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Biochimica

Isolering af lipoprotein partikler fra hønseæg æggeblomme til undersøgelse af bakteriel Patogenchlorid fedtsyre inkorporering i membran Fosfolipids

Published: May 15, 2019
doi:
10.3791/59538
, ,
1Department of Microbiology and Molecular Genetics,Michigan State University, 2Department of Physiology,Michigan State University

Summary

Denne metode giver en ramme for at studere inkorporering af eksogene fedtsyrer fra komplekse vært kilder i bakterielle membraner, især Staphylococcus aureus. For at opnå dette, protokoller for tilsætning af lipoprotein partikler fra hønseæg æggeblomme og efterfølgende fedtsyre profilering af bakterielle fosfolipider udnytter massespektrometri er beskrevet.

Abstract

Staphylococcus aureus og andre gram positive patogener inkorporerer fedtsyrer fra omgivelserne i membran Fosfolipiderne. Under infektion, de fleste af eksogene fedtsyrer er til stede i Host lipoprotein partikler. Der er fortsat usikkerhed med hensyn til reservoirer af værts fedtsyrer og de mekanismer, hvormed bakterier ekstrahere fedtsyrer fra lipoprotein partiklerne. I dette arbejde beskriver vi protokoller for berigelse af low-density lipoprotein (LDL) partikler fra hønseæg æggeblomme og afgøre, om LDLs fungere som fedtsyre reservoirer for S. aureus. Denne metode udnytter upartisk lipidomic analyse og kylling LDLs, en effektiv og økonomisk model for udforskning af interaktioner mellem LDLs og bakterier. Analysen af S. aureus integration af eksogene fedtsyrer fra LDLs udføres ved hjælp af høj opløsning/nøjagtig massespektrometri og tandem massespektrometri, der muliggør karakterisering af fedtsyre sammensætningen af bakterien membran og objektiv identifikation af nye kombinationer af fedtsyrer, som opstår i bakterielle membran lipider ved udsættelse for LDLs. Disse avancerede massespektrometri teknikker tilbyder en uovertruffen perspektiv af fedtsyre inkorporering ved at afsløre de specifikke eksogene fedtsyrer indarbejdet i Fosfolipiderne. De metoder, der er skitseret her, er tilpasningsdygtige til studiet af andre bakterielle patogener og alternative kilder til komplekse fedtsyrer.

Introduction

Methicillin-resistent S. aureus (MRSA) er den førende årsag til sundhedsrelateret infektion, og den associerede antibiotikaresistens er en betydelig klinisk udfordring1,2,3. Derfor er udviklingen af nye terapeutiske strategier en høj prioritet. En lovende behandlingsstrategi for gram-positive patogener hæmmer fedtsyre syntesen, et krav til membran fosfolipid produktion, der i S. aureus, omfatter phosphatidylglycerol (PG), lysyl-PG, og cardiolipin4. I bakterier, fedtsyre produktion sker via fedtsyre syntese II pathway (fasii)5, som er betydeligt forskellig fra eukaryote modstykke, gør fasii et attraktivt mål for antibiotika udvikling5,6 . FASII-hæmmere primært Target FabI, et enzym, der kræves for fedtsyre kulstofkæde forlængelse7. Fabi-hæmmeren triclosan anvendes i vid udstrækning i forbruger-og medicinalvarer8,9. Yderligere Fabi-hæmmere er ved at blive udviklet af flere farmaceutiske virksomheder til behandling af S. aureus infektion10,11,12,13,14 ,15,16,17,18,19,20,21,22,23 ,24,25,26. Men, mange gram-positive patogener, herunder S. aureus, er i stand til at scavenging eksogene fedtsyrer for phospholipid syntese, omgåelse fasii hæmning27,28,29. Således diskuteres det kliniske potentiale af fasii-hæmmere på grund af betydelige huller i vores viden om kilderne til vært fedtsyrer og de mekanismer, hvormed patogener ekstrakt fedtsyrer fra værten27,28. For at afhjælpe disse mangler, vi udviklet en objektiv lipidomisk analysemetode til at overvåge inkorporering af eksogene fedtsyrer fra lipoprotein partikler i membran fosfolipider af S. aureus.

Under sepsis, vært lipoprotein partikler repræsenterer en potentiel kilde til Host-afledte fedtsyrer i Vaskulaturen, som et flertal af vært fedtsyrer er forbundet med partiklerne30. Lipoproteiner består af en hydrofile Shell sammensat af phospholipider og proteiner, som vedlægger en hydrofobe kerne af triglycerider og kolesterol estere31. Fire store klasser af lipoproteiner–chylomicron, meget lav densitet lipoprotein, high-density lipoprotein, og low-density lipoprotein (LDL)-er produceret af værten og fungere som lipid transport køretøjer, leverer fedtsyrer og kolesterol til og fra Host celler via vasculature. LDLs er rigelige i esterificeret fedtsyre, herunder triglycerider og kolesterol estere31. Vi har tidligere påvist, at højt renset humane LDLs er en levedygtig kilde til eksogene fedtsyrer til PG syntese, hvilket giver en mekanisme for FASII-hæmmer bypass32. Rensning af humane ldl’er kan være teknisk udfordrende og tidskrævende, mens kommercielle kilder til rensede humane ldl’er er uoverkommeligt dyre at bruge rutinemæssigt eller til at udføre store bakterie skærme. For at løse disse begrænsninger, har vi ændret en procedure for berigelse af LDLs fra kylling æggeblomme, en rig kilde til lipoprotein partikler33. Vi har med succes brugt umålrettet, høj opløsning/nøjagtig massespektrometri og tandem massespektrometri til at overvåge inkorporering af humane LDL-afledte fedtsyrer i membranen af S. aureus32. I modsætning til tidligere rapporterede metoder, denne fremgangsmåde kan kvantificere individuelle fedtsyrer isomerer for hver af de tre store stafylokok phospholipid typer. Oliesyre (18:1) er en umættet fedtsyre til stede i alle Host lipoprotein partikler, der let inkorporeres i S. aureus fosfolipids29,30,32. S. aureus er ikke i stand til oliesyre syntese29; Derfor fastsætter mængden af phospholipid-indarbejdet oliesyre tilstedeværelsen af vært lipoprotein-afledte fedtsyrer i stafylokoken membran29. Disse phospholipid arter kan identificeres ved den state-of-the-art massespektrometri metode, der er beskrevet her, tilbyder hidtil uset opløsning af membranens sammensætning af S. aureus dyrket i nærværelse af en fedtsyre kilde det sandsynligt møder under infektion.

Protocol

Bemærk: følgende protokol for tilsætning af LDL-partikler fra kyllingægge blomme er afledt af Moussa et al. 200233. 1. fremstilling af hønseæg æggeblomme til berigelse af LDL-partikler Sanitize to store hønseæg ved at vaske skaller med 70% ethanol opløsning og lad lufttørre. Sanitize ægge separatoren ved hjælp af 70% ethanol opløsning og lad lufttørre. Fastgør ægseparatoren på læben på et mellemstort bægerglas. Knæk hv…

Representative Results

Protokollen for tilsætning af LDL fra kyllingægge blomme er illustreret i figur 1. Denne proces begynder ved at fortynde hele æggeblomme med saltvand og adskille æggeblomme tørstof benævnt granulat fra den opløselige eller plasma fraktion indeholdende Ldl’er (figur 1)33. LDL-indholdet i plasma fraktionen er yderligere beriget ved udfældning af ~ 30-40 kDa β-livetiner (figur 2<…

Discussion

S. aureus inkorporerer eksogene fedtsyrer i dets membran fosfolipider27,32,43. Fosfolipid syntese ved hjælp af eksogene fedtsyrer omgår fasii hæmning, men også ændrer de biofysiske egenskaber af membranen27,32,44. Mens inkorporering af eksogene fedtsyrer i phospholipider af gram-positive patogener er veldokumenteret, mangl…

Divulgazioni

The authors have nothing to disclose.

Acknowledgements

Vi takker medlemmerne af hammer laboratoriet for deres kritiske evaluering af manuskriptet og støtte til dette arbejde. Dr. Alex Horswill fra University of Colorado School of Medicine venligt forudsat AH1263. Dr. Chris Waters laboratorium på Michigan State University leverede reagenser. Dette arbejde blev støttet af American Heart Association Grant 16SDG30170026 og start-up midler fra Michigan State University.

Materials

Ammonium sulfate Fisher BP212R-1 ≥99.5% pure
Cell culture incubator Thermo MaxQ 6000
Centrafuge Thermo 75-217-420 Sorvall Legen XTR, rotor F14-6×250 LE
Costar assay plate Corning 3788 96 well
Filter paper Schleicher & Schuell 597
Large chicken egg N/A N/A Common store bought egg
Microplate spectrophotometer BioTek Epoch 2
NaCl Sigma S9625
S. aureus strain AH1263 N/A N/A Provided by Alex Horswill of the University of Colorado
Dialysis tubing Pierce 68700 7,000 MWCO
Tryptone Becton, Dickison and Company 211705
0.5 mm zirconium oxide beads Next Advance ZROB05
Bullet Blender Next Advance BBX24B
Methanol (LC-MS grade) Fisher A4561
Chloroform (reagent grade) Fisher MCX10559
Isopropanol (LC-MS grade) Fisher A4611
Dimyristoyl phosphatidylcholine Avanti Polar Lipids 850345C-25mg
Ammonium bicarbonate Sigma 9830 ≥99.5% pure
Ammonium formate Sigma 70221-25G-F
Xcalibur software Thermo Scientific OPTON-30801
LTQ-Orbitrap Velos mass spectrometer Thermo Scientific high resolution/accurate mass MS
Agilent 1260 capillary HPLC Agilent
SpeedVac Vacuum Concentrators Thermo Scientific
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Tags

Keywords: Lipoprotein ParticlesChicken Egg YolkBacterial PathogenFatty Acid IncorporationMembrane PhospholipidsLipidomic AnalysisStaphylococcus AureusAmmonium SulfateDialysis
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Delekta, P. C., Lydic, T. A., Hammer, N. D. Isolation of Lipoprotein Particles from Chicken Egg Yolk for the Study of Bacterial Pathogen Fatty Acid Incorporation into Membrane Phospholipids. J. Vis. Exp. (147), e59538, doi:10.3791/59538 (2019).
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