The role of resuscitation promoting factors in peptidoglycan hydrolysis and reactivation from dormancy in Mycobacterium smegmatis

The global burden of tuberculosis (TB) has reached an alarming status with numerous countries reporting an incidence of greater than 300 cases per 100 000 population per capita. By far, the most concerning statistic regarding TB is that estimates indicate 2 billion people worldwide are infected with Mycobacterium tuberculosis, the causative agent of TB. Only a small proportion (10%) of these individuals progress to active disease whilst the majority control the infection in an asymptomatic state that is termed latent TB infection (LTBI). These individuals carry a lifetime risk, which is significantly enhanced in the presence of HIV co-infection, of progressing to active disease. It has been hypothesized that during LTBI, the infecting bacterial population adopts a non-growing, dormant state and that progression to active disease is a result of reactivation of these organisms from dormancy. M. tuberculosis encodes for 5 homologues for the resuscitation promoting factor (Rpf), designated rpfA-E which have been shown to be important for reactivation from dormancy and critical for virulence during TB infection. As such, these factors may play an important role in bacterial reactivation during LTBI. In this study, we further characterize Rpf function in Mycobacterium smegmatis, a non-pathogenic relative of M. tuberculosis. M. smegmatis encodes four rpf–like homologues and using sequential deletion mutagenesis, we constructed a panel of deletion mutants that were defective in one, two, three or all four rpf-like genes in M. smegmatis. The successful construction of a quadruple mutant lacking all four rpf-like genes demonstrated that, as observed in M. tuberculosis, the rpf-like genes are collectively dispensable for growth of M. smegmatis. A select group of rpf deficient mutant strains were then characterized further. Our analysis indicates that the double ΔrpfA ΔrpfB, triple ΔrpfA ΔrpfB ΔrpfC, and quadruple ΔrpfA ΔrpfB ΔrpfC ΔrpfE, mutants displayed no growth defects in vitro but were impaired for biofilm formation and formed abnormal colonies that lacked the surface cording which is characteristic of mycobacterial colonies. Genetic complementation reversed these defects. Furthermore, the triple ΔrpfA ΔrpfB ΔrpfC, and quadruple ΔrpfA ΔrpfB ΔrpfC ΔrpfE, mutants displayed increased susceptibility to vancomycin, erythromycin and cephalosporins, suggesting some change in peptidoglycan structure or composition. Microscopic analysis of rpf deletion mutants revealed that the double ΔrpfA ΔrpfB, triple ΔrpfA ΔrpfB ΔrpfC, and quadruple ΔrpfA ΔrpfB ΔrpfC ΔrpfE, mutants displayed cells that were smaller than the wild type. In an interesting development, analysis of growth and survival in an in vitro model of mycobacterial dormancy indicated that the triple ΔrpfA ΔrpfB ΔrpfC, and quadruple ΔrpfA ΔrpfB ΔrpfC ΔrpfE mutants were unable to enter into a viable but non-culturable state. These results point to a novel role for Rpfs in the establishment of the dormant state in mycobacteria. Transmission Electron Microscopy on cells isolated from the dormancy model confirmed the progressive accumulation of inclusion bodies in the double ΔrpfA ΔrpfB, triple ΔrpfA ΔrpfB ΔrpfC, and quadruple ΔrpfA ΔrpfB ΔrpfC ΔrpfE mutants as these stains were passaged through our model of dormancy. Further analysis with Nile Red staining identified these inclusion bodies as lipid bodies. Collectively, our data reveal an important role for Rpfs in regulating essential growth processes and reveal a new role for Rpfs in the ordered shutdown of bacterial growth and establishment of dormancy in M. smegmatis