Cholesterol-dependent actin remodeling via RhoA and Rac1 activation by the Streptococcus pneumoniae toxin pneumolysin
Cholesterol-dependent actin remodeling via RhoA and Rac1 activation by the Streptococcus pneumoniae toxin pneumolysin
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DOI：
10.1073/pnas.0608213104
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发表时间：
2007-02-20
影响因子：
11.1
通讯作者：
Wouters, Fred S.
中科院分区：
文献类型：
--
作者：
Iliev, Asparouh I.;Djannatian, Jasmin Roya;Wouters, Fred S.
肺炎链球菌毒素肺炎溶素属于胆固醇依赖性溶细胞素家族。它在较高浓度时可导致细胞迅速裂解，在较低浓度时则引发细胞凋亡。在细胞膜上，它会形成前孔和孔。在此，我们发现亚裂解浓度的肺炎溶素可快速激活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）特异性抑制剂分别逆转了片状伪足和应力纤维的形成，这表明RhoA和Rac1是该毒素的关键效应因子。活细胞成像排除了大孔形成（通过细胞膜对钙黄绿素的不透性来判断），但显示出非常早期的膜去极化[通过双 -（1,3 - 二丁基巴比妥酸）三甲川氧杂蒽醇染色来判断]，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进行毒素暴露可逆转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。通过甲基 -β - 环糊精消耗细胞胆固醇，或者在细胞处理前将毒素与胆固醇一起孵育，可消除其膜结合以及随后的GTP酶激活。因此，我们的实验表明一种胆固醇依赖性溶细胞素可激活小GTP酶，这提示了一种依赖膜胆固醇的激活机制。
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.