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

Antimicrobial resistance is a serious and growing threat to public health. The Centers for Disease Control estimates that at least 2 million people contract serious infections each year resulting in at least 23,000 deaths each year in the United States alone. To develop more effective treatments, it is important to understand the fundamental biology underlying the mechanisms of antibiotic resistance. Substitutions in the LiaFSR membrane stress response pathway are frequently associated with emergence of antimicrobial peptide resistance in both Enterococcus faecalis and Enterococcus faecium. Cyclic di-AMP (c-di-AMP) is an important signal molecule that affects many aspects of bacterial physiology including stress response. We have previously identified a mutation in a gene (designated yybT) of E. faecalis that was associated with development of daptomycin resistance. The adaptive mutation produced a change at position 440 in the predicted protein (yybTI440S). Here, we show that intracellular cyclic di-AMP signaling is present in enterococci and, based upon in vitro physicochemical characterization, we show that E. faecalis yybT encodes a cyclic dinucleotide phosphodiesterase of the GdpP family that exhibits specific activity toward c-di-AMP by hydrolyzing it to 5’pApA. The E. faecalis GdpPI440S substitution reduces cyclic di-AMP phosphodiesterase activity more than 11-fold leading to further increases in cyclic di-AMP levels. Additionally, deletions of liaR (encoding the response regulator of the LiaFSR system) that lead to daptomycin hypersusceptibility in both E. faecalis and E. faecium also resulted in increased cyclic-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 cyclic di-AMP pools is strongly associated with antibiotic-induced cell membrane stress response via changes in GdpP activity and signaling through the LiaFSR system