Assembly of Staphylococcus aureusγ‐hemolysin into a pore‐forming ring‐shaped complex on the surface of human erythrocytes

Abstract

Staphylococcal y-hemolysin consists of Hlgl (or Luk F) of 34 kDa and Hlg2 of 32 kDa, which cooperatively lyse human erythrocytes. Since y-hemolysin caused swelling of human erythrocytes prior to lysis, we studied pore-forming nature of the toxin by use of polyethylene glycols as osmotic protectants and determined the functional diameter of the pore. To elucidate the molecular architecture of the membrane pore formed by yhemolysin, we solubilized the pore complex with 2% sodium dodecyl sulfate, separated it from erythrocyte membrane proteins by sucrose gradient ultracentrifugation, and observed the isolated complex under an electron microscope. Our data showed that Hlgl and Hlg2 of y-hemolysin assemble into a ring-shaped 195 kDa complex in a molar ratio of 1 : 1, which may form a membrane pore with a functional diameter of 2.1-2.4 nm.

Bacterial Two-component and Hetero-heptameric Pore-forming Cytolytic Toxins: Structures, Pore-forming Mechanism, and Organization of the Genes

“…The data indicated that Hlg forms a 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. 28) It was also shown that the preceding binding of LukF is essential for complex formation as well as for Hlg2 binding under the physiologically relevant conditions. 29) Recently, we found that LukF binds strongly to membranes with a binding constant (K F ) of 2:1 Â 10 À4 m À2 and a large number of binding sites (R F ) of 2:0 Â 10 4 m À2 , and that Hlg2 also binds to membranes with a lower binding constant (K H = 1:2 Â 10 À5 m À2 ) combined with a similar number of binding sites (R H , 1:8 Â 10 4 m À2 ), indicating an approximately 18-fold decrease in the extent of binding of Hlg2 compared with that of LukF.…”

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

“…As shown by white arrowheads, several pores in the cluster are anchored to a part of the membrane. In our previous study, the diameter of Hlg pores was estimated to be approximately 2 nm [5], which is smaller than that of hemoglobin. However, hemoglobin seems to easily pass through the large hole around the cluster.…”

“…Removal of non-bound Hlg and incubation were performed as described in Section 2.1. The amount of potassium ions from the Hlg-treated erythrocytes was measured as described previously [5].…”

“…Membranes were collected and washed as described above (this method is applied in the experiments as shown in Figs. [2][3][4][5].…”

“…We have studied the assembly and pore-forming nature of these toxins [5][6][7][8][9][10] and revealed that Hlg and Luk form a transmembrane pore with a functional diameter of ca. 2 nm [5,6].…”