| Research Abstract |
(1) Pore-forming Nature of Hlg and Luk
When 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
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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 TOXINS
Most 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
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