Investigation into the fluidity of lipopolysaccharide and free lipid A membrane systems by Fourier‐transform infrared spectroscopy and differential scanning calorimetry

Abstract

The phase behaviour, particularly the fluidity within each phase state and the transitions between them, of lipopolysaccharides and of their lipid moiety, free lipid A, of various species of Gram-negative bacteria, especially of Salmonella minnesota and Escherichia coli, has been investigated by applying mainly Fourier-transform infrared spectroscopy and differential scanning calorimetry. For enterobacterial strains, the transition temperatures of the gel-liquid crystalline (P-cI) phase transition of the hydrocarbon chains in dependence on the length of the sugar moiety are highest for free lipids A (around 45 "C) and lowest for deep rough mutant lipopolysaccharides (around 30 "C). Evaluating certain infrared active vibration bands of the hydrocarbon moiety, mainly the symmetric stretching vibration of the methylene groups around 2850 cm-', it was found that, in the gel state, the acyl chains of lipopolysaccharides and free lipid A have a higher fluidity as compared with saturated and the same fluidity as compared with unsaturated phospholipids. This 'partial fluidization' of lipopolysaccharide below the transition temperature correlates with its reduced enthalpy change at that temperature compared to phospholipids with the same chain length. The fluidity depends strongly on ambient conditions, i.e. on the Mg2+ and H + content: higher Mg2+ concentrations and low pH values make the acyl chains of free lipid A and lipopolysaccharide preparations significantly more rigid and also partially increase the transition temperature. The influence of Mg2 + is highest for free lipid A and decreases with increasing length of the sugar side chain within the lipopolysaccharide molecules, whereas the effect of a low pH is similar for all preparations. At basic pH, a fluidization of the lipopolysaccharide and lipid A acyl chains and a decrease in transition temperature take place. Free lipid A and all investigated rough mutant lipopolysaccharides exhibit an extremely strong lyotropic behaviour in the p-a melting enthalpy but not in the value of the transition temperature. The phase transition is distinctly expressed only at water concentrations higher than 50-60%. A further increase of the water content still leads to an increase in the phase-transition enthalpy, particularly for lipopolysaccharides with a more complete sugar moiety.The fluidity of the hydrocarbon chains is shown to be an important parameter with respect to the expression of biological activities. Thus, for the various mutant and the wild-type lipopolysaccharides and free lipid A from S. minnesota in a number of different biological test systems reported in the literature, the rough mutants, especially Re and Rd lipopolysaccharides with the lowest transition temperature, i.e. the highest fluidity at 37"C, induce the highest biological activities; smooth (S) form lipopolysaccharide and free lipid A with their higher transition temperatures, i.e. a lower fluidity at 37 "C, have significantly lower activities.The interaction of lipopolysaccharides, the major amphip...

Yersinia pseudotuberculosis and Yersinia pestis show increased outer membrane permeability to hydrophobic agents which correlates with lipopolysaccharide acyl-chain fluidity

“…Moreover, the same effects could explain the increase in acyl-chain fluidity observed for the LPS of Y . pseudotuberculosis grown at 37 "C. However, as phase transitions have been observed for R LPSs of enterobacteria under conditions similar to those used in this study (Brandenburg & Seydel, 1984, 1990 In a previous study we have shown that the OMS of Y . enterocolitica pathogenic biogroups, but not those of non-pathogenic biogroups, become permeable to hydrophobic agents when grown with Mg2+ but no Ca2+ available (Bengoechea et al, 1996).…”

“…The variations in LPS acyl-chain fluidity (and the concomitant variations in LPS-LPS interactions) must have a structural explanation. Since electrostatic and/or hydrophilic interactions between polysaccharide moieties of LPSs can decrease the overall LPS fluidity (Brandenburg & Seydel, 1984, 1990) and the LPS polysaccharide length decreased in the order Y . enterocolitica-Y.…”

Lipopolysaccharide, a Key Molecule Involved in the Synergism between Temporins in Inhibiting Bacterial Growth and in Endotoxin Neutralization

“…This can be interpreted in part to result from the reaction between the divalent cations and the negatively charged groups, mainly the phosphate groups of the disaccharide moiety of lipid A. Indeed, this should rigidify the structure of LPS to a higher degree than that of LPS with longer sugar chains, to which Mg 2ϩ cations can distribute over a much larger region (64), also increasing the volume occupied by water molecules, in a disordered assembly (63).…”

Molecular Basis of Bacterial Outer Membrane Permeability Revisited

“…Nevertheless, the study of the latter system gives us some interesting pieces of information. In more recent studies (89,604), it was shown that Mg 2ϩ raises the melting temperature of lipid A. Furthermore, the melting temperature of lipid A measured by Fourier transform infrared spectroscopy was about 57°C, in comparison with that of dioleoylglycerophosphate (about 3°C) (89), showing clearly that lipid A hydrocarbons are in a much more strongly ordered state.…”