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The molecular characterisation of the anti-bacterial properties of marine and soil natural products against zoonotic mycobacterial species

Primary Supervisor: Professor Gurdyal S Besra, School of Biosciences

Secondary supervisor: Dr Apoorva Bhatt

PhD project title: The molecular characterisation of the anti-bacterial properties of marine and soil natural products against zoonotic mycobacterial species

University of Registration: University of Birmingham

Project outline:

Aim: The molecular characterisation of the anti-bacterial properties of marine and soil natural products against mycobacterial zoonotic species.

Background: The proposed PhD studentship will exploit the full power of taking a multi-disciplinary approach to address the rising threat of antibiotic resistance; thus, in employing chemistry and biochemistry, molecular and structural biology, and the latest omics technologies, the results generated will impact on researchers working in all of the above fields providing the characterisation of new chemical entities from marine and natural products as leads against novel targets for antibiotic research.

Objectives:

  1. To determine the mode of action and molecular targets of prioritised natural products identified by their activity against mycobacterial zoonotic species.
  2. To validate targets identified by genetic and biochemical approaches.
  3. To purify protein targets, develop suitable robust biochemical assays and crystals for structural studies with most promising natural products.
  4. Chemical synthesis of potential analogues for biochemical and structural studies.

The success of our approach rests on being efficient strategies for elucidating the cellular targets of identified natural products. In this regard, the main aim of this PhD studentship will to determine the mode of action of selected natural products identified from published phenotypic screening campaigns and repositories, which has provided us with 25 novel compounds for mode of action studies. We will prioritise a selection of these molecules for our proposed studies based on their reported in vivo and in vitro activities. This multi-disciplinary project will therefore define new drug targets and lead-molecules at UoB, and identify molecules for further optimisation studies. Simultaneously, this will also provide new insights into fundamental microbial biology, as we've shown previously for EchA6 and KasA [1, 2].

Experimental Methods and Research Plan:

  1. Target identification: We plan to take three parallel approaches, the first two of which we have successfully applied to identify target(s) of novel hits [1-3]. The first approach will involve the isolation of spontaneous resistant mutants generated against the prioritised natural product. Whole genome sequencing will be used to identify SNPs or InDels that are potentially responsible for resistance. Through a series of further genetic and biochemcial studies, the target will be identified [1-3]. The second approach uses our recently published chemical proteomics methodology to identify the respective target [1]. The third approach involves the development of a novel mass spectrometry-based metabolomics platform, which would allow one to monitor directly the metabolic flux of specific target inhibition through a whole cell phenotypic hit.
  2. Target validation: For this purpose we will use CESTET, a tool for testing gene essentiality and studying loss of gene function through metabolic labelling in mycobacteria [1].
  3. Purification of protein targets: For biochemical and structural studies we will express and purify the proteins using a variety of expression vectors and cell lines [1-3]. The resulting recombinant proteins will be used in in vitro biochemical assays and for structural studies with our phenotypic hits [1-4].
  4. Chemical synthesis of analogues: Selected leads will be optimised through the generation of a small array of molecules for subsequent evaluation in our proposed studies.

Expected Outcomes: We will identify novel molecules and their respective drug targets and refresh the antibiotic pipeline.

References:

  1. Cox, J. (2016) Nature Microbiology 1:15006.
  2. Abrahams, K. (2016) Nature Communications 7:12581.
  3. Burke, C. (2019) FASEB Bioadvances 1: 246.
  4. Zhang, L. et al. (2020) Science 368(6496): 1211, eaba9102.

BBSRC Strategic Research Priority: Understanding the Rules of Life: Microbiology

          Techniques that will be undertaken during the project:

          • Growth of bacterial cultures: E. coli and Mycobacterium spp.
          • Molecular biology, gene expression experiments, including construction of plasmids, recombinant proteins and construction of mutants.
          • Protein biochemistry: ligand binding assays and the construction of mutants.
          • Structural biology: crystallography and cryo-EM.
          • Chemical biology: analysis of mutants.
          • Chemical synthesis: structure-activity-relationship of hits.

          Contact: Professor Gurdyal S Besra, University of Birmingham