Increased intracellular cyclic di-AMP levels impair growth and virulence of Bacillus anthracis

Cyclic di-AMP (c-di-AMP) is a recently identified bacterial second messenger that regulates biological processes. In this study, we found that inactivation of two c-di-AMP phosphodiesterases (PDEs), GdpP and PgpH, resulted in accumulation of 3.8 folds higher c-di-AMP levels than the parental strain Sterne in Bacillus anthracis and inhibited bacterial growth. Moreover, c-di-AMP accumulation decreased bacterial toxin expression, increased sensitivity to osmotic stress and detergent, and attenuated virulence in both C57BL/6J and A/J mice. Complementation of the PDE mutant with a plasmid carrying gdpP or pgpHin trans from a Pspac promoter restored bacterial growth, virulence factors expression, and resistant to detergent. Our results indicate that c-di-AMP is a pleiotropic signaling molecule in B. anthracis that is important for host-pathogen interaction. Importance Anthrax is an ancient and deadly disease caused by the spore-forming bacterial pathogen Bacillus anthracis. Vegetative cells of this species produce anthrax toxin proteins and S layer components during infection of mammalian hosts. So far, how the expression of these virulence factors is regulated remains largely unknown. Our results suggest that elevated c-di-AMP levels inhibit bacterial growth, reduce expression of S layer components and anthracis toxins as well as reduce virulence in a mouse model of disease. These results indicate that c-di-AMP signaling plays crucial roles in B. anthracis biology and disease.