Molecular characterization of suicidal phage-resistance mechanisms in bacteria

About the Project

Importance

The proliferation of antibiotic resistance (AMR) through horizontal-gene-transfer (HGT) is a formidable health challenge, accounting for 700,000 deaths annually1. Although the dissemination of AMR through HGT is described by conjugation in E. coli2, there are other clinically important bacterial families where AMR has come to the forefront due to the emergence of severe drug resistance. Perhaps the most important example is gram+ Mycobacteriaceae, which includes M. tuberculosis (M. tb), responsible for significant infectious disease related mortality and AMR due to the emergence of multi-drug (MDR) and extreme drug resistance (XDR)-TB. MDR/XDR-TB is challenging to treat because M. tb resides deep within granulomas evading classical antibiotics. Consequently, programming bacteriophages to target persistent pathogens has recently gained popularity. Although utilizing phage therapies to treat bacterial infections and counter antibiotic resistance is very promising3, there is a risk of encountering or favouring the emergence of phage resistant/insensitive bacterial pathogens. Despite the promise of bacteriophage-based treatments, molecular basis of bacterial phage resistance remains unexplored.

Background

Bacteria resist phage infections by several strategies, most interestingly as a measure of last resort, by committing suicide upon phage attack by abortive infection (Abi)4. Although a plethora of Abi systems are known, RexA/RexB system5 from E. coli remains the most extensively characterized system where a λ-lysogenic prophage encoded RexAB complex inactivates the infection of T4/5/7 phages. Despite being the first identified Abi system, their mode of action remains enigmatic. The RexA protein is thought to sense a poorly characterized T4-phage protein DNA complex and two copies of RexA activate one copy of RexB, an inner membrane ion channel resulting in severe loss of membrane potential and a drop in ATP-levels, eventually leading to cell death.

Objectives

The overarching vision of this research project is to characterize the molecular processes associated with abortive infection in bacteria with the eventual aim of designing robust phage therapy against infectious diseases. This involves characterizing the RexA/RexB from E. coli using a combination of biophysical and structural methods. The immediate goals in this project are to first study the RexAB system in E. coli and decouple the RexA sensing and RexB membrane integrated ion channel functions by studying them separately using X-ray crystallography and cryo-EM.

The key questions in this project are

(1) How does RexA sense phage-DNA-protein complexes?

(2) How does the RexA-phage-DNA-protein complex activate membrane integrated RexB?

(3) How does RexB activation cause pore formation, cation efflux and membrane depolarization?

Environment

The project combines various biophysical and structural methods, utilizing the facilitates available within the University of Leicester's ecosystem (LISCB and Centre for Phage Research). It also leverages multidisciplinary collaborations across institutions for live-cell imaging and eBIC for cryo-FIB, CLEM, and cryo-ET. Additionally, AI/ML methods within RoseTTAFold will also be used to identify targets for stabilization of protein complexes.

Techniques that will be undertaken during the project

• Generation of recombinants using infusion cloning and homologous recombination.
• Native over-expression and purification of recombinant protein-complexes.
• Membrane-protein purification methodologies (using detergents, amphipols and SMALPs), solubilization and stabilisation procedures (using in-vitro/in-vivo crosslinking methods and ultracentrifugation-based fractionating methods).
• Biophysical characterization of protein-complexes using SEC-MALS, SPR, DLS, ITC.
• Biochemical experiments to characterize phageDNA-RexA complex using DNA-foot printing and electrophoretic mobility shift assay (EMSA) alongwith using co-immunoprecipitation and chromatographic methods to purify the larger complex of phageDNA-RexA-RexB.
• Interactome mapping using co-evolution methods employing HMMER, TrROSETTA, AI/ML-led GREMLIN, Alphafold-2 multimer and characterizing them using western blots and cross-linking mass spectrometry (XL-MS).
• Membrane protein X-ray crystallography, data processing and structure determination.
• Single particle electron microscopy (EM) studies (negative stain and cryo-EM), image processing, low resolution model building and integrative structural biology methods.
• In-situ studies on the RexAB system using CLEM, Cryo-FIB and Cryo-ET.
• Functional studies involving site-directed mutagenesis, in-vitro liposome-based assays, patch clamp electrophysiology, radioactive or fluorescently labelled ion flux assays and live-cell imaging experiments.

Enquiries

Project Enquiries to akv10@leicester.ac.uk

To apply please refer to

https://le.ac.uk/study/research-degrees/research-subjects/molecular-and-cell-biology