Characterization of the interactions of the Streptococcus pneumoniae toxin, Pneumolysin, with soluble molecules of the immune system
Characterization of the interactions of the Streptococcus pneumoniae toxin, Pneumolysin, with soluble molecules of the immune system
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发表时间：
2017-11
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通讯作者：
Bayan H. A. Faraj
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作者：
Bayan H. A. Faraj
肺炎链球菌溶血素（Ply）是肺炎链球菌（Pneumococcus pneumoniae）的关键毒力因子。主要功能包括在哺乳动物细胞膜中形成孔和激活补体级联以转移宿主的免疫系统。本论文的目的是在分子水平上研究这些过程，以了解Ply如何促进肺炎球菌的疾病。以前的研究表明，Ply与免疫系统的各种可溶性分子相互作用，包括L-纤维胶凝蛋白和IgG。这些相互作用分别通过凝集素和经典途径激活补体。在这篇论文中，我已经证明了Ply不与天然血清L-纤维胶凝蛋白或在中国仓鼠卵巢细胞中产生的重组人L-纤维胶凝蛋白相互作用。先前的错误报告可能是由于Ply制剂被L-纤维胶凝蛋白配体污染所致。对Ply和IgG之间结合的研究表明，Ply与IgG 2、IgG 3和IgG 4结合，但不与IgG 1结合。通过Ply的结构域1-3与IgG的Fab区之间的相互作用介导结合。本论文的另一个目的是研究孔的形成。在我们的小组中确定的Ply的晶体结构表明，晶体中的Ply单体以类似于它们可能在孔形成之前在细胞表面上组装的方式聚集在一起。基于该结构，产生了一系列突变以在预孔和孔形成期间破坏Ply单体之间的包装。两个突变体，Asp 205 Arg和Asn 339 Arg的活动被完全废除，大多数的突变体有大大降低的活动相比，野生型Ply表明，这些残基在孔形成过程中发挥重要作用。有趣的是，电子显微镜显示Ply Asp 205 Arg在膜上形成链状结构，但不能形成圆孔或弧形。因此，尽管单体仍然自我缔合，但它们不能杀死细胞。相比之下，Ply Asn 339 Arg结合至膜但不寡聚化。在进一步的工作中，晶体结构的膜结合结构域的Ply揭示了构象的变化，在一个色氨酸丰富的环的基础上参与膜结合的毒素。这些变化促进Ply单体之间的新的堆积相互作用，从而促进膜上的低聚。最后，我研究了膜的结构变化，通过光谱监测光学捕获囊泡。从单个脂质体监测非弹性背散射光，通过光学镊子保持并暴露于Ply。磷脂结合增加了膜的流动性，由于在双分子层中的脂质分子的短程顺序的减少。一系列的点突变体的分析表明，这些变化是由关联的Ply单体在形成的前孔，插入跨膜之前。
Pneumolysin (Ply) is a key virulence factor of the bacterium Streptococcus pneumoniae (Pneumococcus). Major functions include forming pores in mammalian cell membranes and activating the complement cascade to divert the host’s immune system. The aim of this thesis was to investigate these processes at the molecular level to understand how Ply facilitates disease by the pneumococcus. Previous studies have suggested that Ply interacts with various soluble molecules of the immune system, including L-ficolin and IgG. These interactions activate complement via the lectin and classical pathways, respectively. In this thesis I have demonstrated that Ply does not interact with either native serum L-ficolin or recombinant human L-ficolin produced in Chinese hamster ovary cells. The previous erroneous report probably arose as a result of contamination of Ply preparations with L-ficolin ligands. Investigation of binding between Ply and IgG showed that Ply binds to IgG2, IgG3 and IgG4 but not to IgG1. Binding is mediated through interactions between domains 1-3 of Ply and the Fab region of the IgGs. An additional aim of this thesis was to investigate pore formation by Ply. The crystal structure of Ply, determined in our group, showed that Ply monomers in the crystal pack together similar to the way in which they are likely to assemble on the cell surface prior to pore formation. Based on the structure, a series of mutations were created to disrupt packing between Ply monomers during pre-pore and pore formation. The activities of two of the mutants, Asp205Arg and Asn339Arg were completely abolished and most of the mutants had greatly reduced activities compared to wild-type Ply indicating that these residues play important roles during pore formation. Interestingly, electron microscopy showed that Ply Asp205Arg forms chain like structures on membranes but cannot form circular pores or arcs. Thus although monomers still self-associated they could not kill cells. By contrast, Ply Asn339Arg, binds to the membrane but does not oligomerize. In further work, crystal structures of the membrane-binding domain of Ply revealed conformational changes in a Trp rich-loop at the base of the toxin involved in membrane binding. These changes promote new packing interactions between Ply monomer thereby promoting oligomerization on the membrane. Finally, I investigated the structural changes of the membrane by spectroscopic monitoring of optically trapped vesicles. The inelastic back-scattered light was monitored from a single liposome, held by optical tweezers and exposed to Ply. Ply binding increased the membrane fluidity due to a decrease in the short-range order of the lipid molecules in the bilayer. Analysis of a series of point mutants suggests that these changes are caused by association of Ply monomers during formation of the pre-pore, prior to insertion across the membrane.