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Genetic, proteomic and molecular tools to aid in the identification of the mechanism of action novel hit molecules active against Mycobacterium tuberculosis

Principal Supervisor: Professor Gurdyal S. Besra - School of Biosciences

Co-supervisor: Dr Apoorva Bhatt

PhD project title: Genetic, proteomic and molecular tools to aid in the identification of the mechanism of action novel hit molecules active against Mycobacterium tuberculosis

University of Registration: Birmingham

Project outline:

Aim: The identification of the mode of action of a novel natural product possessing whole cell activity against Mycobacterium tuberculosis (Mtb)

Project Objectives: 

  1. To determine the mode of action and molecular target(s) of a novel natural product identified by their whole cell activity against Mtb.
  2. To validate targets identified by genetic and biochemical approaches.
  3. To purify protein targets and develop suitable robust biochemical assays and crystals for structural studies.

Background and outline of proposal: Despite the existence of treatments for tuberculosis (TB), TB continues to present a major healthcare challenge, accounting for nearly nine million new infections and over one million deaths per annum; indeed, this global health threat is increasing given the increased susceptibility of HIV-infected individuals to TB, the limited efficacy of the BCG vaccine and the growing prevalence of drug-resistant strains of Mycobacterium tuberculosis, the microorganism causing the disease. Treating TB requires a drug cocktail given for at least six months, and extending to two years for infections caused by multi drug-resistant strains of M. tuberculosis. A largely successful campaign by the WHO to stem the rise of TB has not diminished the urgent need for better drugs and new cellular targets to alleviate the devastating impact of this disease and this PhD project is within the remit of the BBSRC research areas of industrial biotechnology and world-class bioscience aimed at novel approaches to eradicating bacterial pathogens and antibiotic resistance. The principal aim of the mini-project will be to determine the mode of action of a currently studied natural product antibiotic in our laboratory, which was identified from a Mtb whole-cell phenotypic screening campaign through genetic, chemical proteomic, metabolomic and structural studies.

Experimental Approach: 

  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 [2]. The first approach will involve the isolation of spontaneous resistant Mtb mutants generated against the prioritised hit molecule. 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 [2]. The second approach uses our recently published chemical proteomics methodology to identify the respective target [2]. The third approach involves the development of a novel mass spectrometry-based metabolomics platform [3], 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 labeling in mycobacteria [2].
  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 [2]. The resulting recombinant proteins will be used in in vitro biochemical assays and for structural studies with our phenotypic hits [2].

Expected Outcomes: We will identify a novel drug target and refresh the TB drug pipeline.

References:

  1. Rebello-Lopez, M (2015) PLoS ONE, 10(12):e0142293 2. Cox, J. (2016) Nat. Microbiology DOI:10.1038/NMICROBIOL.2015.6;
  2. Abrahams, K. (2016) Nat. Comm., in press
  3. Prosser, S. (2012) PNAS, 109:11354.

BBSRC Strategic Research Priority: Industrial Biotechnology and Bioenergy

Techniques that will be undertaken during the project:

  1. Mycobacterial culture, microbiology and genetics.
  2. Whole genome sequencing and data analysis.
  3. Protein biochemistry.
  4. LC-MS metabolomics fingerprinting.
  5. Structural biology.
  6. The use of the in-house HTS facility, which will allow screening in specialised in vitro assay formats.

Contact: Professor Gurdyal Besra, University of Birmingham