Isolation of Lipoprotein Particles from Chicken Egg Yolk for the Study of Bacterial Pathogen Fatty Acid Incorporation into Membrane Phospholipids

Summary

This method provides a framework for studying incorporation of exogenous fatty acids from complex host sources into bacterial membranes, particularly Staphylococcus aureus. To achieve this, protocols for the enrichment of lipoprotein particles from chicken egg yolk and subsequent fatty acid profiling of bacterial phospholipids utilizing mass spectrometry are described.

Abstract

Staphylococcus aureus and other Gram-positive pathogens incorporate fatty acids from the environment into membrane phospholipids. During infection, the majority of exogenous fatty acids are present within host lipoprotein particles. Uncertainty remains as to the reservoirs of host fatty acids and the mechanisms by which bacteria extract fatty acids from the lipoprotein particles. In this work, we describe protocols for enrichment of low-density lipoprotein (LDL) particles from chicken egg yolk and determining whether LDLs serve as fatty acid reservoirs for S. aureus. This method exploits unbiased lipidomic analysis and chicken LDLs, an effective and economical model for the exploration of interactions between LDLs and bacteria. The analysis of S. aureus integration of exogenous fatty acids from LDLs is performed using high-resolution/accurate mass spectrometry and tandem mass spectrometry, enabling the characterization of the fatty acid composition of the bacterial membrane and unbiased identification of novel combinations of fatty acids that arise in bacterial membrane lipids upon exposure to LDLs. These advanced mass spectrometry techniques offer an unparalleled perspective of fatty acid incorporation by revealing the specific exogenous fatty acids incorporated into the phospholipids. The methods outlined here are adaptable to the study of other bacterial pathogens and alternative sources of complex fatty acids.

Introduction

Methicillin-resistant S. aureus (MRSA) is the leading cause of healthcare-associated infection and the associated antibiotic resistance is a considerable clinical challenge^1,2,3. Therefore, the development of novel therapeutic strategies is a high priority. A promising treatment strategy for Gram-positive pathogens is inhibiting fatty acid synthesis, a requirement for membrane phospholipid production that, in S. aureus, includes phosphatidylglycerol (PG), lysyl-PG, and cardiolipin⁴. In bacteria, fatty acid production occurs via the fatty acid synthesis II pathway (FASII)⁵, which is considerably different from the eukaryotic counterpart, making FASII an attractive target for antibiotic development^5,6. FASII inhibitors primarily target FabI, an enzyme required for fatty acid carbon chain elongation⁷. The FabI inhibitor triclosan is broadly used in consumer and medical goods^8,9. Additional FabI inhibitors are being developed by several pharmaceutical companies for the treatment of S. aureus infection^{10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26}. However, many Gram-positive pathogens, including S. aureus, are capable of scavenging exogenous fatty acids for phospholipid synthesis, bypassing FASII inhibition^27,28,29. Thus, the clinical potential of FASII inhibitors is debated due to considerable gaps in our knowledge of the sources of host fatty acids and the mechanisms by which pathogens extract fatty acids from the host^27,28. To address these gaps, we developed an unbiased lipidomic analysis method to monitor incorporation of exogenous fatty acid from lipoprotein particles into membrane phospholipids of S. aureus.

During sepsis, host lipoprotein particles represent a potential source of host-derived fatty acids within the vasculature, as a majority of host fatty acids are associated with the particles³⁰. Lipoproteins consist of a hydrophilic shell composed of phospholipids and proteins that enclose a hydrophobic core of triglycerides and cholesterol esters³¹. Four major classes of lipoproteins--chylomicron, very low-density lipoprotein, high-density lipoprotein, and low-density lipoprotein (LDL)—are produced by the host and function as lipid transport vehicles, delivering fatty acids and cholesterol to and from host cells via the vasculature. LDLs are abundant in esterified fatty acid including triglycerides and cholesterol esters³¹. We have previously demonstrated that highly purified human LDLs are a viable source of exogenous fatty acids for PG synthesis, thus providing a mechanism for FASII inhibitor bypass³². Purifying human LDLs can be technically challenging and time consuming while commercial sources of purified human LDLs are prohibitively expensive to use on a routine basis or to perform large-scale bacterial screens. To address these limitations, we have modified a procedure for the enrichment of LDLs from chicken egg yolk, a rich source of lipoprotein particles³³. We have successfully used untargeted, high-resolution/accurate mass spectrometry and tandem mass spectrometry to monitor incorporation of human LDL-derived fatty acids into the membrane of S. aureus³². Unlike previously reported methods, this approach can quantify individual fatty acid isomers for each of the three major staphylococcal phospholipid types. Oleic acid (18:1) is an unsaturated fatty acid present within all host lipoprotein particles that is readily incorporated into S. aureus phospholipids^29,30,32. S. aureus is not capable of oleic acid synthesis²⁹; therefore, the quantity of phospholipid-incorporated oleic acid establishes the presence of host lipoprotein-derived fatty acids within the staphylococcal membrane²⁹. These phospholipid species can be identified by the state-of-the-art mass spectrometry method described here, offering unprecedented resolution of the membrane composition of S. aureus cultured in the presence of a fatty acid source it likely encounters during infection.

Protocol

NOTE: The following protocol for enrichment of LDL particles from chicken egg yolk is derived from Moussa et al. 2002³³.

1. Preparation of chicken egg yolk for enrichment of LDL particles

1. Sanitize two large chicken eggs by washing the shells with 70% ethanol solution and allow to air dry.
2. Sanitize the egg separator using 70% ethanol solution and allow to air dry. Attach the egg separator onto the lip of a medium sized beaker.
3. Crack each egg individually into the egg separator and allow the albumen to flow into the beaker. The intact egg yolk will be retained by the separator.
4. Wash the egg yolk twice with 30 mL of sterile phosphate-buffered saline (PBS) to remove residual albumen.
5. Gently place the egg yolk onto filter paper.
6. Puncture the vitelline membrane with a sterile pipette tip and drain the contents of the membrane into a sterile 50 mL conical centrifuge tube. Discard the membrane and filter paper.

2. Fractionation of LDL-containing plasma from chicken egg yolk

1. Add approximately two volumes of 0.17 M NaCl at pH 7.0 to the egg yolk and mix vigorously. Then mix this solution at 4 °C for 60 min.
2. Centrifuge the egg yolk dilution at 10 °C at 10,000 x g for 45 min. Remove the plasma fraction (supernatant) from the granular fraction (pellet) into a sterile 50 mL conical tube.
3. Repeat step 2.2.

3. Isolation of LDL particles from plasma

1. Mix the plasma fraction with 40% ammonium sulfate (w/v) at 4 °C for 60 min.
2. Adjust the pH of the plasma fraction with a 420 mM NaOH solution to 8.7.
3. Centrifuge the egg yolk dilution at 4 °C at 10,000 x g for a duration of 45 min. Remove the upper semisolid yellow fraction into 7 kDa pore size dialysis tubing. Provide room in the tubing to allow for it to swell.
4. Dialyze overnight at 4 °C in 3 L of ultrapure water to remove the ammonium sulfate. Gently agitate the water using a stir bar.
5. Transfer dialyzed solution into a sterile 50 mL conical centrifuge tube.
6. Centrifuge the solution at 4 °C at 10,000 x g for a duration of 45 min. Carefully remove the upper semisolid yellow fraction to a sterile tube and store at 4 °C.

4. Assessment of chicken LDLs as a source of fatty acids

1. Subculture S. aureus cells into 5 mL of fatty acid-free 1% tryptone broth and incubate overnight at 37 °C with shaking (225 rpm). For fatty acid auxotrophs, supplement cultures with a source of fatty acids.
2. Dilute overnight cultures to an optical density (OD) at 600 nm (OD₆₀₀) of 0.1 in 1% tryptone broth. Pipette 50 μL of the cell suspension into each well of a round-bottom 96-well plate.
   NOTE: When working with fatty acid auxotrophs, wash the overnight cultures in two volumes of tryptone broth and resuspend in 5 mL of tryptone broth to limit carryover of fatty acids before determining the OD of the culture.
3. For the wells containing untreated controls, add 50 µL of 1% tryptone broth per well.
4. To the wells containing the experimental cell suspensions, add 50 µL of 1% tryptone broth supplemented with 10% egg yolk-derived LDL, 2 μM triclosan, or a mixture of 10% egg yolk-derived LDL and 2 μM triclosan.
   NOTE: At this point, each well will contain 100 μL and the final concentration of egg yolk-derived LDL and triclosan per well will be 5% and 1 µM, respectively.
5. Measure OD₆₀₀ over time, using a microplate reader set at 37 °C with continuous, linear shaking to monitor growth.

5. Incubation of S. aureus with LDLs for membrane lipid analysis.

1. Culture an isolated colony into 5 mL of fatty acid-free 1% tryptone broth and incubate overnight at 37 °C with shaking (225 rpm).
2. Dilute overnight cultures 1:100 into a sterile 250 mL baffled flask containing 50 mL of 1% tryptone broth. Incubate to mid-log phase (approximately 4 h) at 37 °C with shaking.
3. Transfer 25 mL of culture to a sterile 50 mL centrifuge tube and pellet the cells. Remove the supernatant and resuspend the cell pellet in 750 µL of 1% tryptone broth.
4. Combine the resuspended cells and aliquot 300 µL of the cell suspension into a sterile 1.5 mL centrifuge tube.
5. Add LDLs to the desired final concentration and incubate at 37 °C with shaking (225 rpm) for 4 h.
6. Centrifuge the cultures at 4 °C at 16,000 x g for a duration of 2 min and wash the cell pellets in two volumes of sterile PBS then repeat.
7. Record the weight of each wet cell pellet. Snap-freeze the cell pellets on dry ice or in liquid nitrogen and store at -80 °C or proceed directly to section 6.

6. Extraction of S. aureus membrane lipids

1. Place frozen S. aureus cell pellets on dry ice. Add 0.5 mm zirconium oxide beads on top of each cell pellet, using a volume of beads approximately equal to the volume of the cell pellet.
   NOTE: As an alternative to this method of lipid extraction, researchers can use the well-established Bligh and Dyer or Folch methods for exacting lipids from bacterial cells³⁴.
2. Add 740 µL of 75% methanol (HPLC grade) chilled to -80 °C directly to the cell pellets.
3. Add 2 μL of 50 µM dimyristoyl phosphatidylcholine (prepared in methanol) per 1 mg of cells as an internal standard. Close the test tube and place the 1.5 mL centrifuge tubes containing each sample into an available port in a Bullet Blender tissue homogenizer. Homogenize the samples on low speed, setting 2-3, for 3 min.
4. Visually inspect the samples for homogeneity. If clumps of cells are visible, continue homogenization in the Bullet Blender in 2 min increments.
5. Remove the samples from Bullet Blender and transfer to a chemical fume hood.
6. Add 270 µL of chloroform to each sample tube. Vortex the samples vigorously for 30 min.
   CAUTION: Chloroform is a possible carcinogen.
7. Centrifuge the samples in a benchtop centrifuge for up to 30 min at a minimum 2,000 x g. Faster speeds may be used with compatible centrifuge tubes and the duration of centrifugation may be shortened to 10 min.
8. In a chemical fume hood, collect the monophasic supernatant and transfer to a new test tube, while carefully avoiding the protein pellet at the bottom of the extraction tube.
9. Add 740 µL of 75% methanol (HPLC grade) and 270 µL of chloroform to the protein pellet, and re-extract each sample as described in steps 6.6-6.8 above. Combine the supernatant from the second extraction with the previously collected supernatant for each sample.
10. Evaporate the extraction solvents under a stream of inert gas such as nitrogen or argon, or under vacuum using a centrifuge concentrator (Table of Materials).
11. Wash dried lipid extracts three times with 1.0 mL of aqueous 10 mM ammonium bicarbonate solution and re-dry the samples as in step 6.10.
12. Resuspend the dried lipid extracts in a suitable nonpolar solvent such as isopropanol. Resuspend samples using 20 µL per 1 mg of fresh cell weight determined in step 5.7 Alternatively, if the weight of the cell pellets is unknown, resuspend the samples in 200 µL of isopropanol and proceed to Section 7.

7. Analysis of S. aureus lipid profiles using high resolution/accurate mass spectrometry

1. Prior to conducting a full lipid analysis, select representative test samples from the experimental group(s) and analyze them over a range of sample dilution factors to determine sample dilution ranges in which the total lipid concentrations fall within the linear range of detector response for the mass spectrometer, as previously described³⁵.
2. Evaporate aliquots of each sample lipid extract to be subjected to lipid analysis, by drying the aliquots under inert gas or under vacuum in a centrifuge concentrator (Table of Materials).
3. Resuspend each dried lipid extract in liquid chromatography–mass spectrometry (LC-MS) grade isopropanol:methanol (2:1, v:v) containing 20 mM ammonium formate, using volumes equivalent to an optimal sample dilution factor as determined in step 7.1.
4. For an untargeted lipid analysis, samples may be introduced directly to the high resolution/accurate mass spectrometry platform without the use of chromatography by flow injection or direct infusion of extracts^35,36. Transfer the diluted lipid extracts prepared in step 7.3 to an appropriate autosampler vial or 96-well plate.
5. For flow injection-based analysis, place the autosampler vials into a temperature-controlled (15 °C) autosampler of an HPLC system capable of capillary/low flow applications, such as a HPLC (Table of Materials) equipped with an electronic flow proportioning and flow monitoring system.
6. Fill the HPLC solvent reservoirs with LC-MS grade isopropanol:methanol (2:1, v:v) containing 20 mM ammonium formate.
7. Using Agilent Chemstation software, program the HPLC autosampler to perform 5 µL sample injections. From the Instrument menu, select Set Up Injector, and type 5.0 in the Injection Volume field. The units are given as microliters. Ensure that the HPLC is set to isocratic flow at 1 µL per min of 2:1 (v:v) isopropanol:methanol containing 20 mM ammonium formate.
8. From the Chemstation Instrument menu, select Set Up Pump… and then select the Micro Flow mode toggle switch.
9. In the Timetable fields, enter: Time 0.00, 100% B, Flow 1.0. Hit Enter and create a second row in the Timetable by entering Time 10.0, 100% B, Flow 1.0. Select the OK button at the bottom of the Set Up Pump menu. These settings will enable 10 analytical runs at a flow rate of 1.0 µL per minute.
10. Introduce eluate from the HPLC transfer line to the mass spectrometer using an electrospray ionization source fitted with a low-flow (34 G) metal needle.
11. Using Thermo Tune Plus instrument control software, select the Setup menu and select Heated ESI Source. Set the ionization voltage to 4000 V and sheath gas to 5 (arbitrary units) by typing these values into the corresponding fields of the dialogue box. Similarly, set the Capillary Temp to 150 °C and the S-lens to 50%.
    NOTE: These values need to be optimized for each mass spectrometry platform.
12. For untargeted lipid analysis, use a high resolution/accurate mass MS platform (Table of Materials) as the detector.
13. Using Thermo Tune Plus software, click the Define Scan button, and in the Analyzer menu select FTMS. Set the Mass Range field to Normal and in the Resolution field select 100,000. Ensure that Scan Type is set to Full. Under the Scan Ranges menu, enter 200 in the First Mass (m/z) field, and enter 2000 in the Last Mass (m/z) field.
14. Ensure that negative polarity is utilized to detect the most abundant S. aureus lipids.
15. Repeat the sample analysis using ion mapping tandem mass spectrometry (MS/MS) fragmentation of all lipid ions within a spectral region of interest in order to confirm lipid structures and fatty acid constituents. Alternatively, selected lipid ions of interest may be subjected to MS/MS analysis after initial lipid identifications have been assigned in Section 8.

8. Database searching to identify endogenous S. aureus and exogenous LDL-derived lipids

1. Use Thermo Xcalibur software to further refine observed mass accuracy. In Xcalibur, under the Tools menu, select the Recalibrate Offline. After the Recalibrate Offline window opens, load the mass spectrum file to be recalibrated by selecting the File menu and selecting the Open option.
2. Open the file of interest, toggle the Insert Row button at the top of the view window, to view the total ion chromatogram for the MS run. Average the acquired MS signals by left-clicking the computer mouse on one edge of the observed signal peak in the total ion chromatogram and dragging the mouse across the broadest part of the peak.
3. Under the Scan Filter menu, select the filter corresponding to full scan MS data. Load a reference file containing the theoretical monoisotopic masses of at least three known S. aureus endogenous lipids by selecting the Load Ref… button and selecting the reference file. Check the Use box next to each lipid monoisotopic mass.
4. Click the Search button at the bottom of the viewing window. Re-average the MS signal across the signal peak in the total ion chromatogram as done previously.
5. Click the Convert button near the bottom of the viewing window. When the Convert dialogue box opens, click OK. Omit this step if data was collected on mass spectrometry platforms from vendors other than Thermo Scientific.
6. Using Xcalibur software, export the recalibrated accurate mass peak lists for each untreated or LDL-treated sample to separate worksheets of an Excel file. Select the Qual Browser icon. Open the recalibrated file of interest by selecting the File menu and selecting the Open… option.
7. Average the signal across the broad peak in the total ion chromatogram as described in step 8.2.
8. Right click the thumbtack icon in the mass spectrum viewing window and select View | Spectrum List. From the same menu, select Display Options and then All Peaks toggle box in the Display menu. Click the OK button to close the viewing window.
9. Right click the thumbtack icon in the mass spectrum viewing window again and select the Export | Clipboard (Exact Mass). Paste the exported data cell A1 of the first worksheet into a new Excel spreadsheet.
10. Delete the first 8 rows of text in the exported data file, such that cell A1 of the Excel spreadsheet contains the first mass data point from the mass spectrum. Repeat the exporting of each recalibrated MS file, using a new worksheet in the Excel file for each exported peak list.
11. Using the Lipid Mass Spectral Analysis (LIMSA) software³⁷ Add-In for Excel, construct a database containing molecular formulas of known S. aureus lipid species as described by Hewelt-Belka et al. 2014³⁸, as well as formulas representing lipid species that could hypothetically be present in LDL.
    NOTE: Additionally, care should be taken to include potential molecular formulas in the database for hypothetical bacterial lipids that have incorporated major LDL fatty acids, such as oleic (18:1) and linoleic (18:2) fatty acids³².
12. To construct the database, open a blank Excel spreadsheet. In cell A1 of the first worksheet, type the theoretical/computed monoisotopic mass of the lipid species to be added to the database, corresponding to the mass of the lipid species in the ionic state observed in the mass spectrometer. In cell B1, enter a name for the lipid species, such as PG(34:0).
13. In cell C1, enter the molecular formula for the lipid species, corresponding to the ionic state of the lipid observed in the mass spectrometer. In cell D1, enter the charge of the lipid species as observed in the mass spectrometer. Move to cell A2 to begin a new entry for the next lipid species to be entered in the searchable database.
14. Repeat the steps 8.12 and 8.13 until all desired lipid species have been entered into the database. Save the database file and leave it open in Excel.
15. In Excel, select the Add-Ins menu in. Select LIMSA to start the LIMSA software. From the main menu, click the Compound Library… button. In the new window that appears, click Import Compounds. This will upload the compound database for use by the LIMSA software.
16. Perform accurate-mass based lipid identifications on all MS spectra using the LIMSA software Add-In for Excel according to the vendor’s instructions³⁵. From the LIMSA main menu, select Peak List under the Spectrum type menu. Select Positive mode or Negative mode to correspond with the polarity in which the MS data was acquired.
17. In the Peak fwhm (m/z) window, enter the desired mass search window for peak finding. A mass tolerance search window of 0.003-0.005 m/z is recommended for high resolution/accurate mass MS data.
18. In the Sensitivity window, enter the desired baseline cutoff (for example, 0.01% relative abundance). In the Isotope correction menu, select Linear fit or Subtract algorithms, either of which may be used with peak list data.
19. Highlight lipid compounds to include in the database search by clicking on desired lipid species within the Available Compounds window. Click the Add button to add highlighted lipid species to the search group.
20. Define internal standards by clicking on added compounds, then changing the Concentration window to the corresponding concentration of the selected internal standard.
21. Ensure that the internal standard and selected lipid species to be quantitated belong to the same class name by selecting each added lipid species and internal standard and typing a class name (such as PG or Lipid) in the Class field.
22. To save the searchable group of lipid compounds for future use, click the Save button next to the Groups menu.
23. Ensure that the Excel file containing all exported MS peak lists is open to the sheet corresponding to the first S. aureus MS run, and then click the Search button from the LIMSA main menu.
    NOTE: The output of the LIMSA database search will include a list of mass spectral features matched to lipids present in the database constructed in steps 8.12-8.13, as well as concentrations for each matched feature following normalization to one or more selected internal standards.
24. Use Xcalibur software to examine accurate mass MS/MS spectra for m/z corresponding to lipid ions of interest in order to confirm fatty acid constituents present in each identified lipid molecular species. Select the Qual Browser icon. Open the MS/MS file of interest by selecting the File drop-down menu and selecting the Open… option.
25. Select the scan filter corresponding to MS/MS analysis of a lipid m/z of interest by right mouse-clicking on the thumbtack icon in the mass spectrum viewing window and select Ranges from the menu. In the new window that appears, select the Filter menu to select the scan.
26. Average the signal across the total ion chromatogram as described in step 8.2. Use MetaboAnalyst (www.metaboanalyst.ca) software to perform appropriate statistical tests. Evaluate statistically significant difference in S. aureus lipid composition by comparing normalized lipid abundances across untreated and LDL-treated conditions.

Results

The protocol for the enrichment of LDL from chicken egg yolk is illustrated in Figure 1. This process begins by diluting whole egg yolk with saline and separating the egg yolk solids referred to as granules from the soluble or plasma fraction containing the LDLs (Figure 1)³³. The LDL content of the plasma fraction is further enriched by precipitation of the ~ 30-40 kDa β-livetins (

Discussion

S. aureus incorporates exogenous fatty acids into its membrane phospholipids^27,32,43. Phospholipid synthesis using exogenous fatty acids bypasses FASII inhibition but also alters the biophysical properties of the membrane^27,32,44. While incorporation of exogenous fatty acids into phospholipids of Gram-positive pathogens is well...

Materials

Name	Company	Catalog Number	Comments
Ammonium sulfate	Fisher	BP212R-1	≥99.5% pure
Cell culture incubator	Thermo	MaxQ 6000
Centrafuge	Thermo	75-217-420	Sorvall Legen XTR, rotor F14-6x250 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