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Investigation of Resuscitation-promoting factor dependency in Mycobacterium bovis BCG

Principal Supervisor: Dr Galina Mukamolova, Department of Infection, Immunity and Inflammation

Non-academic partner: Public Health England (PHE)

PhD Project Title: Investigation of Resuscitation-promoting factor dependency in Mycobacterium bovis BCG

Project outline:

Tuberculosis remains a major global threat as it affects humans and animals. Several mycobacterial pathogens, which can cause tuberculosis, share unique biological features and the remarkable ability to persist in vivo in a special dormant state. Certain dormant mycobacteria develop Resuscitation-promoting factor (Rpf) dependency, since they can only be resuscitated to actively growing forms by addition of Rpf. These Rpf-dependent mycobacteria can be detected in high numbers in clinical samples and infected animal tissues; they can be also generated in vitro by exposure to antimicrobial agents and nitric oxide (NO). Rpf-dependent mycobacteria are not detectable by standard tools; moreover they are more resistant to major anti-tuberculosis drugs and apparently enriched in treated patients and animals. Therefore Rpf-dependent mycobacteria have been linked to reactivation of latent infection and persister phenomenon.

Rpf-dependent forms are produced by various mycobacteria, including Mycobacterium bovis, the causative agent on bovine tuberculosis. M. bovis is usually detected in cattle by immunological or molecular techniques but not standard cultures, suggesting generation of Rpf-dependent mycobacteria. Furthermore, preparations of M. bovis BCG vaccine may also contain Rpf-dependent bacteria resulting in varying efficacy of vaccination. There needs to be greater understanding of the phenotypes of the different sub-populations in animals and culture and better methods for enumeration of M. bovis and BCG.

Rpf is a family of secreted peptidoglycan hydrolases produced by growing mycobacteria. Enzymatic activity is critical for resuscitation and growth stimulatory effect of Rpf. However, it is unknown why mycobacteria develop Rpf-dependency and how precisely Rpf resuscitate dormant forms.

The proposed project will be focused on investigating molecular mechanisms of Rpf-mediated resuscitation of mycobacteria. It will utilise our recently established model of NO-induced Rpf-dependency model and Mycobacterium bovis BCG.

Objectives and experimental approaches

  1. Enumerating Rpf-dependent M.bovis BCG populations generated under various environmental conditions by application of molecular (mycobacterial load assay) and microbiological techniques (microscopy, CFU and MPN with and without RPF counts).
  2. Measuring Rpf expression during transition to dormancy and resuscitation by utilisation of reporter strains and qRT-PCR.
  3. Determining the RNA expression profiles of bacteria during their transition between dormancy and resuscitation using RNAseq and identifying RPF-dependency and resuscitation transcriptomic signatures.
  4. Assessing the response of Rpf-dependent M. bovis BCG to anti-tuberculosis drugs for development of systems for validation of drugs targeting Rpf-dependent mycobacteria.
  5. Enabling technology transfer these models to M. tuberculosis at PHE-Porton as a means of understanding drug tolerance and providing a relevant bacterial system for the evaluation of drugs to target these populations.

Closing date for applications: 14th January 2018

Interview dates: February 2018 TBC

Check eligibilty and apply