The Lipid-Modifying Multiple Peptide Resistance Factor Is an Oligomer Consisting of Distinct Interacting Synthase and Flippase Subunits

ABSTRACT 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...

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Autores principales:	Christoph M. Ernst, Sebastian Kuhn, Christoph J. Slavetinsky, Bernhard Krismer, Simon Heilbronner, Cordula Gekeler, Dirk Kraus, Samuel Wagner, Andreas Peschel
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Lenguaje:	EN
Publicado:	American Society for Microbiology 2015
Materias:	Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/eaa22c27ed4c493c9d617225a249e9ec
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spelling	oai:doaj.org-article:eaa22c27ed4c493c9d617225a249e9ec2021-11-15T15:41:19ZThe Lipid-Modifying Multiple Peptide Resistance Factor Is an Oligomer Consisting of Distinct Interacting Synthase and Flippase Subunits10.1128/mBio.02340-142150-7511https://doaj.org/article/eaa22c27ed4c493c9d617225a249e9ec2015-02-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.02340-14https://doaj.org/toc/2150-7511ABSTRACT 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.Christoph M. ErnstSebastian KuhnChristoph J. SlavetinskyBernhard KrismerSimon HeilbronnerCordula GekelerDirk KrausSamuel WagnerAndreas PeschelAmerican Society for MicrobiologyarticleMicrobiologyQR1-502ENmBio, Vol 6, Iss 1 (2015)
institution	DOAJ
collection	DOAJ
language	EN
topic	Microbiology QR1-502
spellingShingle	Microbiology QR1-502 Christoph M. Ernst Sebastian Kuhn Christoph J. Slavetinsky Bernhard Krismer Simon Heilbronner Cordula Gekeler Dirk Kraus Samuel Wagner Andreas Peschel The Lipid-Modifying Multiple Peptide Resistance Factor Is an Oligomer Consisting of Distinct Interacting Synthase and Flippase Subunits
description	ABSTRACT 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.
format	article
author	Christoph M. Ernst Sebastian Kuhn Christoph J. Slavetinsky Bernhard Krismer Simon Heilbronner Cordula Gekeler Dirk Kraus Samuel Wagner Andreas Peschel
author_facet	Christoph M. Ernst Sebastian Kuhn Christoph J. Slavetinsky Bernhard Krismer Simon Heilbronner Cordula Gekeler Dirk Kraus Samuel Wagner Andreas Peschel
author_sort	Christoph M. Ernst
title	The Lipid-Modifying Multiple Peptide Resistance Factor Is an Oligomer Consisting of Distinct Interacting Synthase and Flippase Subunits
title_short	The Lipid-Modifying Multiple Peptide Resistance Factor Is an Oligomer Consisting of Distinct Interacting Synthase and Flippase Subunits
title_full	The Lipid-Modifying Multiple Peptide Resistance Factor Is an Oligomer Consisting of Distinct Interacting Synthase and Flippase Subunits
title_fullStr	The Lipid-Modifying Multiple Peptide Resistance Factor Is an Oligomer Consisting of Distinct Interacting Synthase and Flippase Subunits
title_full_unstemmed	The Lipid-Modifying Multiple Peptide Resistance Factor Is an Oligomer Consisting of Distinct Interacting Synthase and Flippase Subunits
title_sort	lipid-modifying multiple peptide resistance factor is an oligomer consisting of distinct interacting synthase and flippase subunits
publisher	American Society for Microbiology
publishDate	2015
url	https://doaj.org/article/eaa22c27ed4c493c9d617225a249e9ec
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The Lipid-Modifying Multiple Peptide Resistance Factor Is an Oligomer Consisting of Distinct Interacting Synthase and Flippase Subunits

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