Improved Method for Purification of Leukocidin and Gamma-Hemolysin Components from<i>Staphylococcus aureus</i>

Nariya, Hirofumi; Asami, Ikuno; Ozawa, T.; Beppu, Yoshinori; Izaki, Kazuo; Kamio, Yoshiyuki

doi:10.1271/bbb.57.2198

Cited by 13 publications

(9 citation statements)

References 5 publications

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“…Purification of recombinant mutant proteins was carried out as described previously (Nariya et al ., 1993), except that the concentration of the washing and elution buffer for the Hydroxyapatite column was raised to 0.32 M and 0.8 M, respectively, to obtain pure double‐cysteine mutants. Protein concentration was measured by the method of Bradford using bovine serum albumin as a standard.…”

Section: Methodsmentioning

confidence: 99%

Controlling pore assembly of staphylococcal γ‐haemolysin by low temperature and by disulphide bond formation in double‐cysteine LukF mutants

Nguyen

Higuchi

Kamio

2002

Molecular Microbiology

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SummaryStaphylococcal LukF and Hlg2 are water-soluble monomers of g g g g -haemolysin that assemble into oligomeric pores on the erythrocyte membranes. Here, we have created double-cysteine LukF mutants, in which single disulphide bonds connect either the prestem domain and the cap domain (V12C-T136C, CapStem), or two b b b b -strands within the prestem domain (T117C-T136C, Stem-Stem) to control pore assembly of g g g g -haemolysin at intermediate stages. The disulphide-trapped mutants were inactive in erythrocyte lysis, but gained full haemolytic activity if the disulphide bonds were reduced. The disulphide bonds blocked neither the membrane binding ability nor the intermediate prepore oligomerization, but efficiently inhibited the transition from prepores to pores. The prepores of Cap-Stem were dissociated into monomers in 1% SDS. In contrast, the prepores of Stem-Stem were stable in SDS and had ring-shaped structures similar to those of wild-type LukF, as observed by transmission electron microscopy. The transition of both mutants from prepores to pores could even be achieved by reducing disulphide bonds at low temperature (2 ∞ ∞ ∞ ∞ C), whereas prepore oligomerization was effectively inhibited by low temperature. Finally, real-time transition of Stem-Stem from prepores to pores on ghost cells, visualized using a Ca 2 + + + + -sensitive fluorescent indicator (Rhod2), was shown by the sequential appearance of fluorescence spots, indicating pore-opening events. Taken together, these data indicate that the prepores are legitimate intermediates during g g g g -haemolysin pore assembly, and that conformational changes around residues 117 and 136 of the prestem domain are essential for pore formation, but not for membrane binding or prepore oligomerization. We propose a mechanism for g g g ghaemolysin pore assembly based on the demonstrated intermediates.

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Section: Methodsmentioning

confidence: 99%

Controlling pore assembly of staphylococcal γ‐haemolysin by low temperature and by disulphide bond formation in double‐cysteine LukF mutants

Nguyen

Higuchi

Kamio

2002

Molecular Microbiology

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“…γ ‐Hemolysin was purified from the culture supernatant of Staphylococcus aureus 5R Smith strain as described previously [11].…”

Section: Methodsmentioning

confidence: 99%

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

1997

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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.

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“…Den-'sitometry for the protein bands indicated that the fractions 2, 3, 4, 6, 8, and 9 contained LukS and LukF in a molar ratio of 1.00 to O.98-1.03. These results suggested that the fractions [1][2][3][4][5][6][7][8][9] contained Luk complexes of > 300, 300, 205, and 140 kDa, and that the Luk complexes were dissociated to monorneric LukS aRd LukF in a molar ratio of 1:1 upon heating at 1OOOC in the presence of 2% SDS. Immunoblot analysis showed that the fractions 1 1-･16 contained significant amounts of monomeric LukS and LukF but no large-molecular-sized complex of Luk (results not shown).…”

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Assembly ofStaphylococcus aureusLeukocidin into a Pore-Forming Ring-Shaped Oligomer on Human Polymorphonuclear Leukocytes and Rabbit Erythrocytes

Sato

Tomita

Sato

et al. 1999

Bioscience, Biotechnology, and Biochemistry

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Staphylococcal leukecidin consists of two separate proteins, LukS and LukF, which eooperatively lyse hllman a"d rabbit pelymorphenuclear leukocytes and rabbit erythrecytes. Here we studied the pore-forming properties of leukocidin alld the melecular architecture of the leukocidin pere. (1) Leukocidin caused an eMux of potassium iens from rabbit erythrocytes and swelling of the cells before hemolysis. However, ultimate lysis of the toxin-treated swollen erythrocytes did not occur when pelyethylene glycels with hydrodynanic dia!rteters of z2.1nm were present in the extracellular space. (2) Electron micrescopy shewed the presence of a ring-shaped structure with outer and inner diameters of 9 and 3 nm, respectiyely, en 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 ef 1:1. (4) A single ring-shaped toxin complex had a molecular size of 205 kDa. These results indicated that LukS and LukF ftssemble into a ring-shaped oligomer of approximately 200 kDa on the target cells, forming a membrane pore with a f"nctional diameter ef approximately 2 nm.

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Improved Method for Purification of Leukocidin and Gamma-Hemolysin Components fromStaphylococcus aureus

Cited by 13 publications

References 5 publications

Controlling pore assembly of staphylococcal γ‐haemolysin by low temperature and by disulphide bond formation in double‐cysteine LukF mutants

Controlling pore assembly of staphylococcal γ‐haemolysin by low temperature and by disulphide bond formation in double‐cysteine LukF mutants

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

Assembly ofStaphylococcus aureusLeukocidin into a Pore-Forming Ring-Shaped Oligomer on Human Polymorphonuclear Leukocytes and Rabbit Erythrocytes

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