary work with a focus on host-pathogen interactions.

Details on how to apply can be found here: Funded Studentships nearmejobs.eu Aston University