Mycobacterium tuberculosis resuscitation-promoting factors and tuberculous reactivation

Abstract

Mycobacterium tuberculosis is a globally important pathogen responsible for a huge burden of human disease, especially in the Developing World. The most common form of tuberculosis is latent infection. Individuals can harbor latent tuberculosis with no symptoms for years or even decades. Though latent infection can reactivate and cause overt disease, relatively little is known about the bacterial factors which are involved in reactivation.;Mycobacterium tuberculosis expresses five resuscitation-promoting factors (Rpfs), proteins believed to be involved in resuscitation from dormancy and cell wall processes, namely peptidoglycan modification. Our laboratory had previously shown that deletion of rpfB impaired reactivation of M. tuberculosis in a mouse model. We therefore hypothesized that Rpfs play a role in the reactivation of latent infection, possibly through modification of the cell wall or regulation of cell wall processes.;A double knockout, DeltarpfAB, displayed altered cell wall morphology on solid media. Although DeltarpfAB grew comparably to the parental strain in axenic culture, in vivo it exhibited deficiency in reactivation in a number of mouse reactivation models. Secondly, DeltarpfAB showed a persistence defect not seen with the DeltarpfB or DeltarpfA single mutants. The reactivation-associated impaired growth is reflected in primary mouse macrophages. Using the in vitro anaerobic Wayne growth model of dormancy, we found that the double knockout has a persistence defect in this system and a reactivation defect upon inoculation from this state into oxygenated fresh media. Scanning electron microscope imaging showed that under stationary phase growth conditions and during the Wayne model, the DeltarpfAB mutant cells display an altered cell surface and cell shape, suggesting that rpfA and rpfB are involved in persistence and reactivation by directly modifying the cell wall and/or are involved in the regulation of cell wall processes and cell growth. Peptidoglycan-induced sporulation has been identified in Bacillus subtilis, involving homologs of Rpfs and PknB, a M. tuberculosis transmembrane kinase involved in cell division. Together, these data imply that Rpfs play a role in the pathogenesis of M. tuberculosis persistence and reactivation. Rpfs therefore may be considered important in drug and vaccine design to combat this disease in which latency and reactivation play such pivotal roles.