This work was supported by Grants AI064184 and AI76322 from the National Institutes of Health (NIH) and by Grant 61789-MA-MUR from the Army Research Office to M.S.T. Research was also supported by Welch Foundation Grant F1155 and NIH grant R01GM103655 to J.S. B.

All solutions should be prepared with ultrapure water and HPLC grade methanol and chloroform. Prepared solutions that contain organic solvents such as methanol, chloroform, or pyridine and concentrated acids or bases should be prepared and used under a chemical fume hood. All solutions can be stored at RT. Solvents should be measured in a graduated glass cylinder and stored in glass solvent bottles with PTFE lined caps. For long-term storage chloroform-containing solvents should be stored in tinted amber glass bottles to...

No conflicts of interest declared.

In this protocol we have detailed the isolation of lipid A species from whole cells of bacteria, and described TLC or MS based analytical methods to chemically characterize this isolated material. Tandem mass spectrometry is a powerful strategy for de novo structural characterization of biological compounds, and is invaluable for the chemical characterization of the panoply of lipid A molecules observed in nature. CID and UVPD are two complementary activation methods that create different types of product ions t...

Lipopolysaccharide (LPS) is the major outer surface molecule of nearly all gram-negative organisms and consists of three molecular domains: a distal O-antigen polysaccharide, a core oligosaccharide, and the membrane-associated lipid A domain deposited on the outer leaflet of the outer membrane bilayer1,2. The lipid A domain consists of 3-deoxy-D-manno-oct-2-ulosonic (Kdo) residues and a lipid A molecular species, where lipid A can be defined as the chloroform soluble portion of LPS upon mild-acid hydrolysis1,2. The standard lipid A molecule can be chemically defined as a diglucosamine backbone that is hexa-acylated and bis-phosphorylated; consistent with the major lipid A species observed in the model organism Escherichia coli (E. coli)1,2. Nine constitutively expressed genes, conserved throughout gram-negative bacteria, are responsible for the production of the lipid A domain (Figure 1)1,2. Most bacteria have an additional set of genes, which vary in degree of phylogenetic conservation, that participate in further chemical modification of lipid A3. Dephosphorylation, removal of acyl chains, and the addition of chemical moieties such as amino sugars (e.g. aminoarabinose) and/or phosphoethanolamine are the most commonly observed activities (Figure 1). Many of the enzymes responsible for lipid A modification are directly activated by environmental signals, such as divalent cations, or their expression is regulated by two component response-regulator systems3.
Recognition of lipid A species by the host innate immune system is mediated by the Toll-like receptor 4/myeloid differentiation factor 2 (TLR4/MD2) co-receptor4. Hydrophobic forces between MD2 and the lipid A acyl chains, as well as between TLR4 and the 1 and 4 'phosphate groups of lipid A promote the strong association of lipid A with TLR4/MD24,5. Modifications that alter acylation state or the negative charge of lipid A impact TLR4/MD2 based lipid A recognition and downstream stimulation of the innate immune response activators NF-&kappa;B and mediators of inflammation such as TNF&alpha; and IL1-&beta;6,7. Modifications that mask the negative charge of lipid A also prevent bactericidal cationic antimicrobial peptides from binding to gram-negative cell surfaces3,8. Many lipid A modifications are hypothesized to increase bacterial fitness under specific environmental conditions, such as inside the human host or in an ecological niche. For this reason many modification enzymes are attractive targets in the rational development of antimicrobial compounds. The chemical diversity of lipid A structures, with respect to organism and/or environment, and the biological implications of these diverse structures make the structural characterization of lipid A an important endeavor in the study of gram-negative bacteria.
Isolation of lipid A molecules from whole bacteria involves the extraction of LPS from the bacterial cell surface, a hydrolytic step to liberate lipid A, followed by a final purification procedure9-11. The most frequently cited LPS extraction procedure is the hot-phenol water extraction procedure, first introduced by Westphal and Jann10. After extraction whole LPS is subjected to mild-acid hydrolysis, which chemically separates Kdo from the 6'-hydroxyl of the distal glucosamine sugar of lipid A (Figure 1). Numerous pitfalls exist for the hot-phenol water procedure including the use of a high hazard reagent, the need to degrade co-extracted nucleic acids and proteins, and several days are required to complete the protocol10.
Our lab has further developed the extraction and isolation of lipid A as first developed by Caroff and Raetz12,13. Compared to hot-phenol water procedures, the method presented here is more rapid and efficient and accommodates a wide range of culture volumes from 5 ml to multiple liters. Moreover, unlike hot-phenol water extractions, our method does not select for rough- or smooth-types of LPS, providing optimal recovery of lipid A species. In our protocol, chemical lysis of whole bacterial cells is performed using a mixture of chloroform, methanol and water, where LPS can be pelleted by centrifugation. A combination of mild-acid hydrolysis and solvent extractions (Bligh-Dyer) are used to liberate lipid A from covalently attached polysaccharide. The method of Bligh and Dyer was first applied to the extraction of lipid species from a variety of animal and plant tissues14, modified here to separate hydrolyzed polysaccharide from lipid A. In this final separation step, chloroform soluble lipids selectively partition into the lower organic phase. To further purify lipid A, reverse-phase or anionic exchange column chromatography can be used12.
After isolation of lipid A species from whole cells, a number of analytical methods can be used to characterize the chemical structure of the isolated material such as NMR, TLC, and MS-based analysis. NMR allows for non-destructive structural elucidation, and provides structural detail related to glycosidic linkages, unequivocal assignment of acyl chain positions, and assignment of attachment sites for lipid A modifications like aminoarabinose or phosphoethanolamine15-17. NMR analysis of lipid A is not discussed within our protocol, but has been described adequately elsewhere15,16. For rapid analysis TLC based methods are frequently used, but fail to provide direct information regarding fine chemical structure. MS based protocols are the most frequently employed method to characterize lipid A structures18,19. Matrix associated laser desorption ionization (MALDI)-MS is often used to initially survey intact lipid A species. Singly charged ions are generated from analyte prepared according to our extraction procedures. As more fine structural analysis is required, MS/MS based methods prove more informative than MALDI-MS. Coupled to electrospray ionization (ESI) singly or multiply charged lipid A precursor ions are further fragmented by collision induced dissociation (CID) or ultraviolet photodissociation (UVPD), to generate structurally informative product ions18,20,21. Neutral loss products from lipid A precursor ions are also frequently generated during ESI-MS providing an additional layer of structural information.
Tandem mass spectrometry (MS/MS) has proven to be an indispensable and versatile method for the elucidation of lipid A structures. During MS/MS, ions are activated to yield a diagnostic fragmentation pattern that can be used to elucidate the structure of the precursor ion. The most widely available MS/MS method is CID. This method produces fragment ions via collisions of the selected precursor ion with an inert target gas, resulting in energy deposition that leads to dissociation. CID has proven a critical tool in the assignment of lipid A structure for a wide range of bacterial species22-33.
Although CID is the most universally implemented MS/MS method, it generates a limited array of product ions. 193 nm UVPD is an alternative and complementary MS/MS method. This method uses a laser to irradiate ions, and the absorption of photons results in energization of the ions and subsequent dissociation. This higher energy MS/MS technique produces a more diverse array of product ions than CID and thus provides more informative fragmentation patterns. In particular, UVPD affords information about subtle changes in lipid A species based on cleavages at glycosidic, amine, acyl and C-C linked bonds18,21,34.

<table border="1">
		<tbody>
		 <tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		 </tr>
		 <tr>
			<td>Chloroform</td>
			<td>Thermo Fisher Scientific</td>
			<td>C607</td>
			<td>HPLC Grade</td>
		 </tr>
		 <tr>
			<td>Methanol</td>
			<td>Thermo Fisher Scientific</td>
			<td>A452</td>
			<td>HPLC Grade</td>
		 </tr>
		 <tr>
			<td>Teflon FEP Centrifuge Bottles</td>
			<td>Thermo Fisher Scientific</td>
			<td>05-562-21</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Silica Gel 60 TLC Plates</td>
			<td>EMD Biosciences</td>
			<td>5626-6</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Grade No. 3MM Chromatography Paper</td>
			<td>Whatman</td>
			<td>3030700</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Orbitrap Elite</td>
			<td>Thermo Fisher Scientific</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>Mass Spectrometer</td>
 <td></td>
 <td></td>
 <td></td>
		 </tr>
		 <tr>
			<td>ExciStar XS Excimer Lasrer</td>
			<td>Coherent Inc.</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>PicoTip Nanospray ESI emitters</td>
			<td>New Obectives</td>
			<td></td>
			<td>&ge; 30 &mu;m to reduce clogging</td>
		 </tr>
		 <tr>
			<td>Model 505 Pulse/Delay Generator</td>
			<td>Berkeley Nucleonics Corporation</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>Hot Plate Thermoylne 2200</td>
			<td>Barnstead/Thermolyne</td>
			<td>HPA2235MQ</td>
			<td></td>
		 </tr>
		 <tr>
			<td>16x125 mm GPI 15-415 Threaded Disposable Borosilicate Culture Tubes</td>
			<td>Corning Pyrex</td>
			<td>99449-16X</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Reusable Threaded PTFE screw caps GPI 45-415</td>
			<td>Corning </td>
			<td>9999-152</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Personal Molecular Imager System (phosphorimager)</td>
			<td>BioRad</td>
			<td>170-9400</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Autoradiography Cassette</td>
			<td>Thermo Fisher Scientific</td>
			<td>FBCS810</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Phosphorscreen SO230</td>
			<td>Kodak</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>Peptide Mass Standards Kit</td>
			<td>Sequazyme</td>
			<td>P2-3143-00</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Sonifier S250-A</td>
			<td>Branson</td>
			<td>101063196</td>
			<td></td>
		 </tr>
		 <tr>
			<td>1.5 ml 12x32 mm Tapered Base Screw Thread Vial</td>
			<td>Thermo Fisher Scientific</td>
			<td>C4000-V1</td>
			<td></td>
		 </tr>
		</tbody>
 </table>

isolation and chemical characterization of lipid a from gram-negative bacteria

Lipopolysaccharide (LPS) is the major cell surface molecule of gram-negative bacteria, deposited on the outer leaflet of the outer membrane bilayer. LPS can be subdivided into three domains: the distal O-polysaccharide, a core oligosaccharide, and the lipid A domain consisting of a lipid A molecular species and 3-deoxy-D-manno-oct-2-ulosonic acid residues (Kdo). The lipid A domain is the only component essential for bacterial cell survival. Following its synthesis, lipid A is chemically modified in response to environmental stresses such as pH or temperature, to promote resistance to antibiotic compounds, and to evade recognition by mediators of the host innate immune response. The following protocol details the small- and large-scale isolation of lipid A from gram-negative bacteria. Isolated material is then chemically characterized by thin layer chromatography (TLC) or mass-spectrometry (MS). In addition to matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) MS, we also describe tandem MS protocols for analyzing lipid A molecular species using electrospray ionization (ESI) coupled to collision induced dissociation (CID) and newly employed ultraviolet photodissociation (UVPD) methods. Our MS protocols allow for unequivocal determination of chemical structure, paramount to characterization of lipid A molecules that contain unique or novel chemical modifications. We also describe the radioisotopic labeling, and subsequent isolation, of lipid A from bacterial cells for analysis by TLC. Relative to MS-based protocols, TLC provides a more economical and rapid characterization method, but cannot be used to unambiguously assign lipid A chemical structures without the use of standards of known chemical structure. Over the last two decades isolation and characterization of lipid A has led to numerous exciting discoveries that have improved our understanding of the physiology of gram-negative bacteria, mechanisms of antibiotic resistance, the human innate immune response, and have provided many new targets in the development of antibacterial compounds.

Canonical lipid A of E. coli and Salmonella enterica serovar Typhimurium is a hexa-acylated disaccharide of glucosamine with phosphate groups at the 1- and 4 '-positions. During growth in rich media (e.g. Luria Broth) a portion of the lipid A contains a pyrophosphate group at the 1-position yielding a tris-phosphorylated species36 (Figure 1). Kdo (3-deoxy-D-manno-octulosonic acid), is attached at the 6'-hydroxyl and serves as a bridge to link lipid ...

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Isolation and Chemical Characterization of Lipid A from Gram-negative Bacteria

Isolation and characterization of the lipid A domain of lipopolysaccharide (LPS) from gram-negative bacteria provides insight into cell surface based mechanisms of antibiotic resistance, bacterial survival and fitness, and how chemically diverse lipid A molecular species differentially modulate host innate immune responses.

Lipopolysaccharide (LPS) is the major cell surface molecule of gram-negative bacteria, deposited on the outer leaflet of the outer membrane bilayer. LPS ...

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31.4K Views.  The University of Texas at Austin. Isolation and characterization of the lipid A domain of lipopolysaccharide (LPS) from gram-negative bacteria provides insight into cell surface based mechanisms of antibiotic resistance, bacterial survival and fitness, and how chemically diverse lipid A molecular species differentially modulate host innate immune responses.