Structural studies of lipid A from a lipopolysaccharide of the Coxiella burnetii isolate RSA 514 (Crazy).

Institute of Microbiology, v.v.i, CAS, Prague, Czech RepublicCoxiella burnetii is the aetiological agent of Q fever.LPS is a major factor of virulence of the bacterium,and therefore studies of its structure⁄functionrelationship studies are of potential interest. Invirulent phase I, C. burnetii biosynthesises smoothLPS I with an O-specific chain, whereas inavirulent phase II, it synthesises rough LPS II[1]. Both LPSs were isolated [1] from the C.burnetii isolates RSA 493, clone 7, and RSA 439,clone 4, respectively. We investigated an LPSfrom the C. burnetii clonal derivative RSA 514named ‘Crazy’ (Cr), which was isolated from theplacental tissue of a guinea pig infected with theRSA 493 isolate for 343 days [2]. The majoremphasis was put on the lipid A as no data onits structure have been available thus far. It hasbeen recognised recently that variation of thelipid A domain of LPS serves as one strategyutilised [3] by Gram-negative bacteria to promotesurvival by providing resistance to components ofthe innate immune system and helping to evaderecognition by Toll-like receptor 4. Thus, it was ofinterest to see if the long-term survival of themicroorganism in the host led to modifications inits lipid A in comparison with the known struc-tures for lipid A from the C. burnetii isolatePriscilla [4] and also those established mostrecently in the LPS I and LPS II (P.V. Vadovicand R.T. Toman, unpublished results).The isolate Cr underwent large deletions ingenes predicted to function in LPS or lipooligo-saccharide biosynthesis [5]. Thus, it was of inter-est to compare chemical compositions of the lipidA proximal regions in the LPS Cr and the LPS IIfrom the isolate RSA 439, which was also shownto undergo similar deletions [5].The LPS Cr was isolated by the hot phe-nol⁄water method and run on SDS-PAGE. LipidA was obtained on mild acid hydrolysis of theLPS Cr. Sugars were analysed as alditol acetatesand fatty acids were examined directly and aftertrimethylsilylation by GC-MS. For its detailedstructure, the lipid A was analysed by MALDI-TOF- and MALDI-TOF⁄TOF-MS techniques.In contrast to the LPS I and LPS II [1], the LPSCr gave one band at about 14 kDa on SDS-PAGE.Sugar analysis revealed the presence of