Investigating Rpf-dependency in Mycobacteria

Mycobacterium tuberculosis is the causative agent of tuberculosis (TB) in humans and one of the leading causes of death in the world. M. tuberculosis can become dormant, and some dormant bacteria are differentially culturable because they cannot form colonies on agar but resuscitate liquid media. Moreover, resuscitation promoting factors (Rpf), a family of secreted peptidoglycan cleaving enzymes, are required for the resuscitation of certain differentially culturable bacilli. Rpf-dependent M. tuberculosis have been detected in infected mice, individuals with active TB and treated TB patients. These differentially culturable M. tuberculosis bacilli have been linked to TB relapse in mice and humans; however, the precise host factors that trigger Rpf-dependency are currently unknown. The central hypothesis of this study is that nitric oxide (NO), a key signalling molecule, produced by the host during infection, triggered Rpf-dependency in M. tuberculosis.

To test this hypothesis, two novel compounds were synthesised: a NO donor (ND) and a control compound (CC). ND but not CC induced differential culturability in M. tuberculosis and Mycobacterium bovis BCG. The differentially culturable bacteria resuscitated in 7H9 liquid medium, while supplementation with culture supernatant (CSN), obtained from actively growing M. tuberculosis, further improved resuscitation.

Rpf-inhibitor abolished the resuscitation of these bacteria, thus proving that ND generated Rpf-dependent mycobacteria. Furthermore, cell line-based infection models developed in this project revealed that differentially culturable mycobacteria were formed in human THP-1 cells and murine cells (J774A and C57BL/6 bone marrow derived macrophages). Differentially culturable mycobacteria were not detected in iNOS deficient C57BL/6 cells.

To investigate the molecular mechanism of NO-induced Rpf-dependency, the effect of ND on gene expression was evaluated. ND treatment disrupted respiration, lowered the expression of genes involved in cell wall biosynthesis and dramatically downregulated the expression of rpf genes. The findings of this project offer a model for generation of Rpf-dependent M. tuberculosis during infection.