Depleting Cationic Lipids Involved in Antimicrobial Resistance Drives Adaptive Lipid Remodeling in Enterococcus faecalis
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)...
| Main Authors: | , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
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American Society for Microbiology
2023-02-01
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| Series: | mBio |
| Subjects: | |
| Online Access: | https://journals.asm.org/doi/10.1128/mbio.03073-22 |
| _version_ | 1811163339078762496 |
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| author | Rafi Rashid Zeus Jaren Nair Dominic Ming Hao Chia Kelvin Kian Long Chong Amaury Cazenave Gassiot Stewart A. Morley Doug K. Allen Swaine L. Chen Shu Sin Chng Markus R. Wenk Kimberly A. Kline |
| author_facet | Rafi Rashid Zeus Jaren Nair Dominic Ming Hao Chia Kelvin Kian Long Chong Amaury Cazenave Gassiot Stewart A. Morley Doug K. Allen Swaine L. Chen Shu Sin Chng Markus R. Wenk Kimberly A. Kline |
| author_sort | Rafi Rashid |
| collection | DOAJ |
| description | 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 β-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 ΔmprF2 and ΔmprF1 ΔmprF2 mutants completely lacked cationic lysyl-phosphatidylglycerol (L-PG), the ΔmprF1 mutant synthesized ~70% of L-PG compared to the parent. Unexpectedly, we also observed a significant reduction of PG in ΔmprF2 and ΔmprF1 ΔmprF2. In the mprF mutants, particularly ΔmprF1 ΔmprF2, 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 ΔmprF1 ΔmprF2 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 ΔmprF2 and ΔmprF1 ΔmprF2, compared to the wild type and ΔmprF1. 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. |
| first_indexed | 2024-04-10T06:43:43Z |
| format | Article |
| id | doaj.art-32be3af729934681ae993b200c2266d8 |
| institution | Directory Open Access Journal |
| issn | 2150-7511 |
| language | English |
| last_indexed | 2024-04-10T06:43:43Z |
| publishDate | 2023-02-01 |
| publisher | American Society for Microbiology |
| record_format | Article |
| series | mBio |
| spelling | doaj.art-32be3af729934681ae993b200c2266d82023-02-28T14:06:24ZengAmerican Society for MicrobiologymBio2150-75112023-02-0114110.1128/mbio.03073-22Depleting Cationic Lipids Involved in Antimicrobial Resistance Drives Adaptive Lipid Remodeling in Enterococcus faecalisRafi Rashid0Zeus Jaren Nair1Dominic Ming Hao Chia2Kelvin Kian Long Chong3Amaury Cazenave Gassiot4Stewart A. Morley5Doug K. Allen6Swaine L. Chen7Shu Sin Chng8Markus R. Wenk9Kimberly A. Kline10Integrative Sciences & Engineering Programme, National University of Singapore, SingaporeSingapore Centre for Environmental Life Sciences Engineering, Nanyang Technological University, SingaporeSingapore Centre for Environmental Life Sciences Engineering, Nanyang Technological University, SingaporeSingapore Centre for Environmental Life Sciences Engineering, Nanyang Technological University, SingaporeSingapore Lipidomics Incubator (SLING), Life Sciences Institute, National University of Singapore, SingaporeDonald Danforth Plant Science Center, St. Louis, Missouri, USADonald Danforth Plant Science Center, St. Louis, Missouri, USAGERMS and Infectious Disease Group, Genome Institute of Singapore, SingaporeSingapore Centre for Environmental Life Sciences Engineering, Nanyang Technological University, SingaporeSingapore Lipidomics Incubator (SLING), Life Sciences Institute, National University of Singapore, SingaporeSingapore Centre for Environmental Life Sciences Engineering, Nanyang Technological University, SingaporeABSTRACT 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 β-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 ΔmprF2 and ΔmprF1 ΔmprF2 mutants completely lacked cationic lysyl-phosphatidylglycerol (L-PG), the ΔmprF1 mutant synthesized ~70% of L-PG compared to the parent. Unexpectedly, we also observed a significant reduction of PG in ΔmprF2 and ΔmprF1 ΔmprF2. In the mprF mutants, particularly ΔmprF1 ΔmprF2, 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 ΔmprF1 ΔmprF2 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 ΔmprF2 and ΔmprF1 ΔmprF2, compared to the wild type and ΔmprF1. 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.https://journals.asm.org/doi/10.1128/mbio.03073-22multiple peptide resistance factor (MprF)lysyl-phosphatidylglycerol (L-PG)cationic antimicrobial peptides (CAMPs)lipid homeostasislipid metabolismadaptive remodeling |
| spellingShingle | Rafi Rashid Zeus Jaren Nair Dominic Ming Hao Chia Kelvin Kian Long Chong Amaury Cazenave Gassiot Stewart A. Morley Doug K. Allen Swaine L. Chen Shu Sin Chng Markus R. Wenk Kimberly A. Kline Depleting Cationic Lipids Involved in Antimicrobial Resistance Drives Adaptive Lipid Remodeling in Enterococcus faecalis mBio multiple peptide resistance factor (MprF) lysyl-phosphatidylglycerol (L-PG) cationic antimicrobial peptides (CAMPs) lipid homeostasis lipid metabolism adaptive remodeling |
| title | Depleting Cationic Lipids Involved in Antimicrobial Resistance Drives Adaptive Lipid Remodeling in Enterococcus faecalis |
| title_full | Depleting Cationic Lipids Involved in Antimicrobial Resistance Drives Adaptive Lipid Remodeling in Enterococcus faecalis |
| title_fullStr | Depleting Cationic Lipids Involved in Antimicrobial Resistance Drives Adaptive Lipid Remodeling in Enterococcus faecalis |
| title_full_unstemmed | Depleting Cationic Lipids Involved in Antimicrobial Resistance Drives Adaptive Lipid Remodeling in Enterococcus faecalis |
| title_short | Depleting Cationic Lipids Involved in Antimicrobial Resistance Drives Adaptive Lipid Remodeling in Enterococcus faecalis |
| title_sort | depleting cationic lipids involved in antimicrobial resistance drives adaptive lipid remodeling in enterococcus faecalis |
| topic | multiple peptide resistance factor (MprF) lysyl-phosphatidylglycerol (L-PG) cationic antimicrobial peptides (CAMPs) lipid homeostasis lipid metabolism adaptive remodeling |
| url | https://journals.asm.org/doi/10.1128/mbio.03073-22 |
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