A Novel Phosphodiesterase of the GdpP Family Modulates Cyclic di-AMP Levels in Response to Cell Membrane Stress in Daptomycin-Resistant Enterococci

Abstract

Substitutions in the LiaFSR membrane stress pathway are frequently associated with the emergence of antimicrobial peptide resistance in both <i>Enterococcus faecalis</i> and <i>Enterococcus faecium</i> Cyclic di-AMP (c-di-AMP) is an important signal molecule that affects many aspects of bacterial physiology, including stress responses. We have previously identified a mutation in a gene (designated <i>yybT</i>) in <i>E. faecalis</i> that was associated with the development of daptomycin resistance, resulting in a change at position 440 (<i>yybT</i>^I440S) in the predicted protein. Here, we show that intracellular c-di-AMP signaling is present in enterococci, and on the basis of <i>in vitro</i> physicochemical characterization, we show that <i>E. faecalis</i><i>yybT</i> encodes a cyclic dinucleotide phosphodiesterase of the GdpP family that exhibits specific activity toward c-di-AMP by hydrolyzing it to 5'pApA. The <i>E. faecalis</i> GdpP^I440S substitution reduces c-di-AMP phosphodiesterase activity more than 11-fold, leading to further increases in c-di-AMP levels. Additionally, deletions of <i>liaR</i> (encoding the response regulator of the LiaFSR system) that lead to daptomycin hypersusceptibility in both <i>E. faecalis</i> and <i>E. faecium</i> also resulted in increased c-di-AMP levels, suggesting that changes in the LiaFSR stress response pathway are linked to broader physiological changes. Taken together, our data show that modulation of c-di-AMP pools is strongly associated with antibiotic-induced cell membrane stress responses via changes in GdpP activity or signaling through the LiaFSR system.