Iliev Asparouh I, Djannatian Jasmin Roya, Nau Roland, Mitchell Timothy J, Wouters Fred S
Cell Biophysics Group, European Neuroscience Institute Göttingen, Waldweg 33, 37073 Göttingen, Germany.
Proc Natl Acad Sci U S A. 2007 Feb 20;104(8):2897-902. doi: 10.1073/pnas.0608213104. Epub 2007 Feb 14.
The Streptococcus pneumoniae toxin pneumolysin belongs to the group of cholesterol-dependent cytolysins. It produces rapid cell lysis at higher concentrations or apoptosis at lower concentrations. In cell membranes, it forms prepores and pores. Here, we show that sublytic concentrations of pneumolysin produce rapid activation of Rho and Rac GTPases and formation of actin stress fibers, filopodia, and lamellipodia. That Rac1-specific and Rho-associated kinase (ROCK)-specific inhibitors reverted the formation of lamellipodia and stress fibers, respectively, identifies RhoA and Rac1 as key toxin effectors. Live imaging excluded macropore formation (as judged by membrane impermeability toward calcein) but indicated very early membrane depolarization [as judged by bis-(1,3-dibutylbarbituric acid)trimethine oxanol staining], indicative of formation of micropores with ion channel properties. That Rac1-dependent lamellipodia formation was reverted by the voltage-gated calcium channel inhibitor SKF96365 and by toxin exposure in calcium-free medium suggests a role for calcium influx via endogenous calcium channels in the Rac1 activation. Cellular cholesterol depletion by methyl-beta-cyclodextrin or incubation of the toxin with cholesterol before cell treatment eliminated its membrane binding and the subsequent GTPase activation. Thus, that our experiments show small GTPase activation by a cholesterol-dependent cytolysin suggests a membrane cholesterol-dependent activation mechanism.
肺炎链球菌毒素肺炎溶血素属于胆固醇依赖性细胞溶素家族。它在高浓度时可导致细胞快速裂解，在低浓度时可诱导细胞凋亡。在细胞膜中，它可形成前孔和孔道。在此，我们发现亚溶细胞浓度的肺炎溶血素能快速激活Rho和Rac GTP酶，并形成肌动蛋白应力纤维、丝状伪足和片状伪足。Rac1特异性抑制剂和Rho相关激酶（ROCK）特异性抑制剂分别可逆转片状伪足和应力纤维的形成，这表明RhoA和Rac1是关键的毒素效应分子。实时成像排除了大孔的形成（通过钙黄绿素的膜不透性判断），但显示了非常早期的膜去极化（通过双（1,3 - 二丁基巴比妥酸）三甲川氧杂菁染色判断），这表明形成了具有离子通道特性的微孔。电压门控钙通道抑制剂SKF96365以及在无钙培养基中暴露毒素可逆转Rac1依赖性的片状伪足形成，这表明通过内源性钙通道的钙内流在Rac1激活中起作用。用甲基 - β - 环糊精进行细胞胆固醇耗竭或在细胞处理前将毒素与胆固醇一起孵育可消除其与膜的结合以及随后的GTP酶激活。因此，我们的实验表明胆固醇依赖性细胞溶素可激活小GTP酶，这提示了一种膜胆固醇依赖性激活机制。
