Koo S P, Bayer A S, Kagan B L, Yeaman M R
Department of Medicine, Division of Infectious Diseases, St. John's Cardiovascular Research Center, LAC-Harbor UCLA Medical Center, Torrance, California 90509, USA.
Infect Immun. 1999 May;67(5):2475-81. doi: 10.1128/IAI.67.5.2475-2481.1999.
Thrombin-induced platelet microbicidal protein 1 (tPMP-1) is a small, cationic peptide generated from rabbit platelets when they are exposed to thrombin in vitro. It has potent microbicidal activity against a broad spectrum of bacterial and fungal pathogens, including Staphylococcus aureus. Previous in vitro studies involving whole staphylococcal cells and planar lipid bilayers (as artificial bacterial membrane models) suggested that membrane permeabilization by tPMP-1 is voltage dependent (S.-P. Koo, M. R. Yeaman, and A. S. Bayer, Infect. Immun. 64:3758-3764, 1996; M. R. Yeaman, A. S. Bayer, S. P. Koo, W. Foss, and P. M. Sullam, J. Clin. Investig. 101:178-187, 1998). Thus, the aims of the present study were to specifically characterize the electrophysiological events associated with membrane permeabilization by tPMP-1 by using artificial planar lipid bilayer membranes. We assessed the influence of transmembrane voltage polarity and magnitude on the initiation and modulation of tPMP-1 membrane permeabilization at various concentrations of tPMP-1 (range, 1 to 100 ng/ml) added to the cis side of the membranes. The incidence of membrane permeabilization induced by tPMP-1 at all of the concentrations tested was more frequent at -90 mV than at +90 mV. It is noteworthy that membrane permeabilization due to 1-ng/ml tPMP-1 was successfully initiated at -90 mV but not at +90 mV. Further, the mean onset times of induction of tPMP-1 activity were comparable under the various conditions. Modulation of ongoing membrane permeabilization was dependent on voltage and tPMP-1 concentration. Membrane permeabilization at a low tPMP-1 concentration (1 ng/ml) was directly correlated with trans-negative voltages, while a higher tPMP-1 concentration (100 ng/ml) induced conductance which was more dependent on trans-positive voltages. Collectively, these data indicate that the mechanism of tPMP-1 microbicidal activity at the bacterial cytoplasmic membrane may involve distinct induction and propagation stages of membrane permeabilization which, in turn, are modulated by transmembrane potential, as well as peptide concentration.
凝血酶诱导的血小板杀菌蛋白1（tPMP-1）是一种小的阳离子肽，在体外将兔血小板暴露于凝血酶时由其产生。它对包括金黄色葡萄球菌在内的多种细菌和真菌病原体具有强大的杀菌活性。先前涉及完整葡萄球菌细胞和平面脂质双层（作为人工细菌膜模型）的体外研究表明，tPMP-1引起的膜通透性是电压依赖性的（S.-P. Koo、M.R. Yeaman和A.S. Bayer，《感染与免疫》64:3758 - 3764，1996；M.R. Yeaman、A.S. Bayer、S.P. Koo、W. Foss和P.M. Sullam，《临床研究杂志》101:178 - 187，1998）。因此，本研究的目的是通过使用人工平面脂质双层膜来具体表征与tPMP-1引起的膜通透性相关的电生理事件。我们评估了跨膜电压极性和幅度对在膜的顺式侧添加不同浓度（范围为1至100 ng/ml）的tPMP-1时其膜通透性起始和调节的影响。在所有测试浓度下，tPMP-1诱导的膜通透性发生率在 - 90 mV时比在 + 90 mV时更频繁。值得注意的是，1 ng/ml的tPMP-1在 - 90 mV时成功引发了膜通透性，但在 + 90 mV时未引发。此外，在各种条件下，tPMP-1活性诱导的平均起始时间相当。正在进行的膜通透性调节取决于电压和tPMP-1浓度。低浓度tPMP-1（1 ng/ml）时的膜通透性与跨膜负电压直接相关，而较高浓度tPMP-1（100 ng/ml）诱导的电导更依赖于跨膜正电压。总体而言，这些数据表明tPMP-1在细菌细胞质膜上的杀菌活性机制可能涉及膜通透性的不同诱导和传播阶段，而这又反过来受到跨膜电位以及肽浓度的调节。
