Purification and visualization of lipopolysaccharide from Gram-negative bacteria by hot aqueous-phenol extraction.
ABSTRACT 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 cell(1, 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 coli(3-5), 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 virulence(6-8). 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 methods(9). 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 Jann(10), is ideal for LPS extraction.
SourceAvailable from: Alison K Criss[Show abstract] [Hide abstract]
ABSTRACT: The Neisseria gonorrhoeae (Gc) opacity-associated (Opa) proteins mediate bacterial binding and internalization by human epithelial cells and neutrophils (polymorphonuclear leukocytes, PMNs). Investigating the contribution of Opa proteins to gonococcal pathogenesis is complicated by high-frequency phase variation of the opa genes. We therefore engineered a derivative of Gc strain FA1090 in which all opa genes were deleted in-frame. Opaless Gc remained uniformly Opa-negative, whereas cultures of predominantly Opa-negative parental Gc and an intermediate lacking the "translucent" subset of opa genes (ΔopaBEGK) stochastically gave rise to Opa(+) bacterial colonies. Loss of Opa expression did not affect Gc growth. Opaless Gc survived exposure to primary human PMNs and suppressed the PMN oxidative burst akin to parental, Opa(-) bacteria. Notably, unopsonized Opaless Gc was internalized by adherent, chemokine-primed, primary human PMNs, by an actin-dependent process. When a non-phase-variable, in-frame allele of FA1090 opaD was reintroduced into Opaless Gc, the bacteria induced the PMN oxidative burst, and OpaD(+) Gc survived less well after exposure to PMNs compared to Opa(-) bacteria. These derivatives provide a robust system for assessing the role of Opa proteins in Gc biology.Journal of bacteriology 04/2013; DOI:10.1128/JB.00171-13 · 2.69 Impact Factor
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ABSTRACT: Sepsis is often initiated by invasive infection, characterized by overwhelming induction of pro-inflammatory cytokines. The incidence and mortality of sepsis and the associated development of acute kidney injury (AKI) remain high, and lines of research into potential treatments are needed. This study was conducted to investigate effects of alpha-lipoic acid (ALA) on septic AKI in vitro. ALA of 200 or 400 μM was used to pretreat rat HBZY-1 mesangial cells before commencement of 1 μg/mL lipopolysaccharide (LPS). Our data indicated that ALA pretreatment reduced LPS-stimulated release of inflammatory cytokines, such as tumor necrosis factor alpha (TNF-α), interleukin (IL)-1 beta (IL-1β), as well as IL-6, in HBZY-1 cell supernatant. Moreover, LPS-induced expression of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) was inhibited by ALA pretreatment, and consequently, the secretion levels of their respective enzymatic products prostaglandin E2 (PGE2) and nitric oxide (NO) were significantly decreased. LPS-enhanced phosphorylation of nuclear factor kappa B (NF-κB) inhibitor alpha (IκBα) and IκB kinase alpha/beta (IKKα/β) and nuclear translocation of NF-κB subunit p65 in HBZY-1 cells were inhibited by ALA pretreatment. Additionally, the NF-κB inhibitor N-acetylcysteine (NAC) exerted similar inhibitory effects as ALA on COX-2 and iNOS expression. In summary, our study demonstrates that ALA mitigates LPS-induced inflammatory responses in rat mesangial cells probably via inhibition of NF-κB signaling pathway, suggesting a therapeutic potential of ALA in AKI related to sepsis.Inflammation 06/2014; 38(2). DOI:10.1007/s10753-014-9957-3 · 1.92 Impact Factor
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ABSTRACT: Pseudomonas aeruginosa in the lungs of cystic fibrosis patients is characterized by a series of genotypic and phenotypic changes that reflect the transition from acute to chronic infection. These include the overproduction of the exopolysaccharide alginate and the loss of complete lipopolysaccharide (LPS). LPS is a major component of the Gram-negative outer-membrane and is composed of lipid A, core oligosaccharide, and O-antigen. In this report, we show that the LPS-defect of the P. aeruginosa chronic infection isolate strain 2192 is temperature sensitive. When grown at 25°C, 2192 expresses serotype O1 LPS of moderate chain length and reduced amount when compared to a wild-type serotype O1 laboratory strain (stO1). In contrast, 2192 does not express any LPS O antigen when grown at 37°C. This is the first time that a temperature-sensitive defect in O antigen production has been reported. Using complementation analyses with a constructed wbpM deletion mutant of stO1, we demonstrate that the temperature-sensitive O antigen production defect in 2192 is due to a mutation in wbpM, which encodes a UDP-4,6-GlcNAc dehydratase involved in O-antigen synthesis. The mutation, a deletion of a single amino acid (V636) from the extreme C-terminus of WbpM, renders the protein less stable than its wild-type counterpart. This residue of WbpM, which is critical for stability and function, is located outside of the recognized domains of the protein and may provide more insight into the structure-function relationship of this enzyme, which is found in all 20 serotypes of P. aeruginosa. We also identify a promoter of wbpM and map a transcriptional start site of wbpM and show that mucoidy plays a role in the loss of expression of high molecular weight LPS in this CF isolate.Journal of bacteriology 01/2013; DOI:10.1128/JB.01999-12 · 2.69 Impact Factor