Project No.2249
Primary Supervisor
Dr Mark Shepherd – University of Kent
Co-Supervisor(s)
Prof Mark Wass – University of Kent
Dr. Simon Waddell – University of Sussex
Summary
Rationale: Antimicrobial resistance remains, ‘one of the most urgent health threats of our time’, accelerated by indiscriminate antimicrobial use during the COVID pandemic
Tuberculosis accounts for 1 in 3 deaths from antimicrobial resistance. New approaches are urgently needed. The overarching goal of this project is to repurpose existing drugs and identify novel inhibitors to target bacterial protein complexes that are important during infection. The protein complex of interest is the cytochrome bd complex, which is found in a broad range of bacterial pathogens and is not present in humans, making this an excellent choice of drug target. This work is particularly timely given the recent publication of the structures of cytochrome bd from E. coli and Mycobacterium tuberculosis.
Approaches and preliminary work
The Shepherd and Wass labs have developed a computational pipeline for in silico drug screening and have identified hundreds of compounds (from thousands of molecules in a number of libraries) that are likely to target cytochrome bd. Additionally, the host lab has a variety of key mutant strains that are required for drug efficacy assays, which include oxygen electrode measurements, viability assays, and a recently-developed high-throughput fluorescence technique to measure oxygen consumption. The Waddell lab has extensive expertise in Mycobacterium tuberculosis antimicrobial susceptibility testing and the investigation of drug mode of action using transcriptomic and genomic approaches.
The student will employ in silico screening approaches to identify hits and experimentally quantify compound efficacy using the aforementioned suite of techniques. Resistance-causing mutations will also be investigated. We will focus on multidrug-resistant E. coli and M. tuberculosis with scope to expand to other ESKAPE pathogens.
Potential Impact
This work has the potential for transformative change in the treatment of infectious diseases, discovering new inhibitors and repurposing existing drugs to treat bacterial infections that are resistant to all conventional antibiotics.
Training
The student will receive training on in silico drug screening approaches using the molecular docking pipeline, an automated Bash Script approach powered by Autodock Vina that is currently used in the Shepherd lab. Drug susceptibility will be validated using a combination of growth and viability testing on whole cells, along with a direct assay of aerobic respiration in isolated membranes using the Clark type oxygen electrodes in the host lab. Specific respiratory complexes can be interrogated by genetic engineering of the bacterial strain such that it expresses single respiratory oxidases: to be achieved using expression of plasmid-borne recombinant complexes and the l-Red mutagenesis approach that is currently used in the host lab.
At the University of Sussex, the student will be trained to work safely with pathogenic bacteria in a CL3 laboratory and learn mycobacterial culture techniques and antimicrobial drug susceptibility assays. The student will also be trained in RNA extraction, and genomics and transcriptomics (RNAseq, NanoString) approaches to investigate drug mode of action. The Shepherd lab offers expertise in several omics technologies to further support this work.
Publications and relevant techniques
Ribeiro CA, Rahman LA, Holmes LG, et al (2021) Nitric oxide (NO) elicits aminoglycoside tolerance in Escherichia coli but antibiotic resistance gene carriage and NO sensitivity have not co-evolved. Arch Microbiol 203:2541–2550. (Antimicrobial susceptibility testing, genomic analysis of clinical isolates).
Shepherd M, Achard MES, Idris A, et al (2016) The Cytochrome bd-I terminal oxidase of uropathogenic Escherichia coli augments survival during infection. Sci. Rep. 6:35285. (Chromosomal engineering of bacterial clinical isolates using l-Red mutagenesis).
Holyoake, L. V., Hunt, S., Sanguinetti, G., Cook, G. M., Poole, R. K. and Shepherd, M. (2016) CydDC-mediated reductant export in Escherichia coli controls the transcriptional wiring of energy metabolism and combats nitrosative stress. Biochem. J. 473, 693-701. (Spectroscopic quantitation of respiratory complex assembly in whole cells).
Antczak M, Michaelis M*, Wass MN* (2019). Environmental conditions shape the nature of a minimal bacterial genome. Nat Commun 2019;10:3100 (Functional annotation of proteins encoded by the minimal bacterial genome (Use of a diverse range of in silico methods).
Pollo LAE, Martin EF, Machado VR, Cantillon D, Wildner LM, Bazzo ML, Waddell SJ, Biavatti MW and Sandjo LP (2021). Search for antimicrobial activity among fifty-two natural and synthetic compounds identifies anthraquinone and polyacetylene classes that inhibit Mycobacterium tuberculosis. Frontiers in Microbiol; 11:622629. PMID: 33537021. PMCID: PMC7847937. (M.tuberculosis culture, antimicrobial drug sensitivity testing).
Maitra A, Evangelopoulos D, Chrzastek A, Martin LT, Hanrath A, Chapman E, Hailes HC, Lipman M, McHugh TD, Waddell SJ, Bhakta S (2020). Carprofen elicits pleiotropic mechanisms of bactericidal action with the potential to reverse antimicrobial drug resistance in tuberculosis. J Antimicrob Chemother; 75(11):3194-3201. PMID: 32790867. PMCID: PMC7566368. (M.tuberculosis microbiology, antimicrobial drug sensitivity testing, transcriptomics and investigation of mutations to define mechanism of action).