The PhoPR virulence system regulates expression of the universal second messenger c-di-AMP and impacts vaccine safety and efficacy.

Cyclic (di)nucleotides act as universal second messengers endogenously produced by several pathogens. Specifically, the roles of c-di-AMP in immunity and virulence have been largely explored, although its contribution to the safety and efficacy of live tuberculosis vaccines is less understood. In this study, we demonstrate that the synthesis of c-di-AMP is negatively regulated by the PhoPR virulence system. Accordingly, the live attenuated tuberculosis vaccine candidate vaccine (MTBVAC), based on double and deletions, produces more than 25- and 45-fold c-di-AMP levels relative to wild-type or the current vaccine bacille Calmette-Guérin (BCG), respectively. Secretion of this second messenger was exclusively detected in MTBVAC but not in or in BCG. We also demonstrate that c-di-AMP synthesis during cultivation of is a growth-phase- and medium-dependent phenotype. To uncover the role of this metabolite in the vaccine properties of MTBVAC, we constructed and validated knockout and overproducing/oversecreting derivatives by inactivating the or gene, respectively. All MTBVAC derivatives elicited superior interleukin-1β (IL-1β) responses compared with BCG during an infection of human macrophages. However, both vaccines failed to elicit interferon β (IFNβ) activation in this cellular model. We found that increasing c-di-AMP levels remarkably correlated with a safer profile of tuberculosis vaccines in the immunodeficient mouse model. Finally, we demonstrate that overproduction of c-di-AMP due to inactivation resulted in lower protection of MTBVAC, while the absence of c-di-AMP in the MTBVAC derivative maintains the protective efficacy of this vaccine in mice.