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

Abstract

Lipopolysaccharides from phase I (LPSI) Coxiella burnetii Ohio and Nine Mile strains and from phase I1 (LPSII) Nine Mile strain were stained negatively and positively and examined with the electron microscope. The ultrastructure of LPSI and LPSII positively stained with uranyl formate or uranyl acetate was ribbon-like. When negatively stained with uranyl acetate, LPSI was ribbon-like but LPSII exhibited hexagonal lattice structures. However, LPSII stained negatively with sodium phosphotungstate and ammonium molybdate exhibited hexagonal lattice ultrastructures which were not identical to those observed when negatively stained with uranyl acetate. The hexagonal lattice structures formed in vitro were due to the interactions of LPSII and the staining reagents rather than to protein-LPS interactions. The differences in the ultrastructures of LPSI and LPSII are undoubtedly based on variations in their chemical composition.

Right on Q: Genetics begin to Unravel Coxiella Burnetii host cell Interactions

“…During serial passage in immunoincompetent hosts, such as embryonated hen's eggs and tissue culture cells, or synthetic media [21], variants emerge within the population that produce truncated LPS. Extensive passage (∼90 passages) culminates in avirulent organisms with a severely truncated, deep-rough LPS consisting of lipid A and core sugars, but no O-antigen [22][23][24]. This change in LPS structure corresponds with a change in reactivity to postvaccination sera referred to as phase variation.…”

“…Collectively, these observations suggest multiple mutational pathways can lead to phase transition. Nonetheless, a plaque-cloned Phase II variant (NMII) of the Nine Mile Phase I (NMI) strain containing a large chromosomal deletion is widely used by Coxiella researchers, as the strain is considered nonrevertable to full virulence and thus suitable for use at biosafety level-2 [23,30]. All other strains are considered biosafety level-3 organisms.…”

Biochemical and Immunological Comparison of Lipopolysaccharides from Bordetella Species

“…glucoheptose (Sigma) as a reference; KDO was determined by the thiobarbituric acid method of Karkhanis et al (1978) with a small modification (2 M-HCl hydrolysis for 90 min instead of 0.05 M-H,SO, hydrolysis for 30 min) with KDO (Sigma) as a reference; protein was assayed by the Lowry method; total phosphorus was determined by the method of Lowry et al (1954); amino acids and amino compounds, including amino sugars, were analysed in a K202 amino acid autoanalyser (Kyowa Seimitzu Co.) after hydrolysis for 4 h in 4 M-HCl at 100 "C in sealed ampoules (Amano et a/., 1987); fatty acids were analysed as methyl esters in a Hitachi 163 gas chromatograph on a Chromosorb WAW column containing 15% ethylene glycol succinate (Gasukuro Kogyo Co.) as described previously (Amano et a/., 1984). Identification and quantitative analysis of neutral sugars were done in a Hitachi 163 gas chromatograph with the sugars as alditol acetates on a 3% ECNSS-M Gas-Chrom Q glass column (Gasukuro Kogyo Co.) as described previously (Amano & Williams, 1984), and as trimethylsilyl derivatives on a 2.5 % SE-30 Chromosorb WA W column (Gasukuro Kogyo Co.). Laemmli (1970) was used, with the following modifications.…”

Genetic Diversity of the Q Fever Agent,Coxiella burnetii, Assessed by Microarray-Based Whole-Genome Comparisons

“…Serial in vitro passage of phase I C. burnetii in embryonated eggs or tissue culture results in LPS molecules with decreasing molecular weights, culminating in the severely truncated LPS of avirulent phase II organisms. Phase II LPS contains lipid A and some core sugars but is missing O-antigen sugars and appears to represent the minimal LPS structure of C. burnetii (1,30,74). Two cloned LPS variants of the virulent Nine Mile phase I (NMI) isolate have been described: Nine Mile Crazy (NMC; intermediate virulence), producing an intermediate-length LPS, and Nine Mile phase II (NMII; avirulent), producing a severely truncated phase II LPS (30,43).…”