Revival of the Immortals: investigation of molecular mechanisms for mycobacterial resuscitation from dormancy.

Mycobacterial pathogens possess a variety of defences against host-imposed and environmental stressors, making them of high clinical importance. One such mechanism is their ability to enter a differentially culturable state to survive prolonged exposure to NO. In this state, they cannot be grown in standard media and require resuscitation by resuscitation-promoting factor (Rpf) - a murolytic transglycosylase - to resume normal growth. Previous work has shown that Rpf plays an important role in growth, regrowth from dormancy and virulence in Mycobacterium bovis and M. tuberculosis. Furthermore, enzymatic activity of Rpf is essential for resuscitation from NO-induced dormancy; muropeptides cleaved from peptidoglycan by RpfB and RipA revive dormant mycobacteria. NO-treated mycobacteria show a great reduction in expression of rpfA, rfpB and rfpE. Rpf inhibitors then completely prevent resuscitation. However, the molecular mechanisms of Rpf-mediated resuscitation remain unknown. To elucidate this, we will develop a high-throughput resuscitation assay to screen the M. bovis BCG transposon library developed by Dr Moynihan. Mutants showing impaired regrowth after NO-treatment and resuscitation will be identified and analysed through a combination of phenotypic analysis and bioinformatics approaches to establish the key factors involved in Rpf-mediated resuscitation. These will be further assessed by generation and characterisation of complementation mutants. Understanding of these molecular mechanisms is critical for developing strategies against latent bovine and human tuberculosis, which is likewise essential for the eventual elimination of the diseases.