Unraveling the Pore-Forming Steps of Pneumolysin from Streptococcus pneumoniae
Unraveling the Pore-Forming Steps of Pneumolysin from Streptococcus pneumoniae
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DOI：
10.1021/acs.nanolett.6b04219
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发表时间：
2016-12-01
期刊：
影响因子：
10.8
通讯作者：
Yildiz, Oezkan
中科院分区：
文献类型：
--
作者：
van Pee, Katharina;Mulvihill, Estefania;Yildiz, Oezkan
溶肺素（PLY）是引起肺炎、脑膜炎和侵袭性肺炎球菌感染的肺炎链球菌的主要毒力因子。PLY是作为单体生产的，它与含胆固醇的膜结合，在那里它们低聚成大孔。为了研究其成孔机理，我们测定了2.4埃时PLY的晶体结构，并利用其在单体表面设计突变体。与PLY突变体孵育的脂质体电镜显示，一些突变干扰了环的形成。形成不完整环或线性阵列的突变体大大降低了溶血活性。通过高分辨率延时原子力显微镜，我们观察到两种不同的环状配合物。大多数配合物在膜表面上方突出约8nm，而少数配合物在膜表面上方突出约11nm以上。较低的复合物通过其中心存在或不存在脂质双分子层来确定为孔或预孔。较高的配合物是可溶性PLY单体的并排组装，代表了预孔的早期形式。我们的观察结果表明，薄膜附着和孔形成的四步机制，这是在最近的结构模型的背景下讨论的。这些步骤的功能分离对于理解胆固醇依赖性细胞溶解素如何形成孔和溶解细胞是必要的。
Pneumolysin (PLY) is the main virulence factor of Streptococcus pneumoniae that causes pneumonia, meningitis, and invasive pneumococcal infection. PLY is produced as monomers, which bind to cholesterol-containing membranes, where they oligo-merize into large pores. To investigate the pore-forming mechanism, we determined the crystal structure of PLY at 2.4 angstrom and used it to design mutants on the surface of monomers. Electron microscopy of liposomes incubated with PLY mutants revealed that several mutations interfered with ring formation. Mutants that formed incomplete rings or linear arrays had strongly reduced hemolytic activity. By high-resolution time-lapse atomic force microscopy of wild-type PLY, we observed two different ring-shaped complexes. Most of the complexes protruded similar to 8 nm above the membrane surface, while a smaller number protruded similar to 11 nm or more. The lower complexes were identified as pores or prepores by the presence or absence of a lipid bilayer in their center. The taller complexes were side-by-side assemblies of monomers of soluble PLY that represent an early form of the prepore. Our observations suggest a four-step mechanism of membrane attachment and pore formation by PLY, which is discussed in the context of recent structural models. The functional separation of these steps is necessary for the understanding how cholesterol-dependent cytolysins form pores and lyse cells.