Layer formations in the bacteria membrane mimetic DPPE-DPPG/water system induced by sulfadiazine

Abstract

The effect of the frequently used antibiotic sulfadiazine (SD) was studied on a bacteria membrane mimetic model system by using differential scanning calorimetric (DSC), small- and wide-angle X-ray scattering (SWAXS) and freeze-fracture methods. The membrane model system consisted of dipalmitoylphosphatidylethanolamine (DPPE, 0.8 molar ratio) and dipalmitoylphosphatidylglycerol (DPPG, 0.2 molar ratio). The SD molar ratio (relative to the lipids) was varied between 10^− 3 and 1. In the presence of SD, two transitions between the gel and liquid crystalline phases appear at 60.5 °C and about at 65 °C. In the temperature domain of the gel phase, the subcell of the chain packing is strongly temperature dependent indicating the increased dominance of the hydration forces during the first transition and the location of SD molecules in the neighbourhood of the polar lipid head groups. The second transition is accompanied by the changes in the nanometer-scale layer arrangements observed by SAXS and in the μm-scale morphology observed by freeze-fracture. Above the temperature of the second transition, the SD-induced metastable structures undergo further formations to produce a more homogeneous state favoured by the geometrical packing of the cylindrical-shaped lipid molecules.

Introduction

As a group of antimicrobial medicines, antibiotics kill microorganisms or suppress their multiplication and growth. These substances are used not only as human and veterinary medicines for bacterial infections, but they are also applied as feed additives to prevent infections in livestock, for example at poultry and fish farms, to treat the entire flock or herd at risk [1], [2]. These drugs appear in the environment of the treated animals and can also lead to toxic effects in terrestrial and aquatic organisms [3]. Up to now, there has not been any extensive investigation of the pernicious effects of these molecules considering the importance of this risk in the extended application of antibiotics [4].

Sulphonamides are synthetic antimicrobial agents derived from sulfonic acid. These molecules act as competitive inhibitors of para-aminobenzoic acid, which is a necessary intermediate in these organisms for the synthesis of folic acid during DNA synthesis [5]. Sulfadiazine (SD, Fig. 1), a sulpha drug, is one of the most frequently used antibiotics for the therapy of domestic animals and farmed fish [1]. The solubility of sulfadiazine is generally low, but it is higher in water (about 60 mg/dm³) than in organic solvents [6].

The smallest changes in the membrane composition can influence the membrane processes resulting in minor or even drastic changes in the material transport and in other important (for example: signal) features of cell membranes [7], [8], [9]. In spite of the fact that biological membranes consist of a relatively small number of components, the explanation of the effect of antibiotics remains ambiguous because of the complex mechanism of the membranes. Therefore, of the different model systems, vesicles as model membranes are generally studied to obtain information about the effect of toxic molecules like antibiotics on the real membrane structure [10], [11], [12], [13], [14], [15].

The typical main phospholipid components of the bacterial membranes are the phosphatidylethanolamines (PEs). For example, PEs are present in the cytoplasmic membrane of Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Nitromonas europaea, and Salmonella typhimurium, and therefore these phospholipids are frequently used to prepare biologically relevant vesicles [16]. In general, a higher concentration of DPPE is found in the inner membrane of Gram-negative bacteria as compared to Gram-positive bacteria [17], [18]. To obtain reliable structural and thermotropic information on real membranes, mixtures of different lipids are applied in model investigations. In addition to the PEs, the phosphatidylglycerols (PGs) are the most frequently found membrane constituents of Gram-negative bacteria. Indeed, the dipalmitoylphosphatidylethanolamine(DPPE)-dipalmitoylphosphatidylglycerol(DPPG)/water vesicles proved to be the more relevant system than that consisting of only DPPE as was suggested and characterized by Lohner and colleagues [19], [20]. The DPPE-DPPG system exhibits several special features and different domain formations appear to depend on the DPPE molar ratio [19]. In our work, the DPPG was added in a 0.2 DPPG/DPPE + DPPG molar lipid ratio to the system to approximate the complexity of the bacteria membranes. In this case, the calorimetric studies indicate an inhomogeneous state with microdomains resulting in a segregation in the system [21], [22]. According to the chemical character of SD, it is expected to be located in the water shells of the vesicles. The effects induced by the presence of this sulpha drug were examined by using different methods (calorimetry/DSC, simultaneous small- and wide-angle X-ray scattering/SWAXS, freeze-fracture combined with transmission electron microscopy). Of these procedures, freeze-fracture was a powerful method for obtaining unique visual information about the wealth of complex changes in the multilamellar system [23], [24], [25].