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

Summary

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.

Abstract

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.

Introduction

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 bilayer^1,2. 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 hydrolysis^1,2. The standard lipid A molecule can be chemically defined as a diglucosamine backbone that is hexa-acylated and bis-phosphorylated....

Protocol

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 avoid the production of phosgene, a highly reactive acid chloride. PTFE centrifuge tubes and rotary evaporator flasks should be rinsed with methanol ....

Results

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 .......

Discussion

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.......

Materials

List of materials used in this article

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