The three-component regulatory system LiaFSR is a major mediator of resistance against daptomycin (DAP) and antimicrobial peptides (AMP) in enterococci. The main target of the LiaR response regulator is a cluster of three genes (designated liaXYZ). LiaX has distinct C- and N-terminal domains and is highly secreted in DAP-resistant (DAP-R) strains but its role in resistance is unknown. We generated liaX deletions of the full gene (OG1RFΔliaX) and region encoding the C-terminal domain (OG1RFΔliaX289) in E. faecalis (Efs) OG1RF. Mutants were assessed for susceptibility to DAP and by visualization of anionic phospholipids (APLs) using nonyl-acridine orange. To determine whether extracellular LiaX protected DAP-S strains (Efs OG1RF and S613) from the DAP “attack”, we determined MICs (broth macrodilution) in the presence and absence of supernatants recovered from DAP-R strains (R712 and OG1RFΔliaX289). We also assessed expression of the liaFSR and liaXYZ clusters in the liaXmutants and when LiaX was added exogenously. Finally, we investigated the ability of LiaX to bind DAP and LL-37 (human defensin from neutrophils). Deletion of liaX or its C-terminal domain resulted in DAP resistance (MIC 12 μg/ml) and caused cell membrane remodeling of APLs associated with DAP-R. The addition of supernatants of DAP-R R712 and OG1RFΔliaX289 increased the MICs of DAP-S (S613 and OG1RF) ca. three- to five-fold, well above the clinical breakpoint. In contrast, no change in MICs was observed when OG1RFΔliaX supernatant was used. Deletion of LiaX and its C-terminal domain significantly upregulated gene expression of liaFSR and liaXYZ. The same upregulation of those clusters was observed when purified LiaX was added exogenously (concentration of 42 nM) but only in the presence of DAP. LiaX readily bound DAP and the antimicrobial peptide LL-37 with KDs = 0.05 and 8.3 μM, respectively. Our results suggest that LiaX controls cell membrane adaptation by modulating phospholipid remodeling through its C-terminal domain and binding antimicrobial peptides via its N-terminal domain, sensing the presence of attacking molecules and activating the LiaFSR stress response system. This dual function of a single protein to protect against antibiotic attack has no precedent. All authors: No reported disclosures.