Nair, Z. J. | Search bioRxiv

bioRxiv2023

An essential protease, FtsH, influences daptomycin resistance acquisition in Enterococcus faecalis

Daptomycin is a last-line antibiotic commonly used to treat vancomycin resistant Enterococci, but resistance evolves rapidly and further restricts already limited treatment options. While genetic determinants associated with clinical daptomycin resistance (DAPR) have been described, information on factors affecting the speed of DAPR acquisition is limited. The multiple peptide resistance factor (MprF), a phosphatidylglycerol modifying enzyme involved in cationic antimicrobial resistance, is linked to DAPR in pathogens such as methicillin-resistant Staphylococcus aureus. Since Enterococcus faecalis encodes two paralogs of mprF and clinical DAPR mutations do not map to mprF, we hypothesized that functional redundancy between the paralogs prevents mprF-mediated resistance and masks other evolutionary pathways to DAPR. Here we performed in vitro evolution to DAPR in mprF mutant background. We discovered that the absence of mprF results in slowed DAPR evolution and is associated with inactivating mutations in ftsH resulting in the depletion of the chaperone repressor HrcA. We also report that ftsH is essential in the parental, but not in the {Delta}mprF, strain where FtsH depletion results in growth impairment in the parental strain, a phenotype associated with reduced glycolysis and reduced ability for metabolic reduction. This presents FtsH and HrcA as enticing targets for developing anti-resistance strategies. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=198 SRC="FIGDIR/small/551240v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@12ff3adorg.highwire.dtl.DTLVardef@127044aorg.highwire.dtl.DTLVardef@8ab082org.highwire.dtl.DTLVardef@14a4afc_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2022

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

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 {beta}-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 {triangleup}mprF2 and {triangleup}mprF1 {triangleup}mprF2 mutants completely lacked cationic lysyl-phosphatidylglycerol (L-PG), the {triangleup}mprF1 mutant synthesized [~]70% of L-PG compared to the parent. Unexpectedly, we also observed a significant reduction of PG in {triangleup}mprF2 and {triangleup}mprF1 {triangleup}mprF2. In the mprF mutants, particularly {triangleup}mprF1 {triangleup}mprF2, the decrease in L-PG and PG is compensated by an increase in the phosphorus-containing lipid, 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 {triangleup}mprF1 {triangleup}mprF2 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 {triangleup}mprF2 and {triangleup}mprF1 {triangleup}mprF2, compared to wild type and {triangleup}mprF1. 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. Significance StatementThe 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.

microbiology↗