Thermodynamic analysis of the lipopolysaccharide-dependent resistance of gram-negative bacteria against polymyxin B

Abstract

Cationic antimicrobial cationic peptides (CAMP) have been found in recent years to play a decisive role in hosts' defense against microbial infection. They have also been investigated as a new therapeutic tool, necessary in particular due to the increasing resistance of microbiological populations to antibiotics. The structural basis of the activity of CAMPs has only partly been elucidated and may comprise quite different mechanism at the site of the bacterial cell membranes or in their cytoplasm. Polymyxin B (PMB) is a CAMP which is effective in particular against Gram-negative bacteria and has been well studied with the aim to understand its interaction with the outer membrane or isolated membrane components such as lipopolysaccharide (LPS) and to define the mechanism by which the peptides kill bacteria or neutralize LPS. Since PMB resistance of bacteria is a long-known phenomenon and is attributed to structural changes in the LPS moiety of the respective bacteria, we have performed a thermodynamic and biophysical analysis to get insights into the mechanisms of various LPS/PMB interactions in comparison to LPS from sensitive strains. In isothermal titration calorimetric (ITC) experiments considerable differences of PMB binding to sensitive and resistant LPS were found. For sensitive LPS the endothermic enthalpy change in the gel phase of the hydrocarbon chains converts into an exothermic reaction in the liquid crystalline phase. In contrast, for resistant LPS the binding enthalpy change remains endothermic in both phases. As infrared data show, these differences can be explained by steric changes in the headgroup region of the respective LPS.

FIGURE 1

Chemical structures of LPSs from sensitive (S. minnesota R595) and resistant (P. mirabilis R45) strains according to Wiese et al. (3). Both LPS have nonstoichiometric substituents (acyloxyacyl chain C-16 in position 2 of the glucosamine I, amino-arabinose linked to the 4′-phosphate of glucosamine II). LPS R45 has a nonstoichiometric (∼50%) substitution of an amino-arabinose (dark label), which leads to a reduction of the negative net charge as compared to LPS R595. Furthermore, the amide-bound acyl chain in position 2′ consists of a C-14 rather than a C-12 chain. Measured charges are −3.4 for LPS R595 and −3.0 for LPS R45 (3).

FIGURE 2

Isothermal calorimetric titration of LPS (0.05 mM) from P. mirabilis R45 (top) and S. minnesota R595 (bottom) with PMB (3 mM) at 37°C. The LPS dispersion in the calorimetric cell was titrated every 5 min with 3 μl of PMB. The increase in the feedback power indicates an endothermic, the decrease an exothermic process.

FIGURE 3

Enthalpy change of the LPS R45:PMB reaction versus [PMB]/[LPS] molar ratio between 25°C and 50°C, indicating exclusively endothermic reactions.

FIGURE 4

Gel to liquid crystalline (β↔α) phase transition of the hydrocarbon chains of LPS R595 (A) and LPS R45 (B) at different PMB concentrations. The peak position of the symmetric stretching vibration of the methylene groups ν_s(CH₂) is plotted versus temperature.

FIGURE 5

DSC heat capacity curves of mixtures of PMB with LPS R595 (left column) and R45 (right column) in various molar ratios. LPS concentrations were 1 mg/ml.

FIGURE 6

Infrared spectrum of LPS R595 in the region of phosphate and sugar vibrations in the absence (top) and presence (bottom) of an equimolar content of PMB. The band components at 1260–1220 cm⁻¹ correspond to modes from the antisymmetric stretching vibration ), the band components 1178–1030 cm⁻¹ to unspecific sugar ring vibrations (36).

FIGURE 7

Infrared spectrum of LPS R45 in the region of phosphate and sugar vibrations in the absence (top) and presence (bottom) of an equimolar content of PMB.

FIGURE 8

Synchrotron radiation small angle x-ray diffraction of LPS R595 and R45 alone (top) and in the presence of an equimolar content of PMB (bottom). The logarithm of the scattering intensity (log I) is plotted versus scattering vector s = 1/d (d-spacings in nm).

FIGURE 9

Gel to liquid crystalline (β↔α) phase transition of the hydrocarbon chains of LPS B.a. lpcC (A) and LPS B.a.2308 (B) at different PMB concentrations. The peak position of the symmetric stretching vibration of the methylene groups ν_s(CH₂) is plotted versus temperature.

FIGURE 10

DSC heat capacity curves of mixtures of PMB with LPS lpcC (left column) and LPS. 2308 (right column) in various molar ratios. LPS concentrations were 1 mg/ml.

FIGURE 11

Enthalpy change of the LPS 2308/PMB reaction versus [PMB]/[LPS] molar ratio between 25°C and 45°C, indicating exclusively endothermic reactions.

FIGURE 12

Enthalpy change of the LPS lpcC:PMB reaction versus [PMB]/[LPS] molar ratio between 25°C and 55°C, indicating endothermic reactions at 25°C and 35°C and exothermic reactions above.