Gain-of-Function Mutations in the Phospholipid Flippase MprF Confer Specific Daptomycin Resistance

ABSTRACT Daptomycin, a calcium-dependent lipopeptide antibiotic whose full mode of action is still not entirely understood, has become a standard-of-care agent for treating methicillin-resistant Staphylococcus aureus (MRSA) infections. Daptomycin-resistant (DAP-R) S. aureus mutants emerge during the...

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Autores principales:	Christoph M. Ernst, Christoph J. Slavetinsky, Sebastian Kuhn, Janna N. Hauser, Mulugeta Nega, Nagendra N. Mishra, Cordula Gekeler, Arnold S. Bayer, Andreas Peschel
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2018
Materias:	daptomycin MRSA MprF Staphylococcus aureus antibiotic resistance flippase Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/91185988fd2b4f0eadcc6a36be94023e
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spelling	oai:doaj.org-article:91185988fd2b4f0eadcc6a36be94023e2021-11-15T15:52:19ZGain-of-Function Mutations in the Phospholipid Flippase MprF Confer Specific Daptomycin Resistance10.1128/mBio.01659-182150-7511https://doaj.org/article/91185988fd2b4f0eadcc6a36be94023e2018-12-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.01659-18https://doaj.org/toc/2150-7511ABSTRACT Daptomycin, a calcium-dependent lipopeptide antibiotic whose full mode of action is still not entirely understood, has become a standard-of-care agent for treating methicillin-resistant Staphylococcus aureus (MRSA) infections. Daptomycin-resistant (DAP-R) S. aureus mutants emerge during therapy, featuring isolates which in most cases possess point mutations in the mprF gene. MprF is a bifunctional bacterial resistance protein that synthesizes the positively charged lipid lysyl-phosphatidylglycerol (LysPG) and translocates it subsequently from the inner membrane leaflet to the outer membrane leaflet. This process leads to increased positive S. aureus surface charge and reduces susceptibility to cationic antimicrobial peptides and cationic antibiotics. We characterized the most commonly reported MprF mutations in DAP-R S. aureus strains in a defined genetic background and found that only certain mutations, including the frequently reported T345A single nucleotide polymorphism (SNP), can reproducibly cause daptomycin resistance. Surprisingly, T345A did not alter LysPG synthesis, LysPG translocation, or the S. aureus cell surface charge. MprF-mediated DAP-R relied on a functional flippase domain and was restricted to daptomycin and a related cyclic lipopeptide antibiotic, friulimicin B, suggesting that the mutations modulate specific interactions with these two antibiotics. Notably, the T345A mutation led to weakened intramolecular domain interactions of MprF, suggesting that daptomycin and friulimicin resistance-conferring mutations may alter the substrate range of the MprF flippase to directly translocate these lipopeptide antibiotics or other membrane components with crucial roles in the activity of these antimicrobials. Our study points to a new mechanism used by S. aureus to resist calcium-dependent lipopeptide antibiotics and increases our understanding of the bacterial phospholipid flippase MprF. IMPORTANCE Ever since daptomycin was introduced to the clinic, daptomycin-resistant isolates have been reported. In most cases, the resistant isolates harbor point mutations in MprF, which produces and flips the positively charged phospholipid LysPG. This has led to the assumption that the resistance mechanism relies on the overproduction of LysPG, given that increased LysPG production may lead to increased electrostatic repulsion of positively charged antimicrobial compounds, including daptomycin. Here we show that the resistance mechanism is highly specific and relies on a different process that involves a functional MprF flippase, suggesting that the resistance-conferring mutations may enable the flippase to accommodate daptomycin or an unknown component that is crucial for its activity. Our report provides a new perspective on the mechanism of resistance to a major antibiotic.Christoph M. ErnstChristoph J. SlavetinskySebastian KuhnJanna N. HauserMulugeta NegaNagendra N. MishraCordula GekelerArnold S. BayerAndreas PeschelAmerican Society for MicrobiologyarticledaptomycinMRSAMprFStaphylococcus aureusantibiotic resistanceflippaseMicrobiologyQR1-502ENmBio, Vol 9, Iss 6 (2018)
institution	DOAJ
collection	DOAJ
language	EN
topic	daptomycin MRSA MprF Staphylococcus aureus antibiotic resistance flippase Microbiology QR1-502
spellingShingle	daptomycin MRSA MprF Staphylococcus aureus antibiotic resistance flippase Microbiology QR1-502 Christoph M. Ernst Christoph J. Slavetinsky Sebastian Kuhn Janna N. Hauser Mulugeta Nega Nagendra N. Mishra Cordula Gekeler Arnold S. Bayer Andreas Peschel Gain-of-Function Mutations in the Phospholipid Flippase MprF Confer Specific Daptomycin Resistance
description	ABSTRACT Daptomycin, a calcium-dependent lipopeptide antibiotic whose full mode of action is still not entirely understood, has become a standard-of-care agent for treating methicillin-resistant Staphylococcus aureus (MRSA) infections. Daptomycin-resistant (DAP-R) S. aureus mutants emerge during therapy, featuring isolates which in most cases possess point mutations in the mprF gene. MprF is a bifunctional bacterial resistance protein that synthesizes the positively charged lipid lysyl-phosphatidylglycerol (LysPG) and translocates it subsequently from the inner membrane leaflet to the outer membrane leaflet. This process leads to increased positive S. aureus surface charge and reduces susceptibility to cationic antimicrobial peptides and cationic antibiotics. We characterized the most commonly reported MprF mutations in DAP-R S. aureus strains in a defined genetic background and found that only certain mutations, including the frequently reported T345A single nucleotide polymorphism (SNP), can reproducibly cause daptomycin resistance. Surprisingly, T345A did not alter LysPG synthesis, LysPG translocation, or the S. aureus cell surface charge. MprF-mediated DAP-R relied on a functional flippase domain and was restricted to daptomycin and a related cyclic lipopeptide antibiotic, friulimicin B, suggesting that the mutations modulate specific interactions with these two antibiotics. Notably, the T345A mutation led to weakened intramolecular domain interactions of MprF, suggesting that daptomycin and friulimicin resistance-conferring mutations may alter the substrate range of the MprF flippase to directly translocate these lipopeptide antibiotics or other membrane components with crucial roles in the activity of these antimicrobials. Our study points to a new mechanism used by S. aureus to resist calcium-dependent lipopeptide antibiotics and increases our understanding of the bacterial phospholipid flippase MprF. IMPORTANCE Ever since daptomycin was introduced to the clinic, daptomycin-resistant isolates have been reported. In most cases, the resistant isolates harbor point mutations in MprF, which produces and flips the positively charged phospholipid LysPG. This has led to the assumption that the resistance mechanism relies on the overproduction of LysPG, given that increased LysPG production may lead to increased electrostatic repulsion of positively charged antimicrobial compounds, including daptomycin. Here we show that the resistance mechanism is highly specific and relies on a different process that involves a functional MprF flippase, suggesting that the resistance-conferring mutations may enable the flippase to accommodate daptomycin or an unknown component that is crucial for its activity. Our report provides a new perspective on the mechanism of resistance to a major antibiotic.
format	article
author	Christoph M. Ernst Christoph J. Slavetinsky Sebastian Kuhn Janna N. Hauser Mulugeta Nega Nagendra N. Mishra Cordula Gekeler Arnold S. Bayer Andreas Peschel
author_facet	Christoph M. Ernst Christoph J. Slavetinsky Sebastian Kuhn Janna N. Hauser Mulugeta Nega Nagendra N. Mishra Cordula Gekeler Arnold S. Bayer Andreas Peschel
author_sort	Christoph M. Ernst
title	Gain-of-Function Mutations in the Phospholipid Flippase MprF Confer Specific Daptomycin Resistance
title_short	Gain-of-Function Mutations in the Phospholipid Flippase MprF Confer Specific Daptomycin Resistance
title_full	Gain-of-Function Mutations in the Phospholipid Flippase MprF Confer Specific Daptomycin Resistance
title_fullStr	Gain-of-Function Mutations in the Phospholipid Flippase MprF Confer Specific Daptomycin Resistance
title_full_unstemmed	Gain-of-Function Mutations in the Phospholipid Flippase MprF Confer Specific Daptomycin Resistance
title_sort	gain-of-function mutations in the phospholipid flippase mprf confer specific daptomycin resistance
publisher	American Society for Microbiology
publishDate	2018
url	https://doaj.org/article/91185988fd2b4f0eadcc6a36be94023e
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Gain-of-Function Mutations in the Phospholipid Flippase MprF Confer Specific Daptomycin Resistance

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