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

(1) Hlg和luk的成孔性质在相差显微镜下观察Hlg诱导的人红细胞单细胞溶血情况，发现LukF和Hlg2作用10 min后，完整的盘状红细胞肿胀，细胞呈圆形，边缘清晰，肿胀细胞溶解。由于细胞的肿胀通常是由细胞膜的通透性引起的，因此推测Hlg通过形成孔诱导了人红细胞的胶体渗透溶解。这一假设得到以下调查结果的支持：细胞外非电解质（如聚乙二醇）的直径为2.5 nm，可以阻止Hlg诱导的溶血，这表明毒素形成了一个亲水孔，其功能直径约为2.5 nm。电子显微镜下，阴性染色的毒素处理红细胞显示Hlg形成一个环状结构，其外径约为7 nm，内径约为3 nm。因此，我们采用以下方法检测Hlg在人红细胞上的复合物形成：将红细胞膜上的细胞结合毒素用SDS溶解，然后用SDS-聚丙烯酰胺凝胶电泳分析，然后用针对LukF和Hlg2的特异性抗血清进行Western免疫印迹。数据表明，Hlg在人红细胞表面形成约200 kDa的高分子量复合物（es），其中含有LukF和Hlg2，摩尔比为1:1。最近，[LukF-Hlg2]络合物被分离出来。研究还表明，LukF的先前结合对于复合物的形成以及Hlg2的结合是必不可少的。此外，我们最近的数据表明，蛋白酶K可接近的膜组分可能是人红细胞上Hlg复合物形成所必需的。综上所述，Hlg可以在靶膜上组装成环状复合物，形成功能直径约为2.5 nm的跨膜孔。PVL被认为在其溶解白细胞的早期形成膜孔。然而，PVL形成的膜孔的分子结构仍有待研究，并且该孔中是否含有白细胞固有的膜蛋白也有待阐明。我们研究了Luk在人PMNLs和兔红细胞细胞膜上形成的膜孔，结果如下：[1] Luk引起兔红细胞钾离子外排和溶血前细胞肿胀。然而，当水动力直径大于或等于2.1 nm的聚乙二醇存在于细胞外空间时，毒素处理的肿胀红细胞不会发生最终的溶解。电镜显示，luk处理的人PMNLs和兔红细胞上存在外径为9 nm、内径为3 nm的环状结构。[3]从靶细胞中分离出相同尺寸的环状结构，其含有LukS和LukF，摩尔比为1:1。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200 kDa的环状低聚物，形成功能直径约为2 nm的膜孔。(2)装配机理。将LukF水溶性单体和Hla水不溶性七聚体结构的显著特征与野生型和突变型蛋白的研究数据相结合，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）。虽然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）寡聚物和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。Hla七聚体结构是构建孔前组装中间体模型的合理起点，而LukF单体结构可以作为构建Hla、LukS和Hlg2水溶性和膜结合单体模型的起点。这表明，膜结合的单体类似于LukF的水溶性形式，除了与双分子层的相互作用在边缘和茎前区域引起适度的构象变化。此外，膜结合使前茎通过构象改变、通过双层表面的封闭或两者兼而有之而抵抗蛋白水解。图11所示的低聚预孔中间体结构模型的一个重要特征是，富含甘氨酸的预干位于帽域孔内。该模型与以前的模型相反，在以前的模型中，富含甘氨酸的前茎区位于低聚物的外围并与膜表面接触。这一机制解释了毒素如何在水溶液中表现出溶解度，并抵抗组装，直到膜结合触发前孔的形成。在预孔状态下，预茎可能发生了部分重排，氨基锁存器从其β链位置移动，使原聚体与原聚体接触，并且原聚体组装成七聚体，与最终的孔形态相比，七聚体的直径略大。在氨基酸锁存器折叠进入帽结构域的管腔的同时，通过多肽从帽的基部协同“挤压”而将预茎插入膜中。通过形成预孔寡聚物并与所述膜结合，所述预孔可使双分子层变薄，从而便于茎插入。由于LukF、LukS和Hlg2蛋白形成的异构体可能是六体，因此与Hla相比，它们的组装肯定会存在差异。然而，考虑到Hla、LukF、LukS和Hlg2在结构和功能上的相似性，它们无疑具有许多共同的机制特征。尽管图11中所示的机制主要集中在Hla上，但我们预测LukF， LukS和Hlg会像Hla七聚体一样组装成具有帽、边缘和茎结构域的低聚体，Hla和Luk会通过一个低聚中间体组装，其中茎前区域聚集在帽结构域的内部。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，如气溶素和炭疽保护抗原。更一般地说，Hla和LukF的结构研究表明，在这些低聚跨膜毒素的组装中，特定蛋白质和蛋白质-溶剂界面的交换和顺序揭露如何发挥核心作用：水溶性形式通过单个亚基内的相互作用稳定，而低聚形式通过亚基之间以及低聚物与膜之间的相互作用稳定。(3) VITRONECTIN及其片段被纯化为HLG和LUK的血清抑制剂，以及它们与毒素的hg2和LUK的特异性结合最近，从人血清中分离出了75kda的多功能糖蛋白VITRONECTIN及其片段，分别具有62,57和38kda，是一种具有固定HLG和LUK能力的抑制剂。纯化后的玻璃体粘连蛋白及其片段特异性结合到Hlg2和LukS上，分别阻止毒素诱导的人红细胞和人PMNLs的溶解。玻璃体连接蛋白片段和Hlg2（或LukS）形成高分子量复合物，在蔗糖梯度离心中共同沉积，并在天然聚丙烯酰胺凝胶电泳上共同迁移。完整的玻璃体连接蛋白的活性比纯化的抑制剂低15倍，但当与人纤溶蛋白部分消化时，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，玻璃体粘连蛋白及其片段被认为是葡萄球菌感染基因座中Hlg和Luk固定的可能宿主成分。Hlg和Luk的体外连接蛋白结合能力是形成孔的细胞溶解素的一种新功能。由于玻璃体粘连蛋白被认为调节包括补体、凝血和纤溶系统在内的蛋白水解酶级联反应，因此根据宿主防御系统的局部和全身条件，玻璃体粘连蛋白可能作为一种矛盾因子存在：如果Hlg和Luk在葡萄球菌感染位点产生，则Hlg2和Luk将被成纤维细胞和组织巨噬细胞的细胞外基质中的玻璃体连接蛋白及其片段捕获，然后由整合素介导的细胞内吞和降解。胞外基质相关的玻璃体连接蛋白在间质性炎症部位被纤溶酶释放，释放的玻璃体连接蛋白片段会固定和调节Hlg和Luk。[3]然而，Hlg和Luk对玻璃体连接蛋白的消耗会导致凝血、纤溶和补体级联的调节失衡，导致补体末端复合物水平过高和纤溶蛋白过量产生而导致组织损伤。[4] Vitronectin是一种急性期蛋白，主要在肝脏对白细胞介素6的反应中合成，并通过血液循环和内皮细胞的胞吞作用传递到外周组织。一旦细胞外基质相关的玻璃质粘连蛋白被葡萄球菌感染部位的Hlg和Luk消耗，它将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严重预后的皮肤和粘膜感染中起关键作用。玻璃质粘连蛋白已被证明与金黄色葡萄球菌的细胞特异性结合，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通过用Hlg2和/或Luk取代细菌的体外连接蛋白结合位点以及毒素的细胞溶解活性诱导组织结合葡萄球菌脱离和扩散。Hlg2和LukS也能中和可溶性玻璃体连接蛋白的拮抗功能，阻止金黄色葡萄球菌被炎症位点的专业吞噬细胞吞噬。因此，Hlg和Luk不仅具有细胞溶解活性，而且具有胞外连接素结合活性，这是葡萄球菌双组分毒素的病理生理功能。少

(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