2002 Fiscal Year Final Research Report Summary
Staphylococcal pore-forming toxins: Mechanism of pore-forming and recognition of the target cells
| Project/Area Number |
13460034
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
応用微生物学・応用生物化学
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| Research Institution | Tohoku University |
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Principal Investigator |
KAMIO Yoshiyuki Graduate School of Agricultural Science, Tohoku University, Professor, 大学院・農学研究科, 教授 (00109175)
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| Co-Investigator(Kenkyū-buntansha) |
HIGUCHI Hideo Graduate School of Engineering, Tohoku University, Associate Professor, 大学院・工学研究科, 助教授 (90165093)
KANEKO Jun Graduate School of Agricultural Science, Tohoku University, Research Associate, 大学院・農学研究科, 助手 (30221188)
TOMITA Toshio Graduate School of Agricultural Science, Tohoku University, Associate Professor, 大学院・農学研究科, 助教授 (00126129)
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| Project Period (FY) |
2001 – 2002
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| Keywords | Staphylococcus aureus / pore-forming toxin / gamma-hemolysin / staphylococcal toxin / Single-molecule imaging / Assembly of toxin components |
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| Research Abstract |
Staphylococcal LukF and Hlg2 are water-soluble monomers of gamma-hemolysin that assemble into an oligomeric pore on the erythrocyte membrane. The structure of LukF suggests that the pre-stem domain, which folds back toward the cap domain, may insert into the membrane during assembly. Here, we created double-cysteine LukF mutants in which single disulfide bonds connect either the pre-stem domain and the cap domain(V12C-T136C,Cap-Stem), or two b-strands within the pre-stem domain(T117C-T136C,Stem-Stem), to study structural changes of the pre-stem domain in pore assembly. Both mutants are inactive in erythrocyte lysis, however gain full hemolytic activity if the disulfide bonds are reduced. The disulfide bonds block neither the membrane binding ability nor the intermediate pre-pore oligomerization, but efficiently inhibit the transition from pre-pores to functional pores. An SDS-unstable pre-pore formed by the Cap-Stem mutant and the ring-shaped, SDS-stable pre-pore formed by the Stem-Stem mutant are ascribed to "early" and "late" states of pre-pore formation, respectively. The membrane binding and the mercaptoethanol-induced pore formation of both mutants even at 2℃ indicate that only oligomerization is a temperature-dependent step. Thus, the inhibition of pore assembly at various steps by disulfide bond formation and low temperature proves the requirement for structural changes in the pre-stem domain of LukF during pore assembly.
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Research Products
(16 results)
