Koo S P, Yeaman M R, Nast C C, Bayer A S
Department of Medicine, St. John's Cardiovascular Research Center, Los Angeles County-Harbor UCLA Medical Center, Torrance, California 90509, USA.
Infect Immun. 1997 Nov;65(11):4795-800. doi: 10.1128/iai.65.11.4795-4800.1997.
Thrombin-induced platelet microbicidal protein (tPMP-1) is a small, cationic peptide released from rabbit platelets exposed to thrombin in vitro. tPMP-1 is microbicidal against a broad spectrum of bloodstream pathogens, including Staphylococcus aureus. Preliminary evidence suggests that tPMP-1 targets and disrupts the staphylococcal cytoplasmic membrane. However, it is not clear if the cytoplasmic membrane is a direct or indirect target of tPMP-1. Therefore, we assessed the in vitro activity of tPMP-1 versus protoplasts prepared from logarithmic-phase (LOG) or stationary-phase (STAT) cells of the genetically related S. aureus strains 19S and 19R (tPMP-1 susceptible and resistant, respectively). Protoplasts exposed to tPMP-1 (2 microg/ml) for 2 h at 37 degrees C were monitored for lysis (decrease in optical density at 420 nm) and ultrastructural alterations (by transmission electron microscopy [TEM]). Exposure to tPMP-1 resulted in substantial lysis of LOG but not STAT protoplasts of 19S, coinciding with protoplast membrane disruption observed by TEM. Thus, it appears that tPMP-1-induced membrane damage is influenced by the bacterial growth phase but is independent of the staphylococcal cell wall. In contrast to 19S, neither LOG nor STAT protoplasts of 19R were lysed by tPMP-1. tPMP-1-induced membrane damage was further characterized with anionic planar lipid bilayers subjected to various trans-negative voltages. tPMP-1 increased conductance across bilayers at -90 mV but not at -30 mV. Once initiated, a reduction in voltage from -90 to -30 mV diminished conductance magnitude but did not eliminate tPMP-1-mediated membrane permeabilization. Therefore, tPMP-1 appears to directly target the staphylococcal cytoplasmic membrane as a primary event in its mechanism of action. Specifically, tPMP-1 likely leads to staphylococcal death, at least in part by permeabilizing the bacterial membrane in a voltage-dependent manner.
凝血酶诱导的血小板杀菌蛋白（tPMP - 1）是一种小分子阳离子肽，由体外暴露于凝血酶的兔血小板释放。tPMP - 1对包括金黄色葡萄球菌在内的多种血流病原体具有杀菌作用。初步证据表明，tPMP - 1靶向并破坏葡萄球菌细胞质膜。然而，尚不清楚细胞质膜是tPMP - 1的直接还是间接靶点。因此，我们评估了tPMP - 1对由基因相关的金黄色葡萄球菌菌株19S和19R（分别对tPMP - 1敏感和耐药）的对数生长期（LOG）或稳定期（STAT）细胞制备的原生质体的体外活性。在37℃下将暴露于tPMP - 1（2微克/毫升）2小时的原生质体监测其裂解（420纳米处光密度降低）和超微结构改变（通过透射电子显微镜 [TEM]）。暴露于tPMP - 1导致19S的LOG原生质体大量裂解，但STAT原生质体未裂解，这与TEM观察到的原生质体膜破坏一致。因此，似乎tPMP - 1诱导的膜损伤受细菌生长阶段影响，但与葡萄球菌细胞壁无关。与19S相反，19R的LOG和STAT原生质体均未被tPMP - 1裂解。用施加各种跨膜负电压的阴离子平面脂质双层进一步表征tPMP - 1诱导的膜损伤。tPMP - 1在 - 90 mV时增加跨双层的电导，但在 - 30 mV时不增加。一旦启动，电压从 - 90 mV降至 - 30 mV会降低电导幅度，但不会消除tPMP - 1介导的膜通透性。因此，tPMP - 1似乎直接靶向葡萄球菌细胞质膜作为其作用机制的主要事件。具体而言，tPMP - 1可能至少部分通过以电压依赖性方式使细菌膜通透化而导致葡萄球菌死亡。
