Depleting cationic lipids involved in antimicrobial resistance drives adaptive lipid remodeling in Enterococcus faecalis

Authors: RASHID, RAFI - Nanyang Technological University; NAIR, ZEUS - Nanyang Technological University; CHIA, DOMINIC - Nanyang Technological University; CHONG, KELVIN - Nanyang Technological University; GASSIOT, AMAURY - National University Of Singapore; Morley, Stewart; Allen, Douglas - Doug; CHEN, SWAINE - National University Of Singapore; CHNG, SHU - Nanyang Technological University; WENK, MARKUS - National University Of Singapore; KLINE, KIMBERLY - Nanyang Technological University

Submitted to: mBio
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/13/2022
Publication Date: 2/11/2023
Citation: Rashid, R., Nair, Z.J., Chia, D.M., Chong, K.K., Gassiot, A.C., Morley, S.A., Allen, D.K., Chen, S.L., Chng, S.S., Wenk, M.R., Kline, K.A. 2023. Depleting cationic lipids involved in antimicrobial resistance drives adaptive lipid remodeling in Enterococcus faecalis. mBio. 14(1). https://doi.org/10.1128/mbio.03073-22.
DOI: https://doi.org/10.1128/mbio.03073-22

Interpretive Summary: One of the first cellular level lines of defense from a pathogen is the cell membrane. Membranes contain lipids including some with amino acids attached that impact the membrane stability and charge and serve to help provide resistance to pathogens. In this study, lipid profiles were studied in microbial mutants. Differences in lipids, fatty acid content, and fatty acid production by acyl-acyl carrier proteins were considered to explain changes in growth and explained how a mutant alters its membrane composition to accommodate mutations. The work is important in several regards. Microbes investigated here are important to human health, but more broadly the studies depict changes in membranes that are a defense and important to all cells including those in plants. Thus, improving plant resistance to pathogens may benefit from what we learn in other systems.

Technical Abstract: The bacterial cell membrane is an interface for cell envelope synthesis, protein secretion, virulence factor assembly, and a target for host cationic antimicrobial peptides (CAMPs). To resist CAMP killing, several Gram-positive pathogens encode the multiple peptide resistance factor (MprF) enzyme that covalently attaches cationic amino acids to anionic phospholipids in the cell membrane. While E. faecalis encodes two mprF paralogs, MprF2 plays a dominant role in conferring resistance to killing by the CAMP human b-defensin 2 (hBD-2) in E. faecalis strain OG1RF. The goal of the current study is to understand the broader lipidomic and functional roles of E.faecalis mprF. We analyzed the lipid profiles of parental wild-type and mprF mutant strains and show that while DmprF2 and DmprF1 DmprF2 mutants completely lacked cationic lysyl-phosphatidylglycerol (L-PG), the DmprF1 mutant synthesized ;70% of LPG compared to the parent. Unexpectedly, we also observed a significant reduction of PG in DmprF2 and DmprF1 DmprF2. In the mprF mutants, particularly DmprF1 DmprF2, the decrease in L-PG and phosphatidylglycerol (PG) is compensated by an increase in a phosphorus-containing lipid, glycerophospho-diglucosyl-diacylglycerol (GPDGDAG), and D-ala-GPDGDAG. These changes were accompanied by a downregulation of de novo fatty acid biosynthesis and an accumulation of long-chain acyl-acyl carrier proteins (long-chain acyl-ACPs), suggesting that the suppression of fatty acid biosynthesis was mediated by the transcriptional repressor FabT. Growth in chemically defined media lacking fatty acids revealed severe growth defects in the DmprF1 DmprF2 mutant strain, but not the single mutants, which was partially rescued through supplementation with palmitic and stearic acids. Changes in lipid homeostasis correlated with lower membrane fluidity, impaired protein secretion, and increased biofilm formation in both DmprF2 and DmprF1 DmprF2, compared to the wild type and DmprF1. Collectively, our findings reveal a previously unappreciated role for mprF in global lipid regulation and cellular physiology, which could facilitate the development of novel therapeutics targeting MprF. IMPORTANCE The cell membrane plays a pivotal role in protecting bacteria against external threats, such as antibiotics. Cationic phospholipids such as lysyl-phosphatidyglycerol (L-PG) resist the action of cationic antimicrobial peptides through electrostatic repulsion. Here we demonstrate that L-PG depletion has several unexpected consequences in Enterococcus faecalis, including a reduction of phosphatidylglycerol (PG), enrichment of a phosphorus-containing lipid, reduced fatty acid synthesis accompanied by an accumulation of long-chain acyl-acyl carrier proteins (long chain acyl-ACPs), lower membrane fluidity, and impaired secretion. These changes are not deleterious to the organism as long as exogenous fatty acids are available for uptake from the culture medium. Our findings suggest an adaptive mechanism involving compensatory changes across the entire lipidome upon removal of a single phospholipid modification. Such adaptations must be considered when devising antimicrobial strategies that target membrane lipids.