Rapoport N, Smirnov A I, Timoshin A, Pratt A M, Pitt W G
Department of Materials Science and Engineering, University of Utah, Salt Lake City 84112, USA.
Arch Biochem Biophys. 1997 Aug 1;344(1):114-24. doi: 10.1006/abbi.1997.0176.
The objective of this research was to elucidate the factors effecting the permeability of cell membranes of gram-negative bacteria toward hydrophobic compounds. Ultrasound treatment, cell age, and the phase state of phospholipid membranes were considered. Spin-labeling EPR method was used to quantify the penetration and distribution of a lipophilic spin probe, 16-doxylstearic acid (16-DS), in Pseudomonas aeruginosa cell membranes. This bacterium was chosen because of its reported resistance to the action of hydrophobic antibiotics caused by the low permeability of the outer cell membrane for hydrophobic compounds. EPR spectra were collected from cell pellets and cell lysates. The overall spin probe uptake was measured in 10% SDS-cell lysates. Lysis with 0.6% SDS revealed the fraction of the probe located in membrane sites readily accessible to the surfactant. The results indicated a structural heterogeneity of P. aeruginosa membranes, with the presence of structurally "stronger" and "weaker" sites characterized by different susceptibility to the SDS treatment. The intracellular concentration of 16-DS was higher in insonated cells and increased linearly with the sonication power. EPR spectra indicated that ultrasound enhanced the penetration of the probe into the structurally stronger sites of the inner and outer cell membranes. The effect of ultrasound on the cell membranes was transient in that the initial membrane permeability was restored upon termination of the ultrasound treatment. These results suggest that the resistance of gram-negative bacteria to the action of hydrophobic antibiotics was caused by a low permeability of the outer cell membranes. This resistance may be reduced by the simultaneous application of antibiotic and ultrasound. This hypothesis was confirmed in our experiments with P. aeruginosa exposed to erythromycin.
本研究的目的是阐明影响革兰氏阴性菌细胞膜对疏水性化合物通透性的因素。研究考虑了超声处理、细胞年龄和磷脂膜的相态。采用自旋标记电子顺磁共振（EPR）方法来定量亲脂性自旋探针16-脱氧硬脂酸（16-DS）在铜绿假单胞菌细胞膜中的渗透和分布。选择这种细菌是因为据报道其外细胞膜对疏水性化合物的低通透性导致对疏水性抗生素作用具有抗性。从细胞沉淀和细胞裂解物中收集EPR光谱。在10%十二烷基硫酸钠（SDS）细胞裂解物中测量总的自旋探针摄取量。用0.6% SDS裂解可揭示位于表面活性剂易于接近的膜位点的探针部分。结果表明铜绿假单胞菌细胞膜存在结构异质性，存在对SDS处理敏感性不同的结构“更强”和“更弱”的位点。超声处理过的细胞中16-DS的细胞内浓度更高，且随超声功率呈线性增加。EPR光谱表明超声增强了探针向内、外细胞膜结构更强位点的渗透。超声对细胞膜的作用是短暂的，因为超声处理终止后初始膜通透性得以恢复。这些结果表明革兰氏阴性菌对疏水性抗生素作用的抗性是由外细胞膜的低通透性引起的。同时应用抗生素和超声可能会降低这种抗性。在我们用暴露于红霉素的铜绿假单胞菌进行的实验中证实了这一假设。
