Molecular basis of cell membrane adaptation in daptomycin-resistant Enterococcus faecalis
April H. Nguyen, Truc T. Tran, Diana Panesso, Kara S. Hood, Vinathi Polamraju, Rutan Zhang, Ayesha Khan, William R. Miller, Eugenia Mileykovskaya, Yousif Shamoo, Libin Xu, Heidi Vitrac, Cesar A. Arias
April H. Nguyen, Truc T. Tran, Diana Panesso, Kara S. Hood, Vinathi Polamraju, Rutan Zhang, Ayesha Khan, William R. Miller, Eugenia Mileykovskaya, Yousif Shamoo, Libin Xu, Heidi Vitrac, Cesar A. Arias
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Research Article Infectious disease

Molecular basis of cell membrane adaptation in daptomycin-resistant Enterococcus faecalis

Abstract

Daptomycin is a last-resort lipopeptide antibiotic that disrupts cell membrane (CM) and peptidoglycan homeostasis. Enterococcus faecalis has developed a sophisticated mechanism to avoid daptomycin killing by redistributing CM anionic phospholipids away from the septum. The CM changes are orchestrated by a 3-component regulatory system, designated LiaFSR, with a possible contribution of cardiolipin synthase (Cls). However, the mechanism by which LiaFSR controls the CM response and the role of Cls are unknown. Here, we show that cardiolipin synthase activity is essential for anionic phospholipid redistribution and daptomycin resistance since deletion of the 2 genes (cls1 and cls2) encoding Cls abolished CM remodeling. We identified LiaY, a transmembrane protein regulated by LiaFSR, and Cls1 as important mediators of CM remodeling required for redistribution of anionic phospholipid microdomains. Together, our insights provide a mechanistic framework on the enterococcal response to cell envelope antibiotics that could be exploited therapeutically.

Authors

April H. Nguyen, Truc T. Tran, Diana Panesso, Kara S. Hood, Vinathi Polamraju, Rutan Zhang, Ayesha Khan, William R. Miller, Eugenia Mileykovskaya, Yousif Shamoo, Libin Xu, Heidi Vitrac, Cesar A. Arias

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Figure 4

Cls1 localizes in nonseptal anionic phospholipid microdomains in DAP-R E. faecalis.

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Cls1 localizes in nonseptal anionic phospholipid microdomains in DAP-R E...
(A) Left 2 panels: Representative images of NAO staining (top panel), tetracysteine-tagged Cls1 with ReAsH reagent (red fluorescence, middle panel), and overlay of both images (bottom panel). White arrows represent anionic phospholipid microdomains at mid-cell or non–mid-cell locations. Both anionic phospholipid microdomains and Cls1 are shown to relocalize and overlap away from the septum in DAP-R Efs (OG1RFΔliaX) (Pearson Correlation Coefficient, 0.91 [95% CI, 0.74–0.97]) compared with their septal pattern in DAP-S E. faecalis OG1RF (Pearson Correlation Coefficient, 0.94 [95% CI, 0.83–0.98]). Right 2 panels: NAO staining for anionic phospholipid microdomains (green) and tetracysteine tagged-Cls1 localization (red) in E. faecalis OG117ΔliaXΔcls1Δcls2 harboring pMSP3535 carrying the gene expressing the tetracysteine tagged-Cls1 with or without nisin induction at 50 ng/mL, overlap between NAO and ReAsH (Pearson Correlation Coefficient, 0.91 [95% CI, 0.76–0.96]). White arrows represent anionic phospholipid microdomains at mid-cell or non–mid-cell locations. Whole images were adjusted for “Black Balance” per BZ-X800 Image Analysis Software with individual representative selected. (B) Quantification of Cls septal localization for strains, minimum 50 cells counted/strain, n = 3.