3-C-Branched aldoses in lipopolysaccharide of phase I Coxiella buurnetii and their role as immunodominant factors

Abstract

Mild acid hydrolysis with 1% acetic acid (lOO°C, of lipopolysaccharide (LPS) isolated fromCoxiella burnetii phase I cells leads to a drastic decrease in its serological reactivity as shown by the passive hemolysis test. This decrease in reactivity occurs parallel or even prior to the cleavage of LPS into free lipid A and the polysaccharide moiety. During this mild hydrolysis two unusual sugars (X and Y) are released from the LPS, which were obtained in pure state by thin-layer chromatography. Analysis of their alditol acetate derivatives by gas chromatography/mass spectrometry revealed that sugar X is a 6-deoxy-3-C-methyl-hexose and sugar Y a 3-C-(hydroxymethyl)-pentose. Using a range of authentic standards and different thin-layer and gas chromatographic conditions, X could be recognized as 6-deoxy-3-C-methyl-gulose (virenose), very probably as the L form of this sugar (L-virenose). Y has been identified as 3-C-(hydroxymethyl)-lyxose (dihydrohydroxystreptose) by comparigg it with newly synthesized 3-C-(hydroxymethyl)-pentoses (Dahlman, O., Garegg, P. J., Mayer, H., Schramek, S., unpublished results). Both branched sugars are (at least partially) in terminal positions since methylation analysis of LPS afforded (mainly) their permethylated derivatives. This analysis further showed virenose to be linked in C. burnetii phase I LPS as pyranose and dihydro-hydroxystreptose as furanose. The terminal linkage and the chemical nature of X and Y are in accordance with the observed acidlability of the serological determinants.The phenomenon of phase variation exhibited by Coxiella burnetii resembles in many aspects the well-known S+R variation found with many gram-negative bacteria. This phenomenon is connected with changes in the composition and structure of C. burnetii lipopolysaccharide (LPS). Phase I LPS of C. burnetii (smooth type) contains a number of sugars which are lacking in phase I1 LPS (rough type) [l]. Two of them, which can be released from phase I LPS by mild acid hydrolysis and which are involved in the serological activity of the C. burnetii LPS, have been isolated in a pure state and have been characterized as unusual 3-C-branched sugars. MATERIALS AND METHODSPreparation of phase Z LPS. C. burnetii strain Nine Mile, serologically in phase I (yolk sac passage 3, cloned in cell cultures), was propagated in yolk sacs of embryonated hen eggs. The C. burnetii cells were killed by 1% phenol and purified as described previously [2]. The purified rickettsia1 cells were treated with chloroform/methanol [l] and LPS was extracted by the phenol/water procedure and purified further as described previously [3].Mild acid hydrolysis. Liberation of acid-labile-linked sugars from C. burnetii phase I LPS was achieved by mild acid hydrolysis using 1 % or 10% acetic acid at 100 "C for different time intervals. Chromatography and combinedgas liquid chromatography/ mass spectrometry. For separation of the sugars liberated by mild acid hydrolysis, thin-layer chromatography on cellulose plates was used. The developing system wa...

Electron Microscopic Studies of Lipopolysaccharides from Phase I and Phase II Coxiella burnetii

“…These LPSs form bilayer membranes in aqueous solutions (Shands, 1971 ;Shands et al, 1967) with the polysaccharide moiety facing outwards. The hydrophilic and hydrophobic properties of phase I and phase I1 LPS, respectively (Schramek & Mayer, 1982;Amano & Williams, 1984;Schramek et al, 1985) suggest that the carbohydrate side chain contributes to the physico-chemical differences. Both LPSs from C. burnetii also formed the bilayer structures as shown in Fig.…”

Chromosomal DNA Deletions Explain Phenotypic Characteristics of Two Antigenic Variants, Phase II and RSA 514 (Crazy), of theCoxiella burnetiiNine Mile Strain

“…The JB153-7 product has very good homology to fucose synthetase and should complete the synthesis of fucose from the 4-keto sugar by providing the GDP-mannose epimerasereductase (46% identity for most of the protein) activity necessary to effect epimerization of CH 3 at C-5 and epimerization of OH at C-3. However, the presence of fucose in C. burnetii LPS is doubtful, as none of the three independent groups of workers who previously studied the carbohydrate structure of this organism reported the presence of fucose (2,3,8,37). Fucose may be used elsewhere in the organism, such as in a colanic acid-like molecule (20,39,59) or in a capsular polysaccharide.…”

“…Phase II LPS possesses 2-keto-3-deoxyoctulonosic acid (KDO), D-mannose, D-glycero-D-manno-heptose, lipid A or a lipid A analog, and a very complex mixture of fatty acids, many of which are branched (2,36,47,48,58). Phase I LPS also possesses these components but, in addition, has virenose, dihydrohydroxystreptose (3,37,47), and galactosaminuronyl-␣(1,6)-glucosamine (3). These observations are consistent with a smoothto-rough transition analogous to that seen in gram-negative enteric bacteria (2,13,17,36).…”

Antigenic Characteristic of the Lipopolysaccharides of Coxiella burnetii Isolates.

“…However, some studies on the Coxiella LPSs have shown that the sugar composition and structure of polysaccharide moiety and lipid A content produce the unique banding profile on SDS-PAGE, and that three sugars galactosaminuronyl-α(1-6)-glucosamine, dihydrohydroxystreptose and virenose make up the dominant immunogenic and antigenic determinants [1,2,14,15,20]. Collectively, these results may indicate that sugar composition, size and shape of the O-specific polysaccharide chains on the LPSs of four groups are different to each other, although many epitopes are shared.…”