Staphylococcal pore-forming toxins: Mechanism of pore-forming and recognition of the target cells

葡萄球菌成孔毒素：成孔机制和靶细胞识别

• 批准号: 13460034
• 负责人: KAMIO Yoshiyuki
• 金额: $ 11.2万
• 依托单位: Tohoku University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-13460034/
• 关键词: Staphylococcus aureus; pore-forming toxin; gamma-hemolysin; staphylococcal toxin; Single-molecule imaging; Assembly of toxin components; leukocidin; 膜孔形成

Staphylococcal LukF and Hlg2 are water-soluble monomers of gamma-hemolysin that assemble into an oligomeric pore on the erythrocyte membrane. The structure of LukF suggests that the pre-stem domain, which folds back toward the cap domain, may insert into the membrane during assembly. Here, we created double-cysteine LukF mutants in which single disulfide bonds connect either the pre-stem domain and the cap domain(V12C-T136C,Cap-Stem), or two b-strands within the pre-stem domain(T117C-T136C,Stem-Stem), to study structural changes of the pre-stem domain in pore assembly. Both mutants are inactive in erythrocyte lysis, however gain full hemolytic activity if the disulfide bonds are reduced. The disulfide bonds block neither the membrane binding ability nor the intermediate pre-pore oligomerization, but efficiently inhibit the transition from pre-pores to functional pores. An SDS-unstable pre-pore formed by the Cap-Stem mutant and the ring-shaped, SDS-stable pre-pore formed by the Stem-Stem mutant are ascribed to "early" and "late" states of pre-pore formation, respectively. The membrane binding and the mercaptoethanol-induced pore formation of both mutants even at 2℃ indicate that only oligomerization is a temperature-dependent step. Thus, the inhibition of pore assembly at various steps by disulfide bond formation and low temperature proves the requirement for structural changes in the pre-stem domain of LukF during pore assembly.

葡萄球菌LukF和Hlg2是γ-溶血素的水溶性单体，它们聚集在红细胞膜上的一个寡聚孔中。LukF的结构表明，前茎结构域在组装过程中可能插入膜中，该结构域向帽结构域折叠。在这里，我们创建了双半胱氨酸LukF突变体，其中单二硫键连接前茎结构域和帽结构域(V12C-T136C，Cap-Stem)，或前茎结构域内的两条b链(T117C-T136C，Stem-Stem)，以研究前茎结构域在孔组装过程中的结构变化。这两个突变体在红细胞裂解中都没有活性，但如果二硫键被减少，则获得完全的溶血活性。二硫键既不阻碍膜的结合能力，也不阻碍中间的前孔齐聚，但有效地抑制了前孔向功能孔的过渡。由Cap-Stem突变体形成的不稳定的预孔和由突变体形成的环状、稳定的预孔分别归因于预孔形成的“早期”和“晚期”状态。两个突变体的膜结合和巯基乙醇诱导的孔形成表明，只有齐聚是一个温度相关的步骤。因此，二硫键的形成和低温对不同步骤中的孔组装的抑制证明了在孔组装过程中LukF的前茎结构域需要发生结构变化。

项目成果

金子淳, 冨田敏夫, 神尾好是: "黄色ブドウ球菌の2成分性膜孔形成毒素の構造および細胞崩壊機構"蛋白質核酸酵素. 46(4). 497-505 (2001)

Jun Kaneko、Toshio Tomita、Yoshiharu Kamio：“金黄色葡萄球菌双组分成孔毒素的结构和细胞分解机制”蛋白质核酸酶 46(4) (2001)。

Sachiko Narita, Jun Kaneko, Junichi Chiba, Yves Piemont, Sophie Jarraud, Jerome Etienne, Yohiyuki Kamio: "Phage conversion of Panton-Valetine leukocidin in Staphylococcus aureus : molecular analysis of a PVL-converting phage, ΦSLT"Gene. 268. 195-206 (2001

Sachiko Narita、Jun Kaneko、Junichi Chiba、Yves Piemont、Sophie Jarraud、Jerome Etienne、Yohyuki Kamio：“金黄色葡萄球菌中 Panton-Valetine 杀白细胞素的噬菌体转化：PVL 转化噬菌体的分子分析，ΦSLT”基因。 206（2001

V. T. Nguyen, H. Higuchi, Y. Kamio: "Controlling pore assembly of staphylococcal g-hemolysin by low temperature and disulphide bond formation in double-cysteine LukF mutants"Mol. Microbiol.. 45(6). 1485-1498 (2002)

V. T. Nguyen、H. Higuchi、Y. Kamio：“通过低温和双半胱氨酸 LukF 突变体中二硫键的形成来控制葡萄球菌 g-溶血素的孔组装”Mol。

Vananh T.Nguyen, Hideo Higuchi, Yoshiyuki Kamino: "Controlling pore assembly of staphylococcal γ -haemolysin by low temperature and by disulphide bond formation in double-cysteine LukF mutants"Molecular Microbiology. 45(6). 1485-1498 (2002)

Vananh T.Nguyen、Hideo Higuchi、Yoshiyuki Kamino：“通过低温和双半胱氨酸 LukF 突变体中二硫键的形成控制葡萄球菌 γ-溶血素的孔组装”，分子微生物学 45(6)。 ）

Yoshiyuki Kamio, Toshio Tomita, Jun Kaneko: "Staphylococcus aureus Infection and Disease"Kluwer Academic/Plenum Publishers. 330 (2001)

Yoshiyuki Kamio、Toshio Tomita、Jun Kaneko：“金黄色葡萄球菌感染和疾病”Kluwer Academy/Plenum 出版社。