2176. Cardiolipin Synthases Play Redundant Roles And Are Required for Membrane Remodeling in Daptomycin Resistant Enterococcus faecalis

BACKGROUND: Daptomycin (DAP) is a lipopeptide antibiotic targeting anionic phospholipids (APLs) at the division septum. Resistance to DAP (DAP-R) in E. faecalis (Efs) has been associated with activation of the LiaFSR response and redistribution of APL microdomains through the effector protein LiaX. We also identified a novel transmembrane protein (designated LiaY) as essential for changes in APL microdomains which contain cardiolipin (CL). Efs encodes two CL synthase genes, cls1 and cls2. While changes in Cls1 are associated with DAP-R, their link to the LiaFSR system remains unclear. We aim to establish the roles of Cls in DAP-R to provide insights into membrane remodeling associated with DAP-R. METHODS: qRT-PCR was used to study cls expression. Membrane lipid content was analyzed using hydrophilic interaction chromatography-mass spectrometry. Mutants were characterized by DAP minimum inhibitory concentration (MIC) using E-test and localization of APL microdomains with 10-N-nonyl-acridine orange. The bacterial two hybrid system was used to evaluate protein interaction. Fluorescence microscopy evaluated protein co-localization. RESULTS: cls1 was upregulated in DAP-R Efs OG117ΔliaX. Deletion of either cls caused upregulation of the remaining cls gene. Development of DAP-R altered membrane lipid content, namely, an increase in CL and a shift towards species with longer fatty acid chains and higher degree of unsaturation. Upon deletion of both cls, CL content was eliminated, peripheral APL microdomains were abolished, and a decrease in the DAP MIC was observed. LiaY interacted with Cls1, but not Cls2 using the two-hybrid system. The interaction was confirmed with fluorescence microscopy showing co-localization of LiaY and Cls1 at the septum and away from the septum in DAP-S and DAP-R strains, respectively. LiaY and Cls1 were also individually co-localized to APL microdomains. CONCLUSION: While Cls1 is predominantly associated with DAP-R, we show a functional redundancy between Cls1 and Cls2 in both membrane homeostasis and in DAP-R. Cls1 interacts with LiaY, which bridges the resistance mechanism with membrane adaptation, ultimately leading to altered localization of APL microdomains that divert DAP away from the division septum. DISCLOSURES: William R. Miller, M.D., Merck: Grant/Research Support|UpToDate: Honoraria