A Simple and Rapid Microscale Method for Isolating Bacterial Lipopolysaccharides

Abstract

Bacterial endotoxins (lipopolysaccharides (LPSs)) are important mediators of inflammatory processes induced by Gram-negative microorganisms. LPSs are the key inducers of septic shock due to a Gram-negative bacterial infection; thus, the structure and functions of LPSs are of specific interest. Often, highly purified bacterial endotoxins must be isolated from small amounts of biological material. Each of the currently available methods for LPS extraction has certain limitations. Herein, we describe a rapid and simple microscale method for extracting LPSs. The method consists of the following steps: ultrasonic destruction of the bacterial material, LPS extraction via heating, LPS purification with organic solvents, and treatment with proteinase K. LPSs that were extracted by using this method contained less than 2-3% protein and 1% total nucleic acid. We also demonstrated the structural integrity of the O-antigen and lipid A via the sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) methods, respectively. We demonstrated the ability of the extracted LPSs to induce typical secretion of cytokines and chemokines by primary macrophages. Overall, this method may be used to isolate purified LPSs with preserved structures of both the O-antigen and lipid A and unchanged functional activity from small amounts of bacterial biomass.

Keywords: bacterial endotoxin; cell signaling; cytokines; inflammation; lipopolysaccharides; mass spectrometry.

Figure 1

Schematic representation of the described method of bacterial lipopolysaccharide isolation. The bacterial sample (approx. 10–20 mg) was collected in 100 µL of deionized water and centrifuged. The supernatant was discarded. The pellets were resuspended in 100 µL of deionized water and subjected to an ultrasonic bath. After sonication, LPSs were extracted by heating the suspension. Water LPS extract and cell debris were separated by using centrifugation. The supernatant was collected in a clean tube, and LPS extraction from the pellets was repeated via resuspension, sonication, and heating. The prepared water LPS extracts were purified by using chloroform–methanol–water extraction and enzymatic treatment with proteinase K.

Figure 2

The evaluation of O-antigen integrity via electrophoresis in polyacrylamide gel and the subsequent silver staining of LPSs extracted via the herein described method from P. aeruginosa PA103 and E. coli ATCC 25922. Typical O-antigen patterns for these bacteria are shown. The extraction was repeated three times, and the products from each extraction were individually subjected to SDS–PAGE. Commercially available LPS from E. coli O55:B5 was used as a control.

Figure 3

The analysis of lipid A via mass spectrometry. The spectra of lipid A from LPSs extracted from P. aeruginosa PA103 by using the described method and the Westphal method are presented. Peaks with m/z = 1616 correspond to hexa-acylated diphosphorylated lipid A, whereas peaks with m/z = 1536 correspond to hexa-acylated monophosphorylated lipid A. Diphosphorylated penta-acylated lipid A has a peak at m/z = 1446, and monophosphorylated penta-acylated lipid A contains ions with a peak at m/z = 1382 or m/z = 1366. The analysis was performed on negative ions by using MALDI-TOF-TOF UltrafleXtreme. m/z, mass-to-charge ratio.

Figure 4

A comparison of the ability of LPSs extracted by using the described method and the Westphal method to induce the secretion of proinflammatory cytokines and chemokines by cultured primary macrophages. (a) Gating strategy for analyses of primary macrophages before LPS treatment. An FSC-H vs. FSC-A plot was used to exclude doublets or larger aggregates, and cells in this gate were further analyzed with an SSC-A vs. FSC-A dot plot to identify the original total cell population. The gated population was further analyzed for the expression of CD45, and subsequent analysis were performed with the population of CD45+ cells for CD68 and CD206 expression. (b) A heatmap showing the fold increase in the secretion of cytokines and chemokines after the exposure of primary macrophages to the extracted LPSs compared with a nontreated culture.