Virág Eszter, Belagyi Joseph, Gazdag Zoltán, Vágvölgyi Csaba, Pesti Miklós
Department of General and Environmental Microbiology, Faculty of Sciences, University of Pécs, Pécs, Hungary.
Biochim Biophys Acta. 2012 Jan;1818(1):42-8. doi: 10.1016/j.bbamem.2011.09.020. Epub 2011 Sep 29.
The direct interaction of the antibiotic primycin with the plasma membrane was investigated by employing the well-characterized ergosterol-producing, amphotericin B-sensitive parental Candida albicans strain 33erg(+) and its ergosterol-less amphotericin B-resistant plasma membrane mutant erg-2. The growth inhibition concentration in shaken liquid medium was 64 μgml(-1) for 33erg(+) and 128 μgml(-1) for erg-2, suggesting that the plasma membrane composition influences the mode of action of primycin. To determine the primycin-induced changes in the plasma membrane dynamic, electron paramagnetic resonance (EPR) spectroscopy methods were used, the spin-labeled fatty acid 5-(4,4-dimethyloxazolidine-N-oxyl)stearic acid) being applied for the in vivo measurements. The phase transition temperatures of untreated strain 33erg(+) and its mutant erg-2 were 12.5°C and 11°C, respectively. After 128 μgml(-1) primycin treatment, these values increased to 17.5°C and 16°C, revealing a significant reduction in the phospholipid flexibility. Saturation transfer EPR measurements demonstrated that, the rotational correlation times of the spin label molecule for the control samples of 33erg(+) and erg-2 were 60 ns and 100 ns. These correlation times gradually decreased on the addition of increasing primycin concentrations, reaching 8 μs and 1 μs. The results indicate the plasma membrane "rigidizing" effect of primycin, a feature that may stem from its ability to undergo complex formation with membrane constituent fatty acid molecules, causing alterations in the structures of phospholipids in the hydrophobic surface near the fatty acid chain region.
通过使用特征明确的产麦角固醇、对两性霉素B敏感的白色念珠菌亲本菌株33erg(+)及其无麦角固醇、对两性霉素B耐药的质膜突变体erg-2，研究了抗生素原霉素与质膜的直接相互作用。在振荡液体培养基中，33erg(+)的生长抑制浓度为64μg/ml，erg-2的生长抑制浓度为128μg/ml，这表明质膜组成影响原霉素的作用方式。为了确定原霉素诱导的质膜动力学变化，使用了电子顺磁共振（EPR）光谱方法，自旋标记脂肪酸5-(4,4-二甲基恶唑烷-N-氧基)硬脂酸用于体内测量。未处理的菌株33erg(+)及其突变体erg-2的相变温度分别为12.5°C和11°C。用128μg/ml原霉素处理后，这些值分别增加到17.5°C和16°C，表明磷脂柔韧性显著降低。饱和转移EPR测量表明，33erg(+)和erg-2对照样品的自旋标记分子的旋转相关时间分别为60 ns和100 ns。随着原霉素浓度的增加，这些相关时间逐渐缩短，分别达到8μs和1μs。结果表明原霉素对质膜有“硬化”作用，这一特征可能源于其与膜组成脂肪酸分子形成复合物的能力，导致脂肪酸链区域附近疏水表面的磷脂结构发生改变。
