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

基本信息

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

项目摘要

(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引起的人红细胞单细胞溶血时，观察到完整的盘状红细胞溶血后肿胀，细胞呈圆形，边缘清晰。与LukF和Hlg2孵育10分钟，随后肿胀的细胞裂解，因为细胞肿胀通常是由LukF和Hlg2引起的。由于细胞膜的通透性，推测 Hlg 通过孔形成诱导人红细胞的胶体渗透溶解，这一假设得到了以下发现的支持：[1] 细胞外非电解质（例如聚乙二醇）可以防止 Hlg 诱导的溶血。直径>2.5 nm，表明毒素形成了功能直径约为2.5 nm的亲水孔[2]。负染色、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，检查了 Hlg 在人红细胞上的复杂形成。如下：用来自红细胞膜的 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，然后进行 Western 检测使用针对 LukF 和 Hlg2 的特异性抗血清进行免疫印迹。数据表明，Hlg 在人红细胞表面形成约 200 kDa 的高分子复合物，其中包含摩尔比为 1:1 的 LukF 和 Hlg2。最近，[LukF-Hlg2]复合物被分离出来，并且还证明LukF的先前结合对于复合物的形成以及对于复合物是必需的。此外，我们最近的数据表明，可被蛋白酶 K 接触的膜成分可能是 Hlg 在人红细胞上形成复合物所必需的。总而言之，Hlg 可能在目标上组装成环形复合物。膜上形成一个功能直径约为 2.5 nm 的跨膜孔。PVL 已被认为在其白细胞溶解作用的早期阶段就形成了膜孔。 PVL形成的膜孔的结构还有待研究，并且还应该阐明该孔是否含有白细胞的内在膜蛋白。我们研究了Luk在人PMNL和细胞膜中的膜孔形成。兔红细胞和以下发现是明显的 [1] Luk 导致钾离子从兔红细胞中流出并导致溶血前细胞肿胀，然而，毒素处理后最终裂解。当细胞外间隙存在流体动力学直径【大于或等于】2.1 nm的聚乙二醇时，红细胞不会发生肿胀[2]电镜显示存在外径和内径均为9的环形结构。和3 nm，分别在Luk处理的人PMNL和兔红细胞上[3]从靶细胞中分离出相同尺寸的环形结构，并且它们含有。 LukS 和 LukF 的摩尔比为 1:1。 [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 组装成约 200 kDa 的环状寡聚物。靶细胞，形成功能直径约2 nm的膜孔。(2)结合LukF水溶性单体和组装体的显着特征。 Hla 结构的水不溶性七聚体与野生型和突变蛋白研究的数据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 不形成同源七聚体，但结构和功能相似。 LukF 和 Hla 之间以及 Hlg (LukF+Hlg2) 寡聚体和 Hla 七聚体的相似大小预测 LukF 和 Hla 共享结构元素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前茎区域的适度构象变化。此外，膜结合通过构象变化、双层表面封闭或两者都使前茎抵抗蛋白水解，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对比，在之前的模型中，富含甘氨酸的前茎区域位于低聚物的外围并与膜接触。这种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形成。在前孔状态下，前茎可能经历了部分重排，氨基锁已移动。其β-链位置能够产生有效的原聚体-原聚体接触，并且原聚体组装成七聚体，其直径比最终的孔形式稍大。当氨基锁折叠到帽结构域的内腔中的同时，多肽从帽的基部协同“挤出”，通过形成预孔寡聚物并与膜缔合，预孔可以发生。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。虽然 Hla、LukF、LukS 和 Hlg2 之间的功能相似，但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、边缘的低聚物。，以及像 Hla 七聚体这样的茎结构域，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的内部从 LukF 和 Hla 的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 的结构研究表明了如何交换和顺序暴露。特定蛋白质的结构和蛋白质-溶剂界面在这些寡聚跨膜毒素的组装中起着核心作用：水溶性形式通过单个亚基内的相互作用而稳定，而寡聚体形式通过亚基之间以及寡聚体与膜之间的相互作用来稳定。(3) 作为 HLG 和 LUK 血清抑制剂纯化的玻连蛋白及其片段，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 多功能糖蛋白及其带有 62、57 和已从人血清中分离出 38 kDa 的抑制剂，能够固定 Hlg 和 Luk。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。 Hlg2（或 LukS）形成高分子量复合物，在蔗糖梯度离心中共沉淀，并根据这些结果，在天然聚丙烯酰胺凝胶电泳上共迁移的完整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。、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 是成孔溶细胞素的新功能。由于玻连蛋白被认为可以调节蛋白水解酶级联，包括补体、凝血和纤溶系统，因此根据宿主的局部和全身条件，它可以作为宿主的矛盾因子。防御系统 : [1] 如果 Hlg 和 Luk 在葡萄球菌感染位点产生，则 Hlg2 和 LukS 将被玻连蛋白捕获及其片段在成纤维细胞和组织巨噬细胞的细胞外基质中，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纤溶酶在间质炎症部位的作用被释放。释放的玻连蛋白片段会固定并调理 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期蛋白，主要在肝脏中合成。细胞外基质相关的玻连蛋白被 Hlg 和 Luk 消耗后，通过血液循环和内皮细胞转胞吞作用输送到外周组织。葡萄球菌感染时，它会在一段时间内保持在较低水平。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]。金黄色葡萄球菌细胞中，它被认为是细菌的结合分子，金黄色葡萄球菌产生的 Hlg 和 Luk 会诱导分离和分离。通过用 Hlg2 和/或 LukS 替换细菌的玻连蛋白结合位点以及通过毒素 Hlg2 和 LukS 的细胞溶解活性来传播组织结合的葡萄球菌也会中和可溶性玻连蛋白的调理功能，以防止 S 的吞噬作用。因此，不仅具有细胞溶解活性，而且具有玻连蛋白结合活性。 Hlg 和 Luk 是葡萄球菌双组分毒素的假定病理生理功能。