The Lipid-Modifying Multiple Peptide Resistance Factor Is an Oligomer Consisting of Distinct Interacting Synthase and Flippase Subunits
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Phospholipids are synthesized at the inner leaflet of the bacterial cytoplasmic membrane but have to be translocated to the outer leaflet to maintain membrane lipid bilayer composition and structure. Even though phospholipid flippases have been proposed to exist in bacteria, only one such protein, MprF, has been described. MprF is a large integral membrane protein found in several prokaryotic phyla, whose C terminus modifies phosphatidylglycerol (PG), the most common bacterial phospholipid, with lysine or alanine to modulate the membrane surface charge and, as a consequence, confer resistance to cationic antimicrobial agents such as daptomycin. In addition, MprF is a flippase for the resulting lipids, Lys-PG or Ala-PG. Here we demonstrate that the flippase activity resides in the N-terminal 6 to 8 transmembrane segments of the Staphylococcus aureus MprF and that several conserved, charged amino acids and a proline residue are crucial for flippase function. MprF protects S. aureus against the membrane-active antibiotic daptomycin only when both domains are present, but the two parts do not need to be covalently linked and can function in trans . The Lys-PG synthase and flippase domains were each found to homo-oligomerize and also to interact with each other, which illustrates how the two functional domains may act together. Moreover, full-length MprF proteins formed oligomers, indicating that MprF functions as a dimer or larger oligomer. Together our data reveal how bacterial phospholipid flippases may function in the context of lipid biosynthetic processes. IMPORTANCE Bacterial cytoplasmic membranes are crucial for maintaining and protecting cellular integrity. For instance, they have to cope with membrane-damaging agents such as cationic antimicrobial peptides (CAMPs) produced by competing bacteria (bacteriocins), secreted by eukaryotic host cells (defensins), or used as antimicrobial therapy (daptomycin). The MprF protein is found in many Gram-positive, Gram-negative, and even archaeal commensals or pathogens and confers resistance to CAMPs by modifying anionic phospholipids with amino acids, thereby compromising the membrane interaction of CAMPs. Here we describe how MprF does not only modify phospholipids but uses an additional, distinct domain for translocating the resulting lysinylated phospholipids to the outer leaflet of the membrane. We reveal critical details for the structure and function of MprF, the first dedicated prokaryotic phospholipid flippase, which may pave the way for targeting MprF with new antimicrobials that would not kill bacteria but sensitize them to antibiotics and innate host defense molecules.
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MprF harbors two domains: a lysyl-transferase domain that transfers lysine from its tRNA carrier to PG (LPG) in the inner leaflet of the CM, and a flipase domain that translocates newly synthesized LPG from the inner to the outer leaflet of the CM.
“This enzyme harbors two domains, i) a lysyl-transferase domain that transfers the positively-charged amino acid lysine from its tRNA carrier to PG (LPG) in the inner leaflet of the CM, and ii) a flipase domain, through which newly synthesized LPG is translocated from the inner to the outer leaflet of the CM (99).”
From Mechanisms of Antibiotic Resistance (2016) · cites this paper as [99]
In S. aureus, MprF (a lysyl-PG [LPG] synthase) has been one of the most studied enzymes, and inactivation of this protein reversed DAP-R.
“In S. aureus, MprF (a lysyl-PG [LPG] synthase) has been one of the most studied enzymes, and inactivation of this protein reversed DAP-R.”
From Mechanisms of Antibiotic Resistance (2016) · cites this paper as [99]
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- Authors
- Ernst, C. M., Kuhn, S., Slavetinsky, C. J., Krismer, B., Heilbronner, S., Gekeler, C., Kraus, D., Wagner, S., & Peschel, A.
- Affiliations
- German Center for Infection Research (DE)
- Corresponding author
- Ernst, Christoph M.
- ORCID
- Venue
- mBio
- Publisher
- American Society for Microbiology
- Published
- 2015-02-27
- Volume
- 6
- Issue
- 1
- ISSN
- 2150-7511 (ISSN-L)
- 2161-2129
- PMC
- PMC4324311
- OpenAlex
- W2131904584
- Cited by
- 71
- References
- 37
- Citations by year
- 2026: 2 · 2025: 2 · 2024: 2 · 2023: 11 · 2022: 10 · 2021: 3 · 2020: 9 · 2019: 4 · 2018: 4 · 2017: 6 · 2016: 8 · 2015: 10
- FWCI
- 6.42
- Type
- journal article
- Language
- en
- Open access
- gold
- doi.org
- Licence
- Locations
- mBio (publishedVersion, cc-by-nc-sa)
- Europe PMC (PubMed Central) (publishedVersion, cc-by-nc-sa)
- PubMed Central (publishedVersion, cc-by-nc-sa)
- PubMed (publishedVersion)
- DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Bacterial Genetics and Biotechnology
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Lipid Membrane Structure and Behavior
- Life Sciences › Immunology and Microbiology › Microbiology › Antimicrobial Peptides and Activities
- Keywords
- mprF, phospholipid flippase, phosphatidylglycerol, daptomycin resistance, cationic antimicrobial peptides, Staphylococcus aureus, protein oligomerization, lipid asymmetry, transmembrane domain, proline residues
- MeSH terms
- Anti-Bacterial Agents, Bacterial Proteins, Biological Transport, Cell Membrane, Humans, Phospholipids, Staphylococcal Infections, Staphylococcus aureus, Protein Structure, Tertiary, Dimerization, Aminoacyltransferases, Protein Subunits
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