Enterococcus faecalis GdpP Is a Novel Protein with a Potential Role in Stress Signaling and Response through the Essential Second Messenger Cyclic Di‐AMP

Abstract

Enterococci are a leading cause of hospital‐associated infections in the United States. In the absence of good alternatives, the cyclic lipopeptide daptomycin (DAP) has become a key “last‐resort” antimicrobial for the treatment of multidrug‐resistant enterococci infections. However, wider use of DAP leads to the development of resistance. Experimental evolution of a polymorphic population of E. faecalis S613 to DAP resistance using a novel continuous evolution bioreactor identified an adaptive point mutation in GdpP (GGDEF Domain Protein containing Phosphodiesterase), a protein with a potentially important role in bacterial signaling. We are using a combination of biophysical and structural approaches to elucidate the physicochemical basis for E. faecalis GdpP (Efs_GdpP) mediated changes in DAP susceptibility.Efs_GdpP is a multi‐domain protein whose functions remains poorly understood. Using in vitro enzymatic activity assays, we have demonstrated that the GGDEF domain has ATPase activity while the DHH/DHHA1 domain has cyclic di‐AMP (c‐di‐AMP) specific phosphodiesterase (PDE) activity. Our studies reveal that the adaptive mutation I440S strongly diminishes PDE activity. We show for first time that c‐di‐AMP is an important second messenger in enterococci, and is under the regulation of the three component LiaFSR signaling pathway. Moreover, we show that the intracellular c‐di‐AMP levels in E. faecalis wild‐type and DAP‐resistant strains are strongly correlated with GdpP activity and the LiaFSR system. The adaptive mutant Efs_GdpPI440S results in c‐di‐AMP accumulation in E. faecalis which is consistent with observed reduced PDE activity. Bacterial growth rates and transmission electron microscopy images of cell walls suggest upregulated c‐di‐AMP can upregulate the highly cross‐linked peptidoglycan in the cell wall to prevent DAP penetration, and also induce biofilm formation for DAP resistance.Support or Funding InformationThis work is supported by National Institutes of Health Grant [R01AI080714 to Y.S.].