Sterilization by adaptive immunity of a conditionally persistent mutant of mycobacterium tuberculosis

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, can enter into a persistent state that confers resistance to antibacterial agents. Many observations suggest that persistent M. tuberculosis cells also evade the antimycobac-terial immune mechanisms, thereby reducing the effectiveness of the current tuberculosis vaccine. Understanding the factors that contribute to persistence may enable the rational design of vaccines that stimulate effective immune killing mechanisms against persister cells. Independent mutations targeting the methionine and arginine biosyn-thetic pathways are bactericidal for M. tuberculosis in mice. However, in this study, we discovered that the addition of leucine and pantothenate auxotrophy altered the bac-tericidality of methionine auxotrophy. Whereas the leucine/pantothenate/methionine auxotrophic M. tuberculosis strain H37Rv DleuCD DpanCD DmetA was eliminated in immunocompetent mice, this strain persisted in multiple organs of immunodeficient Rag1^-/- mice for at least a year. In contrast, the leucine/pantothenate/arginine auxotroph H37Rv DleuCD DpanCD DargB was eliminated in both immunocompetent and immunodeficient Rag1^-/- mice. Our results showed that leucine and pantothenate starvation metabolically blocked the sterilization mechanisms of methionine starvation but not those of arginine starvation. These triple-auxotrophic strains should be invalu-able tools for unravelling the bacterial and host factors that enable persistence and for vaccine development studies to assess the efficacy of vaccines that boost immune recognition of M. tuberculosis in the persistent state. The sterilization of the DleuCD DpanCD DmetA auxotroph in immunocompetent mice, but not in mice lacking an adaptive immune response, could provide a new system for studying the antimyco-bacterial killing mechanisms of adaptive immunity.