Cellular Localization, qurification, and function of plasmud-determinedrirulence proteins of Salmonella

沙门氏菌质粒决定的毒性蛋白的细胞定位、纯化和功能

• 批准号: 05670264
• 负责人: DANBARA Hirofumi
• 金额: $ 1.34万
• 依托单位: KITASATO,University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1993
• 资助国家: 日本
• 起止时间: 1993 至 1994
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-05670264/
• 关键词: salmonella; Virulence; Plasmid; Regulation of expression; Virulance protein; lmmano-Electron Microscopy; 機能; 精製; 細胞局在性; 発現; 免疫電顕

Our previous studies showed that S.Choleraesuis strain RF-1 isolated from septicemic pig harbors 50kb-virulence plasmid designated pKDSC50, and the 6.4kb SalI-EcoRI region of pKDSC50 encompassing four spv genes, spvR,spvA,spvB,and spvC,is essential to produce systemic disease in pigs and mice. The spvR gene was characterized as a positive activator gene for transcription of the following three genes, spvA,spvB,and spvC.The aim of the present study is to obtain the basic knowledges of Spv proteins which are requird for understanding the Salmonella systemic infection.1. Regulatory function of Spv proteinsSpvR is a positive regulatory protein for the expression of its own gene, spvR.SpvA and SpvBare negative regulatory proteins for the expression of spvR gene. spvR gene is expressed in RpoS-dependent manner at the stationary phase of bacterial growth.2. Isolation, purification, and characterization of Spv proteinsThe four Spv proteins, spvR,spvA,spvB,and spvC were partially purified from S.Choleraesuis strain RF-1. Molecular weight of the purified Spv proteins by SDS-PAGE was determinded as 31 kDa for SpvR,26 kDa for SpvA,73 kDa for SpvB,and 24 kDa for SpvC.It was demonstrated that SpvB is a hydrophobic protein, and SpvC is a basic protein.3. Immunoelectron microscopic localization of Spv proteinsA method for the localizatiom of Spv proteins by immunoelectron microscopy was established. By using cryo ultra-thin section of S.Choleraesuis strain RF-1 fixed with periodete lysin-paraformaldehyde, SpvC was revealed to be a cytoplasmic and outer menbrane protein. The localization of SpvR,SpvA,and SpvB proteins is under investigation.

本实验室前期研究表明，猪霍乱沙门氏菌RF-1株携带50 kb毒力质粒pKDSC 50，其6.4kb的SalI-EcoRI区包含spvR、spvA、spvB和spvC四个spv基因，是引起猪和小鼠全身性疾病的关键。spvR基因被鉴定为一个能激活spvA、spvB和spvC三个基因转录的阳性激活基因。本研究的目的是获得了解沙门氏菌系统性感染所需的Spv蛋白的基本知识。Spv蛋白的调控功能SpvR是其自身基因spvR表达的正调控蛋白，SpvA和SpvB是其自身基因spvR表达的负调控蛋白。spvR基因在细菌生长的稳定期以RpoS依赖的方式表达.从猪霍乱沙门氏菌RF-1菌株中部分纯化了四种Spv蛋白，spvR，spvA，spvB和spvC。经SDS-PAGE分析，纯化的Spv蛋白的分子量为：SpvR为31 kDa，SpvA为26 kDa，SpvB为73 kDa，SpvC为24 kDa，表明SpvB为疏水蛋白，SpvC为碱性蛋白. Spv蛋白的免疫电镜定位建立了Spv蛋白的免疫电镜定位方法。用高碘酸溶素-多聚甲醛固定的猪霍乱沙门氏菌RF-1株的冷冻超薄切片，发现SpvC是一种胞质和外膜蛋白。SpvR、SpvA和SpvB蛋白的定位正在研究中。

项目成果

Masahiko Endoh et.al: "lsolation, Purification and Characterization of Salmonella plasmid virulence proteins of Salmonella Choleraesuis" Infection and Immunology. (in preparation).

Masahiko Endoh 等人：“猪霍乱沙门氏菌质粒毒力蛋白的分离、纯化和表征”感染和免疫学。

Akio Abe et al: "Regulation of Spv gene expression of Salmonella virulence plasmid pkDSC50 in Salmonella choleraesuis serovar Choleraesuis" Molecular Microbiology. 12. 779-787 (1994)

Akio Abe 等人：“猪霍乱沙门氏菌血清型猪霍乱中沙门氏菌毒力质粒 pkDSC50 的 Spv 基因表达的调节”分子微生物学。

Masahiko Endoh: "Isolation,purification,and characterization of Salmonella Plasmid virulence proteins of Salmonella Choleraesuis" Infection and Immunology. (in preparation).

Masahiko Endoh：“猪霍乱沙门氏菌质粒毒力蛋白的分离、纯化和表征”感染和免疫学。

Kachiko Sekiya: "Immunoelectron microscopic localization of Salmonella Plasmid virulence proteins of Salmonella Choleraesuis" Infection and Immunology. (in preparation).

Kachiko Sekiya：“猪霍乱沙门氏菌质粒毒力蛋白的免疫电镜定位”感染与免疫学。

Masahiro Endoh et al: "Isolation,Purification,and Charavterization of Salmonella Plasmid virulence proteins of Salmonella Choleraesuis" Infection and Immunology. (in preparation).

Masahiro Endoh 等人：“猪霍乱沙门氏菌质粒毒力蛋白的分离、纯化和表征”感染和免疫学。