Physicochemical characterization of the endotoxins from Coxiella burnetii strain Priscilla in relation to their bioactivities

Abstract

Background: Coxiella burnetii is the etiological agent of Q fever found worldwide. The microorganism has like other Gram-negative bacteria a lipopolysaccharide (LPS, endotoxin) in its outer membrane, which is important for the pathogenicity of the bacteria. In order to understand the biological activity of LPS, a detailed physico-chemical analysis of LPS is of utmost importance.

Results: The lipid A moiety of LPS is tetraacylated and has longer (C-16) acyl chains than most other lipid A from enterobacterial strains. The two ester-linked 3-OH fatty acids found in the latter are lacking. The acyl chains of the C. burnetii endotoxins exhibit a broad melting range between 5 and 25 degrees C for LPS and 10 and 40 degrees C for lipid A. The lipid A moiety has a cubic inverted aggregate structure, and the inclination angle of the D-glucosamine disaccharide backbone plane of the lipid A part with respect to the membrane normal is around 40 degrees. Furthermore, the endotoxins readily intercalate into phospholipid liposomes mediated by the lipopolysaccharide-binding protein (LBP). The endotoxin-induced tumor necrosis factor alpha (TNFalpha) production in human mononuclear cells is one order of magnitude lower than that found for endotoxins from enterobacterial strains, whereas the same activity as in the latter compounds is found in the clotting reaction of the Limulus amebocyte lysate assay.

Conclusions: Despite a considerably different chemical primary structure of the C. burnetii lipid A in comparison with enterobacterial lipid A, the data can be well understood by applying the previously presented conformational concept of endotoxicity, a conical shape of the lipid A moiety of LPS and a sufficiently high inclination of the sugar backbone plane with respect to the membrane plane. Importantly, the role of the acyl chain fluidity in modulating endotoxicity now becomes more evident.

Figure 1

Chemical structures of two major molecular species present in the lipid A from C. burnetii strain Priscilla

Figure 2

Peak position of the symmetric stretching vibration of the methylene groups versus temperature for lipid A and LPS from C. burnetii strain Priscilla. In the gel phase of the hydrocarbon chains, wavenumber values are in the range 2850.0 to 2850.5 cm^-1, in the liquid crystalline phase the respective values are 2852.5 to 2853.0 cm^-1

Figure 3

Heat capacity curves (1^st to 3^rd heating-scan, h.s.) of lipid A from C. burnetii strain Priscilla from differential scanning calorimetry. The phase transition enthalpies can be calculated from the areas of the transition and are 9, 7, and 6 kJ/mole for the respective scans.

Figure 4

0° and 90° polarized infrared spectra of lipid A from C. burnetii strain Priscilla in the wavenumber range 1500 to 900 cm^-1. The vibrational bands at 1171 and 1040 cm^-1 correspond to diglucosamine ring vibrations.

Figure 5

Synchrotron radiation X-ray diffraction pattern of lipid A from C. burnetii strain Priscilla at 40°C and 90 % water content. The logarithm of the scattering intensity log I is plotted versus the scattering vector s = 1/d = 2 sin θ / λ (2θ = scattering angle, λ = wavelength = 0.15 nm)

Figure 6

LBP-mediated intercalation of lipid A from C. burnetii strain Priscilla and S. minnesota strain R595 into phosphatidylserine liposomes from the ratio of the donor fluorescence intensity I_D to that of the acceptor I_A. At 50 s, lipid A was added to the liposomes, and after 100 s, LBP was added.

Figure 7

Production of tumor-necrosis-factor α (TNFα) of human mononuclear cells induced by different concentrations of LPS and lipid A from Salmonella minnesota strain R595 and C. burnetii strain Priscilla. The data result from one representative experiment. The mean and standard deviation are based on the data from the determination of TNFα in duplicate at two different dilutions. A repetition of the experiments yielded the same dependences except for the absolute amount of TNFα-production which may vary significantly between different donors.