The lipopolysaccharide (LPS) of Chlamydia psittaci was extracted from yolk sac-grown elementary bodies, purified, and characterized chemically, immunochemically, and biologically. The LPS contained Dgalactosamine, D-glucosamine, phosphorus, long-chain fatty acids, and 3-deoxy-D-manno-2-octulosonic acid in the molar ratio of approximately 1:2:2:6:5. The antigenic properties of the isolated LPS were compared with those of the LPS from Chiamydia trachomatis and Salmonella minnesota Re by the passive hemolysis and passive hemolysis inhibition tests, absorption, hydrolysis kinetics, and Western blot analysis with rabbit polyclonal antisera against chlamydiae and with a mouse monoclonal antibody recognizing a genus-specific epitope of chlamydial LPS. Two antigenic determinants were identified, one of which was chlamydia specific and the other of which was cross-reactive with Re LPS. Both determinants were destroyed during acid hydrolysis, whereby a third antigen specificity was exposed which was indistinguishable from the lipid A antigenicity. In rabbit polyclonal antisera prepared against Formalin-killed elementary bodies or detergent-solubilized membranes, two antibody specificities were differentiated. One of these was chlamydia specific, and the other was cross-reactive with Re LPS. The LPS of C. psittaci was inactive within typical endotoxin parameters (lethal toxicity, pyrogenicity, local Shwartzman reactivity); it was, however, active in some in vitro assays, such as those testing for mouse B-cell mitogenicity and the induction of prostaglandin E2 in mouse peritoneal macrophages.

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...