Bacterial lipopolysaccharides : extraction with phenol-water and further applications of the procedure
01 Jan 1965-Vol. 5, pp 83-87
About: The article was published on 1965-01-01 and is currently open access. It has received 2741 citations till now. The article focuses on the topics: Extraction (chemistry).
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TL;DR: Biochemical variation in LPS composition can be detected in silver-stained polyacrylamide gels and could be used to preliminarily characterize the LPS chemotype before purification.
Abstract: The morphological heterogeneity of lipopolysaccharides (LPSs) among salmonella mutants with different LPS chemotypes was analyzed in silver-stained polyacrylamide gels. The biochemical differences in the LPS chemotypes were reflected in the unique profiles of the purified LPSs. The LPS profiles in the whole-cell lysates were also unique for each chemotype. (Whole-cell lysates were assessed by a method which preferentially silver stains LPS and by a proteinase K digest of whole-cell lysates. The silver-stained LPS profiles of proteinase K-digested lysates were similar to the homologous purified LPS and could be used to preliminarily characterize the LPS chemotype before purification.) In summary, biochemical variation in LPS composition can be detected in silver-stained polyacrylamide gels.
2,003 citations
870 citations
TL;DR: It is shown that for intact L. interrogans, it is LPS, not lipoprotein, that constitutes the predominant signaling component for macrophages through a TLR2 pathway, providing a basis for understanding the innate immune response caused by leptospirosis.
Abstract: Leptospira interrogans are zoonotic pathogens that have been linked to a recent increased incidence of morbidity and mortality in highly populated tropical urban centers. They are unique among invasive spirochetes in that they contain outer membrane lipopolysaccharide (LPS) as well as lipoproteins. Here we show that both these leptospiral outer membrane constituents activate macrophages through CD14 and the Toll-like receptor 2 (TLR2). Conversely, it seems that TLR4, a central component for recognition of Gram-negative LPS, is not involved in cellular responses to L. interrogans. We also show that for intact L. interrogans, it is LPS, not lipoprotein, that constitutes the predominant signaling component for macrophages through a TLR2 pathway. These data provide a basis for understanding the innate immune response caused by leptospirosis and demonstrate a new ligand specificity for TLR2.
696 citations
TL;DR: A LPS isolation procedure which is effective in extracting both smooth and rough LPS in high yields and with a high degree of purity is developed and demonstrated by comparing LPS preparations obtained from wild-type and mutant strains of P. aeruginosa.
Abstract: Lipopolysaccharide (LPS) is a major component of the outer membrane of gram-negative bacteria. It is now well established that within a single organism, size heterogeneity of this molecule can exist. We have developed a LPS isolation procedure which is effective in extracting both smooth and rough LPS in high yields (51 to 81% of the LPS present in whole cells as quantitated by using hydroxy fatty acid, heptose, and 2-keto-3-deoxyoctonate yields) and with a high degree of purity. The contamination by protein (0.1% by weight of LPS), nucleic acids (1%), lipids (2 to 5%), and other bacterial products was low. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the LPS demonstrated the presence of a high degree of size heterogeneity in the isolated smooth LPS as well as the presence of significant amounts of rough-type LPS. The Pseudomonas aeruginosa LPS interacted well with a monoclonal antibody in a variety of immunochemical analyses. The usefulness of the procedure was demonstrated by comparing LPS preparations obtained from wild-type and mutant strains of P. aeruginosa and Salmonella typhimurium. For example, it was shown that the LPS of an antibiotic supersusceptible mutant Z61 of P. aeruginosa, which was previously characterized as identical to wild type with respect to the ratio of smooth to rough LPS molecules isolated by the phenol-water procedure, actually contained only a small proportion of O-antigenic side chains.
652 citations
TL;DR: It is shown that A. baumannii can rapidly develop resistance to polymyxin antibiotics by complete loss of the initial binding target, the lipid A component of lipopolysaccharide (LPS), which has long been considered to be essential for the viability of Gram-negative bacteria.
Abstract: Infections caused by multidrug-resistant (MDR) Gram-negative bacteria represent a major global health problem. Polymyxin antibiotics such as colistin have resurfaced as effective last-resort antimicrobials for use against MDR Gram-negative pathogens, including Acinetobacter baumannii. Here we show that A. baumannii can rapidly develop resistance to polymyxin antibiotics by complete loss of the initial binding target, the lipid A component of lipopolysaccharide (LPS), which has long been considered to be essential for the viability of Gram-negative bacteria. We characterized 13 independent colistin-resistant derivatives of A. baumannii type strain ATCC 19606 and showed that all contained mutations within one of the first three genes of the lipid A biosynthesis pathway: lpxA, lpxC, and lpxD. All of these mutations resulted in the complete loss of LPS production. Furthermore, we showed that loss of LPS occurs in a colistin-resistant clinical isolate of A. baumannii. This is the first report of a spontaneously occurring, lipopolysaccharide-deficient, Gram-negative bacterium.
645 citations
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...LPS was purified from overnight cultures of A. baumannii using the previously described method of Westphal and Jann (35)....
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...To assess the ability of the A. baumannii wild-type, lpxA mutant, and complemented lpxA mutant strains to produce LPS, we purified carbohydrate from each strain using the hot phenol LPS extraction method of Westphal and Jann (36) and analyzed the purified material using PAGE, followed by carbohydrate-specific silver staining (Fig....
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...baumannii wild-type, lpxA mutant, and complemented lpxA mutant strains to produce LPS, we purified carbohydrate from each strain using the hot phenol LPS extraction method of Westphal and Jann (36) and analyzed the purified material using PAGE, followed by carbohydrate-specific silver staining (Fig....
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