This protocol provides a framework for studying the incorporation of exogenous fatty acids from complex host sources into bacterial membranes, particularly staphylococcus aureus. The protocol utilizes unbiased lipidomic analysis and lipoprotein particles isolated from chicken egg yolks as an economic source of complex fatty acids to quantify fatty acid incorporation into bacterial lipids. The mass spectrometry techniques describes here offer an unparalleled perspective of the fatty acid profile of staphylococcus aureus lipids, but can be adapted to study other bacterial membranes.
When performing this protocol, care should be taken that each fractionation step to identify and retain the LDL-containing fraction. Removal of the ammonia sulfate from the plasma fraction is critical for downstream applications of the LDLs. Provide ample room in the tubing and change the water as needed to permit effective dialysis.
To begin, wash two egg yolks twice with 30 milliliters of sterile phosphate buffered saline to remove residual albumin. Puncture the vitelline membrane with a sterile pipette tip and drain the contents of the membrane into a sterile 50 milliliter conical centrifuge tube. Add approximately 24 to 30 milliliters or 17 molar sodium chloride solution at pH seven to the egg yolk and mix vigorously.
Then place the tube on a rocking shaker to mix at four degrees Celsius for 60 minutes. Centrifuge the egg yolk dilution at 10 degrees Celsius at 10, 000 times g for 45 minutes. Transfer the supernatant plasma fraction into a sterile 50 milliliter conical tube, leaving behind the pelleted granular fraction.
And centrifuge again. First, mix the plasma fraction with 40 mass volume percent ammonium sulfate on a rocking shaker or similar device at four degrees Celsius for 60 minutes. After adjusting the pH, centrifuge the plasma fraction at four degrees Celsius and 10, 000 time g for a duration of 45 minutes.
Remove the upper semisolid yellow fraction into seven kilodalton pore size dialysis tubing. Provide a minimum of 25%of extra room in the tubing to allow for it to swell. Place the dialysis tubing in three liters of ultrapure water to dialyze overnight at three degrees Celsius to remove the ammonium sulfate.
After dialysis, centrifuge the solution at four degrees Celsius as previously described. Carefully remove the upper, semisolid yellow fraction to a sterile tube and store at four degrees Celsius. After overnight incubation of the culture, transfer 500 microliters into two sterile 250 milliliter baffled flasks containing 49.5 milliliters of 1%tryptone broth.
Incubate for approximately four hours at 37 degrees Celsius with shaking to reach mid-long phase. Transfer 100 milliliters of culture in 25 milliliter increments to four sterile 50 milliliter centrifuge tubes and centrifuge the four tubes at 10, 000 times g to pellet the cells. Remove the supernatant and re-suspend each cell pellet in 750 microliters of 1%tryptone broth.
Combine the four 750 microliter cell suspensions into a single 15 milliliter tube. And aliquot 350 microliters of the cell suspension into six sterile 1.5 milliliter centrifuge tubes. Use a pipette to add 30 microliters of LDLs into the 1.5 milliliter centrifuge tube to achieve a desired final concentration of 5%And incubate at 37 degrees Celsius with shaking for four hours.
Centrifuge the cultures at four degrees Celsius at 16, 000 times g for a duration of two minutes. And re-suspend the cell pellets in 330 microliters of sterile phosphate-buffered saline to wash. Repeat this wash one more time.
Record the weight of the six wet cell pellets by subtracting the weight of the empty tube. Snap freeze the cell pellets on dry ice. First add one scoop of 5 millimeter zirconium oxide beads on top of each cell pellet.
Add 740 microliters of 75%HPLC grade methanol chilled to minus 80 degrees Celsius directly into each tube. Then add two microliters of 50 micromolar dimyristoyl phosphatidylcholine as an internal standard. Place the sample containing 1.5 milliliter centrifuge tubes into an available port on a bullet blender tissue homogenizer.
Homogenize the samples on low speed. Setting two to three for three minutes. Visually inspect the samples for homogeneity.
If clumps of cells are visible, continue homogenization in the bullet blender in two minute increments. Next add 270 microliters of chloroform to each sample in a chemical fume hood. Vortex the samples vigorously for 30 minutes.
Centrifuge the samples in a bench-top centrifuge for up to 30 minutes at minimum of 2, 000 times g. In the chemical fume hood, collect the monophasic supernatant and transfer it to a new test tube while carefully avoiding the protein pellet at the bottom of the extraction tube. Transfer the diluted lipid extracts previously prepared to an appropriate autosampler vial.
For flow injection-based analysis, place the autosampler vials into a temperature-controlled autosampler of an HPLC system which is capable of capillary flow applications. Set up the HPLC system according to the manuscript. The eluent is introduced from the HPLC transfer line to the mass spectrometer through an electrospray ionization source fitted with a low-flow 34 gauge metal needle.
In the Thermo Tune Plus instrument control software, set the ionization voltage to 4, 000 volts and sheath gas to five. Similarly, set the capillary temperature to 150 degrees Celsius and the S-lens to 50%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 field and enter 2, 000 in the last mass field. After further defining observed mass accuracy, average the signal across the broad peak in the total ion chromatogram.
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 okay button to close the viewing window.
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 the new Excel spreadsheet and proceed according to the manuscript. To perform accurate mass-based lipid identifications, open the LIMSA software.
From the main menu, select peak list under the spectrum type menu. Select positive mode or negative mode to correspond with the polarity in which the mass spectrometry data was acquired. In the peak full width at half maximum and over z window, enter the desired mass search window for peak finding.
In this protocol, the LDL content of the plasma fraction was further enriched by precipitation of the 30 to 40 kilodalton beta levitins. The presence of protein bands at 140, 80, 65, 60, and 15 kilodaltons correlate with the apoproteins of LDLs. Treatment with triclosan inhibits growth of staph aureus in fatty acid-free medium.
While supplementing cultures with egg yolk plasma as exogenous fatty acid sources overcomes triclosan-induced growth inhibition. Similarly, supplementation of triclosan-treated cultures with enriched egg yolk LDL restores growth. Further, addition of egg yolk LDLs supports the growth of a previously characterized S.aureus fatty acid, auxotroph.
Pre-fatty acid content was measured in 1%tryptone broth or chicken egg yolk LDL by employing flow injection high resolution accurate mass spectrometry. Minimal quantities of free fatty acid were found. Orthogonal partial least squares discriminant analysis of abundant staph aureus membrane phospholipids demonstrated clear class separation, with a fatty acid composition of untreated and chicken egg yolk LDL treated conditions.
Enrichment of LDLs from chicken egg yolk provides an inexpensive source of complex fatty acids for investigating the interactions between bacterial pathogens and host lipoproteins. Chloroform and methanol used in the preparation of lipid extracts are hazardous. Please be sure to use these reagents in a chemical fume hood and contain all waste streams.
