https://mmbr.asm.org/content/mmbr/47/3/410.full.pdf

Linkage map of Salmonella typhimurium, edition VII.

CHAPTER 4 – Isolation and Chemical and Immunological Characterization of Bacterial Lipopolysaccharides

The lipid-A component of lipopolysaccharides, when presented in a suitable form, acts as an immunogen in rabbits, giving rise to the production of circulating antibodies. Highest anti-lipid-A titers were obtained on immunization with acid-treated bacterial cells coated with lipid A. Anti-lipid-A activity of the immune sera could be assayed by passive hemolysis of lipid-A-sensitized erythrocytes in the presence of complement. On filtration of the immune globulins on Sephadex G-200, antibodies against lipid A were detected in the IgM and IgG fractions. Lipid A preparations derived from various Salmonella and one Echerichica coli strain exhibited strong serological cross reactions with anti-lipid-A antisera. Furthermore, lipid-A antiserum also cross-reacted with all lipopolysaccharides tested, although in some cases to a low degree. On the other hand, only 8 out of 13 antibacterial antisera interacted with lipid-A-coated red cells. Finally, it was found that anti-lipid-A antibodies are effective in sensitizing E. coli 0111 bacteria for intraperitoneal phagocytosis.

Preparation and Properties of Antisera against the Lipid‐A component of Bacterial Lipopolysaccharides

A number of lipopolysaccharides derived from Salmonella and Escherichia coli S and R mutant strains were tested for toxicity and anticomplementary activity in the absence of added antiserum. Although all preparations were toxic, only a few exhibited high anticomplementary activity, while others proved to be of low or negligible activity. It was found that isolated lipid A from both active and inactive lipopolysaccharides was strongly anticomplementary as well as toxic, when made water-soluble with the aid of suitable carriers. Treatment of R form lipopoly-saccharide with Mg2+ or Ca2+ led to complete precipitation of the lipopolysaccharide with consequent loss of toxicity and anticomplementary activity. This treatment had practically no effect on the anticomplementary activity and toxicity of S form lipopolysaccharides. When lipopolysaccharide, after reaction with complement, was reisolated and purified, the resulting preparation was found to be non-toxic and of negligible anticomplementary activity. No detectable alterations in either the sugar or the fatty acid composition could be detected. The only significant change was the loss of solubility in water. Treatment of the reisolated lipopolysaccharide with EDTA completely restored solubility in water, toxicity, and anti-complementary activity.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1971.tb01298.x

Interaction of Lipopolysaccharides and Lipid A with Complement

We have cloned two open reading frames (orf6 and orf8) from the Escherichia coli K-12 rfb region. The genes were expressed in E. coli under control of the T7lac promoter, producing large quantities of recombinant protein, most of which accumulated in insoluble inclusion bodies. Sufficient soluble protein was obtained, however, for use in a radiometric assay designed to detect UDP-galactopyranose mutase activity (the conversion of UDP-galactopyranose to UDP-galactofuranose). The assay is based upon high-pressure liquid chromatography separation of sugar phosphates released from both forms of UDP-galactose by phosphodiesterase treatment. The crude orf6 gene product converted UDP-[alpha-D-U-14C]-galactopyranose to a product which upon phosphodiesterase treatment gave a compound with a retention time identical to that of synthetic alpha-galactofuranose-1-phosphate. No mutase activity was detected in extracts from cells lacking the orf6 expression plasmid or from orf8-expressing cells. The orf6 gene product was purified by anion-exchange chromatography and hydrophobic interaction chromatography. Both the crude extract and the purified protein converted 6 to 9% of the UDP-galactopyranose to the furanose form. The enzyme was also shown to catalyze the reverse reaction; in this case an approximately 86% furanose-to-pyranose conversion was observed. These observations strongly suggest that orf6 encodes UDP-galactopyranose mutase (EC 5.4.99.9), and we propose that the gene be designated glf accordingly. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified UDP-galactopyranose mutase revealed one major band, and analysis by electrospray mass spectrometry indicated a single major species with a molecular weight of 42,960 +/- 8, in accordance with that calculated for the Glf protein. N-terminal sequencing revealed that the first 15 amino acids of the recombinant protein corresponded to those expected from the published sequence. UV-visible spectra of purified recombinant enzyme indicated that the protein contains a flavin cofactor, which we have identified as flavin adenine dinucleotide.

Galactofuranose biosynthesis in Escherichia coli K-12: identification and cloning of UDP-galactopyranose mutase.

The structure of the lipopolysaccharide from Salmonella friedenau T1 form has been investigated. The T1-specific chains are composed of almost equal amounts of β-d-ribofuranose and β-d-galactofuranose residues. The former are linked to the 2-position, the latter to the 3-position or, to a smaller extent, to the 6-position. A low percentage of branching, through β-d-galactofuranose residues, substituted in the 3- and 6-position, is demonstrated. The core region of the T1 lipopolysaccharide seems to have the same general structure as that of other Salmonella lipopolysaccharides.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1969.tb00779.x

Structural Investigations on T1 Lipopolysaccharides

The composition of lipopolysaccharides derived from various Salmonella T forms was studied. All T1-form lipopolysaccharides examined contained 14 to 22% each of both d-galactose and pentose in addition to 4 to 9% each of ketodeoxyoctonic acid, heptose, d-glucosamine, and d-glucose. The pentose was identified as d-ribose. The T2-form lipopolysaccharide examined did not contain a significant amount of pentose, nor more than the usual amounts of d-galactose. Periodate oxidation of T1 (lipo) polysaccharides followed by NaBH4 reduction revealed that ribose was almost quantitatively protected, galactose was destroyed, and threitol and mannose were newly formed. The latter two products probably originated from 4-linked galactose and heptose, respectively. Ribose and galactose were found in specific precipitates of T1 lipopolysaccharide with anti-T1 antiserum but were not found in specific precipitates of alkali-treated T1 lipopolysaccharide and of Freeman degraded polysaccharide with anti-T1 serum Ribose and galactose are present in these degraded preparations in the form of nondialyzable polymers. The T1-form mutant lipopolysaccharides lacked the O-specific sugars constituting the side-chains in the wild-type antigens. They did not produce the soluble O-specific haptenic polysaccharide known to be accumulated in RI strains. With these properties, T1 lipopolysaccharides resemble RII lipopolysaccharides. Like RII degraded polysaccharides, T1-degraded polysaccharides also contained glucosamine. Furthermore, strong cross-reactions were found to exist between T1 and RII lipopolysaccharides in both hemagglutination inhibition assays and in precipitation tests. It is proposed that T1 lipopolysaccharides represent RII lipopolysaccharides to which polymers consisting of ribose and galactose are attached.

M. Berst

Papers

Structural Investigations on T1 Lipopolysaccharides

Lipopolysaccharides of Salmonella T Mutants