Elucidating the role of mycobacterial Resuscitation-promoting factors in stress response and metal acquisition

Tuberculosis (TB) is an infectious disease caused by members of the Mycobacterium tuberculosis complex. TB remains a global health threat. It has been estimated that one-fourth of the world’s population is infected with Mycobacterium tuberculosis. The bacteria’s ability to enter a dormant state makes it an exceptionally successful human pathogen, and results in latent infection. Resuscitation-promoting factors (Rpfs) are cell wall cleaving enzymes that have been postulated to reactivate dormant bacteria via peptidoglycan remodelling; however, the precise molecular mechanism of resuscitation remains unknown. This project was focused on investigation of the roles of Rpfs in Mycobacterium marinum during growth in normal and stressful conditions. While all Rpfs were dispensable for growth of M. marinum in optimal conditions, rpfA had a specific role in the osmotic stress response and showed impaired growth in media containg 550mM NaCl, with a pronounced growth defect on 7H10 agar. Moreover, the mutant had impaired survival in 2 M NaCl. A series of ΔrpfA strains complemented with M. tuberculosis rpfA were generated to investigate the role of the ydaO riboswitch in osmotic tolerance of M. marinum. The M. marinum mutant with the mutated ydaO riboswitch which could not bind the ligand (cyclic di-AMP) had a profound growth defect on 7H10 agar and impaired survival in 2 M NaCl. RpfA was detected in culture supernatant of M. marinum using western blot and mass-spectrometry approaches.

Several double and one triple rpf deletion mutants were generated and their growth was investigated. Finding of this project suggest that a double rpfAB deletion mutant had a growth defect in Sauton’s medium which was ameliorated by addition of haemin and ZnSO4. Genetic complementation of this growth defect was achieved with either rpfA (from M. tuberculosis or M. marinum) or rpfB from M. marinum, suggesting functional redundancy.

Generation of M. marinum ΔrpfAEB was only possible when selection of mutant was done in media supplemented with haemin and ZnSO4 . The mutant showed delayed colony formation on agar, impaired growth in Sauton’s medium and the inability to reach high optical densities compared to wild type M. marinum. We were able to both genetically and metabolically complement the growth defect of ΔrpfAEB using rpfB, haemin, ZnSO4 and culture supernatant. The need for both haemin and ZnSO4 for generation and complementation of the triple mutant suggests a possible role for Rpfs in metal acquisition. Thus, findings within this project confirmed the distinct roles of M. marinum Rpfs in osmotic tolerance and growth in suboptimal conditions.