Name: isolation of lipoprotein particles from chicken egg yolk for the study of bacterial pathogen fatty acid incorporation into membrane phospholipids
Uploaded: 2019-05-15T14:30:00
Description: Watch this Scientific Journal Video about Isolation of Lipoprotein Particles from Chicken Egg Yolk for the Study of Bacterial Pathogen Fatty Acid Incorporation into Membrane Phospholipids at JoVE.com

We thank members of the Hammer laboratory for their critical evaluation of the manuscript and support of this work. Dr. Alex Horswill of the University of Colorado School of Medicine kindly provided AH1263. Dr. Chris Waters laboratory at Michigan State University provided reagents. This work was supported by American Heart Association grant 16SDG30170026 and start-up funds provide by Michigan State University.

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
<ol>
	<li>Sanitize two large chicken eggs by washing the shells with 70% ethanol solution and allow to air dry.</li>
	<li>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.</li>
	<li>Crack each e...

S. aureus incorporates exogenous fatty acids into its membrane phospholipids27,32,43. Phospholipid synthesis using exogenous fatty acids bypasses FASII inhibition but also alters the biophysical properties of the membrane27,32,44. While incorporation of exogenous fatty acids into phospholipids of Gram-positive pathogens is well...

Methicillin-resistant S. aureus (MRSA) is the leading cause of healthcare-associated infection and the associated antibiotic resistance is a considerable clinical challenge1,2,3. 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 development5,6. 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 goods8,9. Additional FabI inhibitors are being developed by several pharmaceutical companies for the treatment of S. aureus infection10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26. However, many Gram-positive pathogens, including S. aureus, are capable of scavenging exogenous fatty acids for phospholipid synthesis, bypassing FASII inhibition27,28,29. 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 host27,28. 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)&#8212;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 phospholipids29,30,32. 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.

<table>
	<tbody>
		<tr>
			<td>Ammonium sulfate</td>
			<td>Fisher</td>
			<td>BP212R-1</td>
			<td>&#8805;99.5% pure</td>
		</tr>
		<tr>
			<td>Cell culture incubator</td>
			<td>Thermo</td>
			<td>MaxQ 6000</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrafuge</td>
			<td>Thermo</td>
			<td>75-217-420</td>
			<td>Sorvall Legen XTR, rotor F14-6x250 LE</td>
		</tr>
		<tr>
			<td>Costar assay plate</td>
			<td>Corning</td>
			<td>3788</td>
			<td>96 well</td>
		</tr>
		<tr>
			<td>Filter paper</td>
			<td>Schleicher &#38; Schuell</td>
			<td>597</td>
			<td></td>
		</tr>
		<tr>
			<td>Large chicken egg</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Common store bought egg</td>
		</tr>
		<tr>
			<td>Microplate spectrophotometer</td>
			<td>BioTek</td>
			<td>Epoch 2</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma</td>
			<td>S9625</td>
			<td></td>
		</tr>
		<tr>
			<td>S. aureus strain AH1263</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Provided by Alex Horswill of the University of Colorado</td>
		</tr>
		<tr>
			<td>Dialysis tubing</td>
			<td>Pierce</td>
			<td>68700</td>
			<td>7,000 MWCO</td>
		</tr>
		<tr>
			<td>Tryptone</td>
			<td>Becton, Dickison and Company</td>
			<td>211705</td>
			<td></td>
		</tr>
		<tr>
			<td>0.5 mm zirconium oxide beads</td>
			<td>Next Advance</td>
			<td>ZROB05</td>
			<td></td>
		</tr>
		<tr>
			<td>Bullet Blender</td>
			<td>Next Advance</td>
			<td>BBX24B</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol (LC-MS grade)</td>
			<td>Fisher</td>
			<td>A4561</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloroform (reagent grade)</td>
			<td>Fisher</td>
			<td>MCX10559</td>
			<td></td>
		</tr>
		<tr>
			<td>Isopropanol (LC-MS grade)</td>
			<td>Fisher</td>
			<td>A4611</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimyristoyl phosphatidylcholine</td>
			<td>Avanti Polar Lipids</td>
			<td>850345C-25mg</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium bicarbonate</td>
			<td>Sigma</td>
			<td>9830</td>
			<td>&#8805;99.5% pure</td>
		</tr>
		<tr>
			<td>Ammonium formate</td>
			<td>Sigma</td>
			<td>70221-25G-F</td>
			<td></td>
		</tr>
		<tr>
			<td>Xcalibur software</td>
			<td>Thermo Scientific</td>
			<td>OPTON-30801</td>
			<td></td>
		</tr>
		<tr>
			<td>LTQ-Orbitrap Velos mass spectrometer</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td>high resolution/accurate mass MS</td>
		</tr>
		<tr>
			<td>Agilent 1260 capillary HPLC</td>
			<td>Agilent</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SpeedVac Vacuum Concentrators</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation of lipoprotein particles from chicken egg yolk for the study of bacterial pathogen fatty acid incorporation into membrane phospholipids

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.

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 &#946;-livetins (<strong class="xfig"...

Watch this Scientific Journal Video about Isolation of Lipoprotein Particles from Chicken Egg Yolk for the Study of Bacterial Pathogen Fatty Acid Incorporation into Membrane Phospholipids at JoVE.com

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

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.

Staphylococcus aureus and other Gram-positive pathogens incorporate fatty acids from the environment into membrane phospholipids. During infection, the ...

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