Vitronectin and its fragments purified as serum inhibitors of Staphylococcus aureus γ-hemolysin and leukocidin, and their specific binding to the Hlg2 and the LukS components of the toxins

Staphylococcal γ-hemolysin and leukocidin are bi-component cytolysins, consisting of LukF (or Hlg1)/Hlg2 and LukF/LukS, respectively. Here, we purified serum inhibitors of γ-hemolysin and leukocidin from human plasma. Protein sequencing showed that the purified inhibitors of 62, 57, 50 and 38 kDa were the vitronectin fragments with truncation(s) of the C-terminal or both N- and C-terminal regions. The purified vitronectin fragments specifically bound to the Hlg2 component of γ-hemolysin and the LukS component of leukocidin to form high-molecular-weight complexes with them, leading to inhibition of the toxin-induced lysis of human erythrocytes and human polymorphonuclear leukocytes, respectively. Intact vitronectin also showed inhibitory activity to the toxins. The ability of γ-hemolysin and leukocidin to bind vitronectin and its fragments is a novel function of the pore-forming cytolysins.     

Tyrosine72 residue at the bottom of rim domain in LukF crucial for the sequential binding of the staphylococcal gamma-hemolysin to human erythrocytes

Staphylococcal bi-component cytotoxins, leukocidin (Luk), Panton-Valentine leukocidin (PVL), and gamma-hemolysin (Hlg) consist of LukF and LukS, LukF-PV and LukS-PV, and LukF and Hlg2, respectively, and Luk and Hlg share LukF. LukF-PV can not substitute for LukF for Hlg, despite 73% identity in amino acid sequence and close similarity in the 3-dimensional structure between them. Here, we demonstrated that the absence of hemolytic activity of LukF-PV in cooperation with Hlg2 is due to the failure of the binding of LukF-PV to human erythrocytes. We identified Y72 residue at the bottom of rim domain in LukF as the crucial residue for its binding, which is a prerequisite to the subsequent binding of Hlg2 to human erythrocytes. The data obtained showed that a mutant of LukF-PV in which T71 residue was replaced by the corresponding residue of LukF, Y72, endowed LukF-PV with the binding capability to human erythrocytes which was accompanied by its hemolytic activity in the presence of Hlg2.     

Bacterial two-component and hetero-heptameric pore-forming cytolytic toxins: structures, pore-forming mechanism, and organization of the genes

Staphylococcal gamma-hemolysin (Hlg), leukocidin (Luk), and Panton-Valentine leukocidin (PVL) are two-component and hetero-oligomeric pore-forming cytolytic toxins (or cytolysin), that were first identified in bacteria. No information on the existence of hetero-oligomeric pore-forming cytolytic toxins in bacteria except for staphylococcal strains is available so far. Hlg (Hlg1 of 34 kDa/Hlg2 of 32 kDa) effectively lyses erythrocytes from human and other mammalian species. Luk (LukF of 34 kDa/LukS of 33 kDa) is cytolytic toward human and rabbit polymorphonuclear leukocytes and rabbit erythrocytes, and PVL (LukF-PV of 34 kDa/LukS-PV of 33 kDa) reveals cytolytic activity with a high cell specificity to leukocytes. Hlg1 is identical to LukF and that the cell specificities of the cytolysins are determined by Hlg2 and LukS. Based on the primary and 3-dimensional structures of the toxin components, Hlg, Luk, and PVL are thought to form a family of proteins. In the first chapter of this article, we describe the molecular basis of the membrane pore-forming nature of Hlg, Luk, and PVL. We also describe a requirement of the phosphorylation of LukS and LukS-PV by protein kinase for their leukocytolytic activity besides their pore formation on human leukocytes.Recently, the assembly mechanism of the LukF and Hlg2 monomers into pore-forming hetero-oligomers of Hlg on human erythrocyte membranes has been clarified for the first time by our study using a single-molecular fluorescence imaging technique. We estimated 11 sequential equilibrium constants for the assembly pathway which includes the beginning with membrane binding of monomers, proceeds through single pore oligomerization, and culminates in the formation of clusters of the pores. In the second chapter of this article, we refer to an assembly mechanism of LukF and Hlg2 on human erythrocytes as well as the roles of the membranes of the target cells in pore formation by Hlg.The LukF, LukS, and Hlg2 proteins are derived from the Hlg locus (hlg), and have been found in 99% of clinical isolates of Staphylococcus aureus. In contrast, LukF-PV and LukS-PV are derived from the PVL locus (pvl) which is distinct from the hlg locus, and only a small percentage of clinically isolated S. aureus strains carries pvl. Recently, we discovered pvl on the genome of lysogenic bacteriophages, psiPVL, and determined the entire gene of the phage. We also demonstrated the phage conversion of S. aureus leading to the production of PVL through the discovery of a PVL-carrying temperate phage, psiSLT, from a clinical isolate of S. aureus. In the third chapter of this article, we discuss genetic analyses of the Hlg, Luk, and PVL genes. We also discuss the current status of knowledge of the genetic organization of PVL-converting phages in order to achieve an understanding of their molecular evolution.     

Tyrosine72 Residue at the Bottom of Rim Domain in LukF Crucial for the Sequential Binding of the Staphylococcal γ-Hemolysin to Human Erythrocytes

Staphylococcal bi-component cytotoxins, leukocidin (Luk), Panton-Valentine leukocidin (PVL), and γ-hemolysin (Hlg) consist of LukF and LukS, LukF-PV and LukS-PV, and LukF and Hlg2, respectively, and Luk and Hlg share LukF. LukF-PV can not substitute for LukF for Hlg, despite 73% identity in amino acid sequence and close similarity in the 3-dimensional structure between them. Here, we demonstrated that the absence of hemolytic activity of LukF-PV in cooperation with Hlg2 is due to the failure of the binding of LukF-PV to human erythrocytes. We identified Y72 residue at the bottom of rim domain in LukF as the crucial residue for its binding, which is a prerequisite to the subsequent binding of Hlg2 to human erythrocytes. The data obtained showed that a mutant of LukF-PV in which T71 residue was replaced by the corresponding residue of LukF, Y72, endowed LukF-PV with the binding capability to human erythrocytes which was accompanied by its hemolytic activity in the presence of Hlg2.     

Assembly of Staphylococcus aureus leukocidin into a pore-forming ring-shaped oligomer on human polymorphonuclear leukocytes and rabbit erythrocytes.

Staphylococcal leukocidin consists of two separate proteins, LukS and LukF, which cooperatively lyse human and rabbit polymorphonuclear leukocytes and rabbit erythrocytes. Here we studied the pore-forming properties of leukocidin and the molecular architecture of the leukocidin pore. (1) Leukocidin caused an efflux of potassium ions from rabbit erythrocytes and swelling of the cells before hemolysis. However, ultimate lysis of the toxin-treated swollen erythrocytes did not occur when polyethylene glycols with hydrodynamic diameters of > or = 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 leukocidin-treated human polymorphonuclear leukocytes and rabbit erythrocytes. (3) Ring-shaped structures of the same dimensions 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 205 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 of approximately 2 nm.     

Stochastic assembly of two-component staphylococcal gamma-hemolysin into heteroheptameric transmembrane pores with alternate subunit arrangements in ratios of 3:4 and 4:3

Self-assembling, pore-forming toxins from Staphylococcus aureus are illustrative molecules for the study of the assembly and membrane insertion of oligomeric transmembrane proteins. On the basis of previous studies, we have shown that the two-component gamma-hemolysin assembles from LukF (or Hlg1, 34 kDa) and Hlg2 (32 kDa) to form ring-shaped transmembrane pores of ca. 200 kDa. Here we show that LukF and Hlg2 assemble in a stochastic manner to form alternate complexes with subunit stoichiometries of 3:4 and 4:3. High-resolution electron microscopic images of negatively stained pore complexes clearly revealed a heptameric structure. When adjacent monomers in the pore complexes were randomly cross-linked by using glutaraldehyde, LukF-LukF, LukF-Hlg2, and Hlg2-Hlg2 dimers were detected in an approximate ratio of 1:12:1, suggesting that LukF and Hlg2 were alternately arranged in the pore complex in molar ratios of 3:4 and 4:3. The alternate arrangements of LukF and Hlg2 in molar ratios of 3:4 and 4:3 were also visualized under electron microscope with the pore complexes consisting of glutathione S-transferase fusion protein of LukF or Hlg2 and wild-type protein of Hlg2 or LukF, respectively.     

Assembly of the Bi-component leukocidin pore examined by truncation mutagenesis

Staphylococcal leukocidin (Luk) and alpha-hemolysin (alphaHL) are members of the same family of beta barrel pore-forming toxins (betaPFTs). Although the alphaHL pore is a homoheptamer, the Luk pore is formed by the co-assembly of four copies each of the two distantly related polypeptides, LukF and LukS, to form an octamer. Here, we examine N- and C-terminal truncation mutants of LukF and LukS. LukF subunits missing up to nineteen N-terminal amino acids are capable of producing stable, functional hetero-oligomers with WT LukS. LukS subunits missing up to fourteen N-terminal amino acids perform similarly in combination with WT LukF. Further, the simultaneous truncation of both LukF and LukS is tolerated. Both Luk subunits are vulnerable to short deletions at the C terminus. Interestingly, the N terminus of the LukS polypeptide becomes resistant to proteolytic digestion in the fully assembled Luk pore while the N terminus of LukF remains in an exposed conformation. The results from this work and related experiments on alphaHL suggest that, although the N termini of betaPFTs may undergo reorganization during assembly, they are dispensable for the formation of functional pores.     

An N-terminal Region of LukF of Staphylococcal Leukocidin/γ-Hemolysin Crucial for the Biological Activity of the Toxin

The two staphylococcal bi-component toxins, leukocidin and γ-hemolysin share LukF [Kamio et al, FEBS Lett., 321, 15-18 (1993)]. This report identifies the pivotal amino acid residues in the N-terminal region of LukF for the leukocytolytic and hemolytic activities in the presence of LukS and Hlg2, respectively, measuring the toxin activiy of a series of LukF mutants with truncated N-terminals. The data obtained showed that the LukF mutant TF21, lacking 20 amino acid residues at the N-terminus of LukF, failed to have any hemolytic activity and had less 10% leukocytolytic activity than that of the intact LukF, while 16-residue truncations retained both toxin activities without loss. The LukF mutants lacking 18- through 19-residue segments from the N-terminus showed low toxin activity on both target cells. All mutants having no toxin activity were also not capable of binding to the human erythrocytes. It can thus be concluded that the 3-residue segment, L¹⁸Y¹⁹K²⁰ of LukF is crucial for the biological activity of the toxin.     

Construction of a LukS-PV mutant of a staphylococcal Panton-Valentine leukocidin component having a high LukS-like function

A 2-residue (D12I13) segment of LukS of a staphylococcal leukocidin component is an essential region for the hemolytic function of LukS towards rabbit erythrocytes in the presence of LukF. Here, we report that insertion of D, I, or AA residue(s) between A11 and E12 residues of LukS-PV, in which the 2-residue D12I13 segment in LukS was absent, confers the full LukS function on LukS-PV, which has only 4% hemolytic activity of that of LukS towards rabbit erythrocytes.     

Assembly of staphylococcal leukocidin into a pore-forming oligomer on detergent-resistant membrane microdomains, lipid rafts, in human polymorphonuclear leukocytes

Staphylococcal leukocidin (Luk) consists of LukS and LukF, which cooperatively lyse human polymorphonuclear leukocytes (HPMNLs), monocytes, and macrophages. Here we found that LukS and LukF assembles into hetero-oligomeric pore complexes on the detergent-resistant membrane microdomains, lipid rafts of HPMNLs. When HPMNLs were treated with LukS alone, 24% of the added LukS was localized in lipid rafts. Furthermore, in HPMNLs treated with both LukS and LukF simultaneously, about 90% of high molecular-mass complexes of 100 kDa, which consists of LukS and LukF, were detected in the lipid raft fractions. In contrast, in HPMNLs treated with LukF alone, LukF was not localized in lipid rafts despite binding to the target cell membranes. Ten mM methyl-beta-cyclodextrin, a dysfunctioning agent of lipid rafts, completely inhibited assembly of Luk on lipid rafts, and resulted in null leukocytolytic activity of Luk. Hence, we concluded that assembly of LukS and LukF into the pore-complex occurs in lipid rafts in HPMNLs and that LukF can bind to LukS, which had already bound to lipid rafts, to assemble into hetero-oligomers.     

Essential residues, W177 and R198, of LukF for phosphatidylcholine-binding and pore-formation by staphylococcal gamma-hemolysin on human erythrocyte membranes

LukF and Hlg2 of staphylococcal gamma-hemolysin assemble into hetero-oligomeric pores on human red blood cells (HRBC). Here, we demonstrate, using a single-molecule imaging technique, that a W177T/R198T mutant of LukF, which exhibits no binding activity toward phosphatidylcholine, could form intermediate oligomers with Hlg2, including dimers, tetramers, and hexamer/heptamers, on HRBC. But, the mutant neither caused K(+) efflux nor lysed HRBC, indicating that functional pores were not formed. Hence, we conclude that the W177 and R198 residues are essential for proper pore-formation by staphylococcal gamma-hemolysin. We also suggest that the interaction between the W177 and R198 residues, and phosphatidylcholine on membranes is the key to the formation of functional pores.     

Assembly of Staphylococcal Leukocidin into a Pore-Forming Oligomer on Detergent-Resistant Membrane Microdomains,Lipid Rafts,in Human Polymorphonuclear Leukocytes

Staphylococcal leukocidin (Luk) consists of LukS and LukF, which cooperatively lyse human polymorphonuclear leukocytes (HPMNLs), monocytes, and macrophages. Here we found that LukS and LukF assembles into hetero-oligomeric pore complexes on the detergent-resistant membrane microdomains, lipid rafts of HPMNLs. When HPMNLs were treated with LukS alone, 24% of the added LukS was localized in lipid rafts. Furthermore, in HPMNLs treated with both LukS and LukF simultaneously, about 90% of high molecular-mass complexes of 100 kDa, which consists of LukS and LukF, were detected in the lipid raft fractions. In contrast, in HPMNLs treated with LukF alone, LukF was not localized in lipid rafts despite binding to the target cell membranes. Ten mM methyl-β-cyclodextrin, a dysfunctioning agent of lipid rafts, completely inhibited assembly of Luk on lipid rafts, and resulted in null leukocytolytic activity of Luk. Hence, we concluded that assembly of LukS and LukF into the pore-complex occurs in lipid rafts in HPMNLs and that LukF can bind to LukS, which had already bound to lipid rafts, to assemble into hetero-oligomers.     

Phosphorylation of LukS by Protein Kinase A is Crucial for the LukS-Specific Function of the Staphylococcal Leukocidin on Human Polymorphonuclear Leukocytes

Staphylococcal leukocidin (Luk) consists of two protein components, LukF and LukS, which cooperatively lyse human and rabbit polymorphonuclear leukocytes. Here, we demonstrate that the phosphorylation of LukS by protein kinase A is crucial for the LukS-specific leukocytolytic function of Luk on HPMNLs by using N-[2(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89), which is a potent and selective inhibitor of protein kinase A. At 0.5 μM H-89 completely prevented the Luk-induced cell lysis accompanied by blocking of the incorporation of exogenous ³²P-H₃PO₄ into LukS on HPMNLs. However, with LukS and LukF together, 0.5 μM H-89 did not inhibit the cell swelling which takes place before the cell lysis. HPMNLs also became swollen upon treating with both LukF and LukS mutants which could not be phosphorylated.     

Substitution of Lysine for Arginine in the N-Terminal 217th Amino Acid Residue of the HγII of Staphylococcall γ-Hemolysin Lowers the Activity of the Toxin

The staphyloccocal toxin γ-hemolysin consists of two protein components, LukF and HyII. Staphylococcus aureus P83 was found to have five components, LukF, LukF-PV, LukM, LukS, and HγII for leukocidin or γ-hemolysin. HγII of S. aureus P83 was demonstrated to be a naturally-occurring analogous molecule of HγII [HγII(P83)], in which the 217th arginine residue was replaced by lysine. The HγII(P83) showed about 50% of the hemolytic activity of normal HyII in the presence of LukF.     

Single-molecule visualization of environment-sensitive fluorophores inserted into cell membranes by staphylococcal gamma-hemolysin

Single-molecule imaging of the entrance of a protein into the hydrophobic environment of a cell membrane was investigated. The pre-stem of LukF, one of the two components of the pore-forming toxin staphylococcal gamma-hemolysin, was specifically labeled with 6-bromoacetyl-2-dimethylaminonaphthalene (Badan), an environment-sensitive fluorophore. Incubation of this derivative with erythrocyte ghost membranes resulted in a pronounced increase in fluorescence indicating insertion of Badan into the hydrophobic interior of the lipid bilayers. However, the increase in fluorescence was completely dependent on the interaction of Badan-labeled LukF with the gamma-hemolysin second component. Individual spots of Badan fluorescence on erythrocyte membranes were visualized that were associated with single pores. Analyses of the intensities of these fluorescent spots and their photobleaching independently showed that a single pore contained 3-4 LukF molecules. Thus, environment-sensitive fluorophore signals can be used to study the insertion of specific protein domains into cell membranes at the single-molecule lever, and the use of this approach in the present study revealed that a single gamma-hemolysin pore opening contains at least three LukF molecules.     

Cluster-forming property correlated with hemolytic activity by staphylococcal γ-hemolysin transmembrane pores

Staphylococcal γ-hemolysin (Hlg) is a pore-forming toxin consisting of two separate components, LukF (34kDa) and Hlg2 (32kDa). Here we show that Hlg pores aggregate and form clusters on human erythrocyte membranes in association with increasing hemolytic activity. Quantitative analysis using transmission electron microscopy and image processing revealed that the formation of single pores and clusters is related to the release of potassium ions and of hemoglobin from erythrocytes, respectively. This is the first study to suggest a novel and unique property which can facilitate hemolysis by the cluster formation of Hlg pores.     

Toll-like receptor 4-dependent activation of myeloid dendritic cells by leukocidin of Staphylococcus aureus

Leukocidin (Luk), an exotoxin of Staphylococcus aureus consisting of LukF and LukS, is a hetero-oligomeric pore-forming cytolytic toxin toward human and rabbit polymorphonuclear leukocytes. However, it is uncertain how Luk affects the host immune response. In the present study, we investigated whether Luk has the ability to stimulate mouse bone marrow-derived myeloid dendritic cells (BM-DCs). LukF activated BM-DCs to generate IL-12p40 mRNA, induce intracellular expression and extracellular secretion of this cytokine and express CD40 on their surface, whereas LukS showed a much lower or marginal ability in the activation of BM-DCs than its counterpart component. Similarly, TNF-α was secreted by BM-DCs upon stimulation with these components. Combined addition of these components did not lead to a further increase in IL-12p40 secretion. IL-12p40 production caused by LukF was completely abrogated in BM-DCs from TLR4-deficient mice similarly to the response to lipopolysaccharide (LPS). Polymixin B did not affect the LukF-induced IL-12p40 production, although the same treatment completely inhibited the LPS-induced response. Boiling significantly inhibited the response caused by LukF, but not by LPS. Finally, in a luciferase reporter assay, LukF induced the activation of NF-κB in HEK293T cells transfected with TLR4, MD2 and CD14, whereas LukS did not show such activity. These results demonstrate that LukF caused the activation of BM-DCs by triggering a TLR4-dependent signaling pathway and suggests that Luk may affect the host inflammatory response as well as show a cytolytic effect on leukocytes.     

Subunit composition of a bicomponent toxin: staphylococcal leukocidin forms an octameric transmembrane pore

Staphylococcal leukocidin pores are formed by the obligatory interaction of two distinct polypeptides, one of class F and one of class S, making them unique in the family of beta-barrel pore-forming toxins (beta-PFTs). By contrast, other beta-PFTs form homo-oligomeric pores; for example, the staphylococcal alpha-hemolysin (alpha HL) pore is a homoheptamer. Here, we deduce the subunit composition of a leukocidin pore by two independent methods: gel shift electrophoresis and site-specific chemical modification during single-channel recording. Four LukF and four LukS subunits coassemble to form an octamer. This result in part explains properties of the leukocidin pore, such as its high conductance compared to the alpha HL pore. It is also pertinent to the mechanism of assembly of beta-PFT pores and suggests new possibilities for engineering these proteins.     

alpha-Hemolysin, gamma-hemolysin, and leukocidin from Staphylococcus aureus: distant in sequence but similar in structure.

alpha-Hemolysin from Staphylococcus aureus assembles from a water-soluble, monomeric species to a membrane-bound heptamer on the surface of target cells, creating water-filled channels that lead to cell death and lysis. Staphylococcus aureus also produces the gamma-hemolysin and leukocidin toxins, which function as two component toxins in the disruption and lysis of erythrocytes and leukocytes. Analysis of the aligned sequences of alpha-hemolysin, gamma-hemolysin, and leukocidin in the context of the alpha-hemolysin heptamer structure supports the conclusion that even though the level of sequence identity between alpha-hemolysin and the gamma-hemolysin and leukocidin toxins is in the so-called twilight zone, the three-dimensional structures of the protomers are probably conserved. By analogy with alpha-hemolysin, gamma-hemolysin and leukocidin may also form oligomeric, transmembrane channels in which an antiparallel beta-barrel constitutes the primary membrane-embedded domain.