Staphylococcal pore-forming toxins, g-hemolysin and leukocidin : Mechanism of pore-forming and expression of the toxins activities on the target cells

葡萄球菌成孔毒素、g-溶血素和杀白细胞素：成孔机制和毒素活性在靶细胞上的表达

• 批准号: 11460034
• 负责人: KAMIO Yoshiyuki
• 金额: $ 9.54万
• 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: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2000
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-11460034/
• 关键词: Staphylococcus aureus; pore-forming toxin; g-hemolysin; leukocidin; staphylococcal gemone; phage conversion; 3次元構造; x線回折; 三次元構造; LukF成分; LukS

(1) Pore-forming Nature of Hlg and LukWhen monitored the Hlg-induced hemolysis for single cells of human erythrocytes under a phase contrast microscope, it was observed that intact, disc-shaped erythrocytes became swollen and round-shaped cells with clear edge after the incubation with LukF and Hlg2 for 10 min, and the swollen cells lysed thereafter. Since swelling of cells is generally caused by the permeabilization of cell membranes, it was presumed that Hlg induced colloid osmotic lysis of human erythrocytes through pore formation. This assumption was supported by the following findings : [1] Hlg-induced hemolysis was prevented by the extracellular nonelectrolytes (such as polyethylene glycols) with the diameters of>2.5 nm, suggesting that the toxin forms a hydrophilic pore with a functional diameter of approximately 2.5 nm. [2] Electron microscopy of the negatively-stained, toxin-treated erythrocytes revealed that Hlg forms a ring-shaped structure, whose outer and inner diameters a … More re approximately 7 and 3 nm, respectively. Therefore, the complex formation of Hlg on human erythrocytes was examined as follows : Cell-bound toxin was solubilized with SDS from erythrocyte membranes and it was then analyzed by SDS-polyacrylamide gel electrophoresis, followed by Western immunoblot using specific antisera raised against LukF and Hlg2. The data indicated that Hlg forms high-molecular-sized complex (es) of approximately 200 kDa, which contain LukF and Hlg2 at a molar ratio of 1 : 1 on the surface of human erythrocytes. Recently, the [LukF-Hlg2] complex was isolated. It was also demonstrated that the preceding binding of LukF is essential for the complex formation as well as for the Hlg2 binding. Furthermore, our recent data suggested that the membrane component (s), which are accessible by proteinase K, may be required for the complex formation of Hlg on human erythrocytes. Taken together, Hlg may assemble into a annular complex on target membranes, forming a transmembrane pore with a functional diameters of approximately 2.5 nm.PVL has been suggested to form membrane pores in the early stage its leukocytolytic action. However, molecular architecture of the membrane pore formed by PVL remained to be studied, and it should be also be elucidated whether or not the pore contains intrinsic membrane protein (s) of leukocytes. We studied membrane pore formation by Luk in the cell membrane of human PMNLs and rabbit erythrocytes and the following findings are evident. [1] Luk caused efflux of potassium ions from rabbit erythrocytes and swelling of the cells before hemolysis. However, ultimate lysis of the toxin-treated swollen erythro-cytes did not occur when polyethylene glycols with hydrodynamic diameters of 【greater than or equal】2.1 nm were present in the extracellular space. [2] Electron microscopy showed the presence of a ring-shaped structure with outer and inner diameters of 9 and 3 nm, respectively, on the Luk-treated human PMNLs and rabbit erythrocytes. [3] Ring-shaped structures of the same dimension were isolated from the target cells, and they contained LukS and LukF in a molar ratio of 1 : 1. [4] A single ring-shaped toxin complex had a molecular size of approximately 200 kDa. These results indicated that LukS and LukF assemble into a ring-shaped oligomer of approximately 200 kDa on the target cells, forming a membrane pore with a functional diameter approximately 2 nm.(2) Mechanism of Assembly. Combining salient features from the water-soluble monomer of LukF and the water-insoluble heptamer of Hla structures with data from studies of wild-type and mutant proteins provides molecular detail to and assembly mechanism for staphylococcal channel-fomming proteins (Figure 11). Although LukF does not form a homoheptamer, the similarity in structure and function between LukF and Hla and the similar size of the Hlg (LukF+Hlg2) oligomer and the Hla heptamer predict that LukF and Hla share elements of structure and mechanism. The Hla heptamer structure is a reasonable starting point from which to buld a model of the pre-pore assembly intermediate and the LukF monomer structure may serve as a starting point for models of the Hla, LukS and Hlg2 water soluble and membrane-bound monomers. It is suggested that the membrane-bound monomer resembles the water soluble form of LukF excetp that interaction with the bilayer induces modest conformational changes in the rim and pre-stem regions. In addition, membrane binding renders the pre-stem resistant to proteolysis either through conformational changes, occlusion via the bilayer surface, or both. An important feature of the model shown in Figure 11 for the structure of the oligomeric pre-pore intermediate is that the glycine-rich pre-stem is located within the cap domain pore. This model stands in contrast to previous models in which the glycine-rich pre-stem region is located on the periphery of the oligomer and in contact with the membrane surface.This mechanism explains how the toxins exhibit solubility in aqueous solution and resist assembly until membrane binding triggers formation of the pre-pore. In the pre-pore state, the pre-stem has probably undergone partial rearrangement, the amino latch has moved from its β-strand position to enable productive protomer-protomer contact and the pro-tomers assemble to a heptamer which is somewhat large in diameter compared to the final pore form. Insertion of the pre-stem into the membrane may occur by a cooperative "extrusion" of the polypeptide from the base of the cap at the same time as the amino latch folds into the lumen of the cap domain. By forming a pre-pore oligmer and associating with the membrane the pre-pore may thin the bilayer and thus facilitate stem insertion.Since the LukF, LukS, and Hlg2 proteins form heteromers that may be hexames, there will certainly be differences in their assembly compared to Hla. However, given the structural and functional similarities among Hla, LukF, LukS and Hlg2, they will undoubtedly share many mechanistic features in common. Although the mechanism shown in Figure 11 is focused on Hla, we predict that LukF LukS and Hlg will assemble to form oligomers that have cap, rim, and stem domains like the Hla heptamer and that Hla and Luk will assemble via an oligmeric intermediate in which the pre-stem regions are clustered in the interior of the cap domain. Insights obtained from the studies of LukF and Hla may also be applicable to other non-staphylococcal channel forming toxins such as aerolysin and anthrax protective antigen. In more general terms, structural studies of Hla and LukF have shown how the exchange and sequential unmasking of specific protein in and protein-solvent interfaces plays a central role in the assembly of these oligomeric transmembrane toxins : the water soluble form is stabilized by interactions within a single subunit while the oligomeric form is stabilized by interactions between subunits and between the oligomer and the membrane.(3) VITRONECTIN AND ITS FRAGMENTS PURIFIED AS SERUM INHIBITORS OF HLG AND LUK, AND THEIR SPECIFIC BINDING TO HLG2 AND LUKS OF THE TOXINSMost recently, vitronectin which is a 75-kDa multifunctional glycoprotein and its frag-ments with 62, 57, and 38 kDa have been isolated from human serum as an inhibitor with an ability to fix Hlg and Luk. The purified vitronectin and its fragments specifically bound to Hlg2 and LukS to prevent the toxin-induced lysis of human erythrocytes and human PMNLs, respectively. The vitronectin fragments and Hlg2 (or LukS) formed high-molecular weight complexes that cosedimented in a sucrose gradient centrifugation and co-migraged on a native polyacrylamide gel electrophoresis. Intact vitronectin was 15-fold less active than the purified inhibitors, but its inhibitory activity was raised to a comparable level to that of the purified inhibitors when partially digested with human plasmin. Based on these results, vitronectin and its fragments are considered to be possible host components for fixation of Hlg and Luk in the loci of stapylococcal infections. The vitronectin-binding ability of Hlg and Luk is a novel function of the pore-forming cytolysins.Since vitronectin is considered to regulate proteolytic enzyme cascades including the complement, coagulation and fibrinolysis systems, it would act as an ambivalent factor for hosts depending on the local and the systemic conditions of defense systems : [1] Provided Hlg and Luk are produced in the loci of staphylococcal infections, Hlg2 and LukS would be captured by vitronectin and its fragments in the extracellular matrix of fibroblasts and tissue macrophages, followed by integrin-mediated endocytosis and degradation by the cells. [2] Extracellular-matrix-associated vitronectins would be liberated by the action of plasmin in the sites of interstitial inflammation, and the liberated vitronectin fragments would fix and opsonize Hlg and Luk. [3] However, consumption of vitronectin by Hlg and Luk would cause an inbalance in the regulation of coagulation, fibrinolysis, and complement cascade, leading to tissue injuries by an excess level of terminal complex of complement and hyperproduction of plasmin. [4] Vitronectin is an acute phase protein, and it is synthesized predominantly in liver in response to interleukin 6, and delivered to peripheral tissues through blood circulation and transcytosis by the endothelial cells. Once extracellular-matrix-associated vitronectin is consumed by Hlg and Luk in the sites of staphylococcal infections, it would remain at lower levels there for a while. In the circumstances, staphylococcal cytolysins including Hlg and Luk might play a key role in skin and mucosal infections with severe prognosis. [5] Vitronectin has been shown to bind specifically to the cells of S.aureus, and it is considered to be a binding molecule for the bacterium. Production of Hlg and Luk by S.aureus would induce detachment and spreading of tissue-bound staphylococci by replacing the vitronectin-binding sites of the bacteria with Hlg2 and/or LukS as well as by the cytolytic activity of the toxins. Hlg2 and LukS would also neutralize the opsonic function of soluble vitronectin to prevent phagocytosis of S.aureus by professional phagocytes in the loci of inflammation. Thus, not only the cytolytic activity but also the vitronectin-binding activity of Hlg and Luk are the putative pathophysiological functions of the staphylococcal bi-component toxins. Less

(1) Hlg和Luk的成孔性质在相差显微镜下监测HLg诱导的人红细胞单细胞溶血情况，观察到完整的盘状红细胞与LukF和Hlg2孵育10分钟后变得肿胀，细胞呈边缘清晰的圆形，随后肿胀的细胞裂解。由于细胞肿胀通常是由细胞膜的透化引起的，因此推测 Hlg 通过孔的形成诱导人红细胞的胶体渗透裂解。这一假设得到以下发现的支持：[1] Hlg 诱导的溶血被直径>2.5 nm 的细胞外非电解质（例如聚乙二醇）阻止，表明毒素形成功能直径约为 2.5 nm 的亲水孔。 [2] 负染、毒素处理的红细胞的电子显微镜显示，HLg 形成环形结构，其外径和内径分别约为 7 和 3 nm。因此，对人红细胞上 Hlg 复合物的形成进行了如下检查：用红细胞膜上的 SDS 溶解细胞结合毒素，然后通过 SDS-聚丙烯酰胺凝胶电泳进行分析，然后使用针对 LukF 和 Hlg2 产生的特异性抗血清进行蛋白质印迹分析。数据表明，Hlg在人红细胞表面形成约200kDa的高分子复合物(es)，其含有摩尔比为1:1的LukF和Hlg2。最近，[LukF-Hlg2]复合物被分离出来。还证明 LukF 的先前结合对于复合物形成以及 Hlg2 结合至关重要。此外，我们最近的数据表明，蛋白酶 K 可接触到的膜成分可能是人红细胞上 Hlg 复杂形成所必需的。总之，HLg可以在靶膜上组装成环形复合物，形成功能直径约为2.5 nm的跨膜孔。PVL被认为在其白细胞溶解作用的早期阶段形成膜孔。然而，PVL形成的膜孔的分子结构仍有待研究，并且还应阐明该孔是否含有白细胞内在的膜蛋白。我们研究了 Luk 在人 PMNL 和兔红细胞细胞膜上的膜孔形成，以下结果是显而易见的。 [1] Luk引起兔红细胞中钾离子的流出以及溶血前细胞的肿胀。然而，当细胞外空间中存在流体动力学直径【大于或等于】2.1 nm的聚乙二醇时，经毒素处理的肿胀红细胞最终并未发生裂解。 [2] 电子显微镜显示，Luk 处理的人 PMNL 和兔红细胞上存在外径和内径分别为 9 和 3 nm 的环形结构。 [3]从靶细胞中分离出相同尺寸的环状结构，其中含有摩尔比为1:1的LukS和LukF。 [4]单个环状毒素复合物的分子大小约为200 kDa。这些结果表明LukS和LukF在靶细胞上组装成约200kDa的环状寡聚物，形成功能直径约2nm的膜孔。(2)组装机制。将 LukF 的水溶性单体和 Hla 结构的水不溶性七聚体的显着特征与野生型和突变蛋白研究的数据相结合，提供了葡萄球菌通道形成蛋白的分子细节和组装机制（图 11）。尽管LukF不形成同七聚体，但LukF和Hla在结构和功能上的相似性以及Hlg(LukF+Hlg2)寡聚体和Hla七聚体的相似大小预示着LukF和Hla具有相同的结构和机制要素。 Hla 七聚体结构是构建预孔组装中间体模型的合理起点，而 LukF 单体结构可以作为 Hla、LukS 和 Hlg2 水溶性和膜结合单体模型的起点。这表明膜结合单体类似于 LukF 的水溶性形式，但与双层的相互作用会引起边缘和茎前区域的适度构象变化。此外，膜结合通过构象变化、通过双层表面的闭塞或两者兼而有之，使前茎对蛋白水解具有抵抗力。图 11 所示的寡聚前孔中间体结构模型的一个重要特征是富含甘氨酸的前茎位于帽域孔内。该模型与之前的模型形成鲜明对比，之前的模型中富含甘氨酸的前茎区域位于低聚物的外围并与膜表面接触。该机制解释了毒素如何在水溶液中表现出溶解性并抵抗组装，直到膜结合触发前孔的形成。在前孔状态下，前茎可能经历了部分重排，氨基锁已从其β链位置移动，以实现有效的原聚体-原聚体接触，并且原聚体组装成七聚体，其直径与最终的孔形式相比稍大。前茎插入膜中可以通过在氨基锁折叠到帽结构域的内腔中的同时将多肽从帽的基部协同“挤出”来进行。通过形成预孔寡聚体并与膜结合，预孔可以使双层变薄，从而促进茎插入。由于LukF、LukS和HLg2蛋白形成可能是六聚体的异聚体，因此与Hla相比，它们的组装肯定会存在差异。然而，鉴于 Hla、LukF、LukS 和 Hlg2 之间结构和功能的相似性，它们无疑具有许多共同的机制特征。尽管图11所示的机制集中于Hla，但我们预测LukF LukS和Hlg将组装形成具有帽、边缘和茎结构域的寡聚物，如Hla七聚体，并且Hla和Luk将通过寡聚中间体组装，其中前茎区域聚集在帽结构域的内部。从 LukF 和 Hla 研究中获得的见解也可能适用于其他非葡萄球菌通道形成毒素，例如气溶素和炭疽保护性抗原。更一般地说，Hla 和 LukF 的结构研究表明，蛋白质-溶剂界面中特定蛋白质的交换和顺序暴露如何在这些寡聚跨膜毒素的组装中发挥核心作用：水溶性形式通过单个亚基内的相互作用来稳定，而寡聚形式通过亚基之间以及寡聚体与毒素之间的相互作用来稳定。 (3) 玻连蛋白及其片段纯化为 HLG 和 LUK 的血清抑制剂，以及它们与毒素的 HLG2 和 LUKS 的特异性结合最近，玻连蛋白是一种 75 kDa 的多功能糖蛋白及其 62、57 和 38 kDa 的片段，已从人血清中分离出来，作为具有固定能力的抑制剂。 Hlg 和 卢克。纯化的玻连蛋白及其片段特异性结合 Hlg2 和 LukS，分别防止毒素诱导的人红细胞和人 PMNL 裂解。玻连蛋白片段和 Hlg2（或 LukS）形成高分子量复合物，在蔗糖梯度离心中共同沉淀，并在天然聚丙烯酰胺凝胶电泳上共同迁移。完整的玻连蛋白的活性比纯化的抑制剂低 15 倍，但当用人纤溶酶部分消化时，其抑制活性提高到与纯化的抑制剂相当的水平。基于这些结果，玻连蛋白及其片段被认为是在葡萄球菌感染位点固定 Hlg 和 Luk 的可能宿主成分。 Hlg 和 Luk 的玻连蛋白结合能力是成孔溶细胞素的一项新功能。由于玻连蛋白被认为可以调节蛋白水解酶级联，包括补体、凝血和纤维蛋白溶解系统，因此根据防御系统的局部和全身条件，它可以作为宿主的矛盾因子：[1] 假设 Hlg 和 Luk 是在 葡萄球菌感染时，HLg2 和 LukS 会被成纤维细胞和组织巨噬细胞的细胞外基质中的玻连蛋白及其片段捕获，然后由细胞整合素介导的内吞作用和降解。 [2] 细胞外基质相关的玻连蛋白将通过间质炎症部位纤溶酶的作用被释放，释放的玻连蛋白片段将固定并调理 Hlg 和 Luk。 [3] 然而，HLg和Luk消耗玻连蛋白会导致凝血、纤溶和补体级联的调节失衡，导致补体末端复合物水平过量和纤溶酶过度产生而导致组织损伤。 [4] 玻连蛋白是一种急性时相蛋白，主要在肝脏中响应白细胞介素6合成，并通过血液循环和内皮细胞的转胞吞作用递送至外周组织。一旦细胞外基质相关的玻连蛋白在葡萄球菌感染部位被 Hlg 和 Luk 消耗，它会在一段时间内保持较低水平。在这种情况下，包括 Hlg 和 Luk 在内的葡萄球菌溶细胞素可能在预后严重的皮肤和粘膜感染中发挥关键作用。 [5] 玻连蛋白已被证明可以与金黄色葡萄球菌细胞特异性结合，并且被认为是该细菌的结合分子。金黄色葡萄球菌产生的 Hlg 和 Luk 将通过用 Hlg2 和/或 LukS 替换细菌的玻连蛋白结合位点以及毒素的细胞溶解活性来诱导组织结合的葡萄球菌的分离和扩散。 Hlg2 和 LukS 还会中和可溶性玻连蛋白的调理功能，以防止炎症部位的专业吞噬细胞对金黄色葡萄球菌的吞噬作用。因此，Hlg 和 Luk 的细胞溶解活性和玻连蛋白结合活性都是葡萄球菌双组分毒素的假定病理生理功能。较少的

项目成果

N.Sugawara,T.Tomita,T.Sato,and Y.Kamio: "Assembly of Staphylococcus aureus leukocidin into a pore-forming ring-shaped oligomer on human polymorphonuclear leukocytes and rabbit erythrocytes"Biosci.Biotechnol.Biochem.. 63(5). 884-891 (1999)

N.Sukawara、T.Tomita、T.Sato 和 Y.Kamio：“将金黄色葡萄球菌杀白细胞素组装成人多形核白细胞和兔红细胞上的成孔环状寡聚物”Biosci.Biotechnol.Biochem.. 63(5

金子淳: "黄色ブドウ球菌の二成分細胞崩壊毒素のファージ変換及び標的細胞との作用に関する研究"日本農芸化学会誌. 75巻(印刷中). (2001)

Jun Kaneko：“金黄色葡萄球菌二元溶细胞毒素的噬菌体转化及其与靶细胞的相互作用”，日本农业化学学会杂志，第 75 卷（出版中）。

D.Zou,J.Kaneko,S.Narita,and Y.Kamio: "Prophage φPV83-pro,carrying Panton-Valentine leukocidin genes, on the Staphylococcus aureus P83 chromosome : comparative analysis of the genome structures of φPV83-pro, φPVL,φ11,and other phages"Biosci.Biotechnol.Bioc

D. Zou、J. Kaneko、S. Narita 和 Y. Kamio：“金黄色葡萄球菌 P83 染色体上携带 Panton-Valentine 杀白细胞素基因的原噬菌体 φPV83-pro：φPV83-pro、φPVL、 φ11，和其他噬菌体“Biosci.Biotechnol.Bioc

H.Katsumi, T.Tomita, J.Kaneko, and Y.Kamio: "Vitronectin and its fragments purified as serum inhibitors of Staphylococcus aureus gammahemolysin and leukocidin, and their specific binding to the Hlg2 and the LukS components of the toxins"FEBS Lett.. 460. 4

H.Katsumi、T.Tomita、J.Kaneko 和 Y.Kamio：“玻连蛋白及其片段纯化为金黄色葡萄球菌伽玛溶血素和杀白细胞素的血清抑制剂，以及它们与毒素的 Hlg2 和 LukS 成分的特异性结合”FEBS Lett

Y.Kamio: "TRENDS in PROTEIN RESEARCH"Scientific Publishers OWN. 150 (1999)

Y.Kamio：《蛋白质研究趋势》科学出版社拥有。