Molecular basis of cell membrane adaptation in daptomycin-resistant Enterococcus faecalis
April H. Nguyen, … , Heidi Vitrac, Cesar A. Arias
April H. Nguyen, … , Heidi Vitrac, Cesar A. Arias
Published October 15, 2024
Citation Information: JCI Insight. 2024;9(22):e173836. https://doi.org/10.1172/jci.insight.173836.
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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 6

LiaY bridges the LiaFSR response with changes in membrane architecture via Cls.

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LiaY bridges the LiaFSR response with changes in membrane architecture v...
(A) Protein-protein interactions between LiaY and Cls1 using the bacterial 2-hybrid system. Proteins were tagged at either the N- or C-terminus, cotransformed into E. coli BTH101, and activity recorded via a β-galactosidase assay. A leucine zipper interaction was used as the positive control (T18-zip/T25-zip, red bar) with 2 nontagged empty vectors used as negative controls (T18/T25). n = 3–8, ****P < 0.001, 1-way ANOVA with multiple comparisons with (-) against LiaY/Cls1 combinations. (B) Protein-to-protein interactions between LiaZ and Cls1 using the bacterial 2-hybrid system and following the same methodology as in A. n = 4–7. (C) Representative images of NAO staining (top row), mCherry (second row), bright-field (third row), and overlay (bottom row) in DAP-S (Efs OG1RFΔliaY) (Pearson Correlation Coefficient, 0.67 [95% CI, 0.24–0.88]) and DAP-R (Efs OG117ΔliaX) (Pearson Correlation Coefficient, 0.91 [95% CI, 0.75–0.97]). Both strains were transformed with pMSP3535::liaY-mCherry. White arrows represent anionic phospholipid microdomains at mid-cell or non–mid-cell locations. Scale bar: 2 μM. (D) Representative images of Cls1-GFP (top row), LiaY-mCherry (second row), bright-field (third row), and overlay (bottom row) of LiaY-mCherry and Cls1-GFP via fluorescence microscopy in Efs OG117 (Pearson Correlation Coefficient, 0.69 [95% CI, 0.28–0.89]) and Efs OG117ΔliaX GFP-Cls1 (Pearson Correlation Coefficient, 0.86 [95% CI, 0.63–0.95]) (with gfp-cls1 introduced in the chromosome) transformed with pMSP3535::liaY-mCherry. White arrows represent anionic phospholipid microdomains at mid-cell or non–mid-cell locations. Scale bar: 2 μM. Whole images were adjusted for “Black Balance” per BZ-X800 Image Analysis Software with individual representative selected. (E and F) Quantification of septal localization, minimum 50 cells counted per strain. n = 3, ***P < 0.001, 2-tailed t test.