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 5

LiaY, a member of the LiaFSR system, mediates changes in phospholipid architecture associated with DAP resistance.

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LiaY, a member of the LiaFSR system, mediates changes in phospholipid ar...
(A) Representative images of anionic phospholipid localization by NAO staining (top row), bright-field (middle row), and overlay (bottom row) in DAP-R Efs OG1RFΔliaX*289 and liaYZ mutants. DAP MICs (under images). White arrows represent anionic phospholipid microdomains at mid-cell or non–mid-cell locations. Scale bar: 2 μM. (B) Quantification of septal localization of anionic phospholipid microdomains by NAO staining in Efs OG1RF and liaXYZ mutants from A (>50 cells counted per strain per replicate, n = 3–4 replicates). ***P < 0.001 via 1-way ANOVA with multiple comparisons. (C) Representative images of anionic phospholipid localization by NAO staining (top row), bright-field (middle row), and overlay (bottom row) in Efs OG1RF liaX*289ΔliaYZ mutant transformed with pAT392 and derivatives. DAP MICs (under images). White arrows represent anionic phospholipid microdomains at mid-cell or non–mid-cell locations. Scale bar: 2 μM. (D) Quantification of septal localization of anionic phospholipid microdomains by NAO staining of Efs OG1RF liaX*289ΔliaYZ mutant complemented with pAT392 and derivatives from C (>50 cells counted per strain per replicate, n = 6–9 replicates). *P < 0.05; **P < 0.001, 1-way ANOVA with multiple comparisons. Whole images were adjusted for “Black Balance” per BZ-X800 Image Analysis Software with individual representative selected.