Department of Microbiology, Immunology, & Cancer Biology, University of Virginia Health System
This article is a part ofJoVE Immunology and Infection. If you think this article would be useful for your research, please recommend JoVE to your institution's librarian.Recommend JoVE to Your Librarian
Current Access Through Your IP Address
Current Access Through Your Registered Email Address
Davis, Jr., M. R., Goldberg, J. B. Purification and Visualization of Lipopolysaccharide from Gram-negative Bacteria by Hot Aqueous-phenol Extraction. J. Vis. Exp. (63), e3916, doi:10.3791/3916 (2012).
Lipopolysaccharide (LPS) is a major component of Gram-negative bacterial outer membranes. It is a tripartite molecule consisting of lipid A, which is embedded in the outer membrane, a core oligosaccharide and repeating O-antigen units that extend outward from the surface of the cell1, 2. LPS is an immunodominant molecule that is important for the virulence and pathogenesis of many bacterial species, including Pseudomonas aeruginosa, Salmonella species, and Escherichia coli3-5, and differences in LPS O-antigen composition form the basis for serotyping of strains. LPS is involved in attachment to host cells at the initiation of infection and provides protection from complement-mediated killing; strains that lack LPS can be attenuated for virulence6-8. For these reasons, it is important to visualize LPS, particularly from clinical isolates. Visualizing LPS banding patterns and recognition by specific antibodies can be useful tools to identify strain lineages and to characterize various mutants.
In this report, we describe a hot aqueous-phenol method for the isolation and purification of LPS from Gram-negative bacterial cells. This protocol allows for the extraction of LPS away from nucleic acids and proteins that can interfere with visualization of LPS that occurs with shorter, less intensive extraction methods9. LPS prepared this way can be separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and directly stained using carbohydrate/glycoprotein stains or standard silver staining methods. Many anti-sera to LPS contain antibodies that cross-react with outer membrane proteins or other antigenic targets that can hinder reactivity observed following Western immunoblot of SDS-PAGE-separated crude cell lysates. Protease treatment of crude cell lysates alone is not always an effective way of removing this background using this or other visualization methods. Further, extensive protease treatment in an attempt to remove this background can lead to poor quality LPS that is not well resolved by any of the aforementioned methods. For these reasons, we believe that the following protocol, adapted from Westpahl and Jann10, is ideal for LPS extraction.
1. Preparation of Bacteria for LPS Extraction
2. Extraction of LPS
3. Representative Results
LPS samples prepared as above can be visualized by direct staining following separation on SDS-PAGE using a standard silver-staining protocol or a commercially available LPS staining kit. Alternatively, LPS separated on a polyacrylamide gel may be transferred to a nitrocellulose membrane and subjected to Western immunoblotting using LPS-specific anti-sera. For this protocol, we used the Pro-Q Emerald 300 Lipopolysaccharide Gel Stain Kit (Molecular Probes), and followed the manufacturer's instructions.
Shown in Figure 1 is a Pro-Q Emerald 300 stained 12% SDS-polyacrylamide gel. Each lane contains 15 μl of LPS prepared from different strains of Burkholderia dolosa isolated from sputum samples of cystic fibrosis patients. Different LPS banding ladder patterns, reflective of different numbers O-antigen repeating units attached to core oligosaccharide, are evident using this method; for example, samples in lane 1 and 6 have similar banding patterns to each other (boxed in red).
Figure 1. Pro-Q Emerald 300 stained LPS from Burkholderia dolosa clinical isolates. LPS from seven B. dolosa strains from an outbreak in cystic fibrosis patients were isolated as described in this protocol. Fifteen μl were separated on a 12% SDS-polyacrylamide gel, and stained with Pro-Q Emerald 300, per the manufacturer's instructions. LPS core is indicated an arrow, and LPS showing similar banding patterns of O-antigen repeats are boxed in red. M = Molecular weight marker.
We have described a method of purifying LPS away from other cellular components, including nucleic acids and proteins. This method provides high-quality LPS that can be used in a number of different visualization methods, including carbohydrate staining of SDS-PAGE gels, as shown in Figure 1. This method can be used to serotype LPS from a variety of strains, using specific anti-sera, or to show relatedness between isolates by direct visualization. For example, a recent genome-wide sequencing project in combination with LPS characterization from an outbreak of B. dolosa led to the discovery of a single nucleotide polymorphism (SNP) that correlates with the presence and absence of LPS in these strains11. We believe that this method is preferable to the protease treatment of whole-cell lysates described by Hitchcock and Brown9, as it is a relatively quick, yet rigorous enough to yield high-quality LPS for further analyses.
While we only show an example using the Burkholderia dolosa, this protocol can be adapted to other Gram-negative species as well. We have successfully used this protocol to extract and visualize LPS from other Burkholderia spp., Escherichia coli, Helicobacter pylori, Pseudomonas aeruginosa and Salmonella spp. If the protocol results in little or no LPS yield, it may be that the LPS fractionates to a different layer in the extraction steps, 2.7-2.9. To troubleshoot this, repeat the protocol and save a sample of each layer during the extraction steps, these can be visualized by staining an SDS-PAGE gel in order to determine where the LPS fractionates, and the protocol can be modified accordingly. It should also be noted that this protocol, while ideal for analytical purposes, does not yield LPS that is appropriate for other applications, such as structural analyses.
No conflicts of interest declared.
This work was supported by grants from the National Institutes of Health and the Cystic Fibrosis Foundation.
|DNase I recombinant, RNase-free||Roche Group||04716728001|
|RNase A||Roche Group||10109169001|
|Proteinase K||Fisher Scientific||BP1700|
|Tris-Saturated Phenol||Fisher Scientific||BP1750-100|
|Diethyl Ether||Thomas Scientific||C313K31|
|Pro-Q Emerald 300 Lipopolysaccharide Gel Stain Kit||Molecular Probes, Life Technologies||P20495|