Studies on the secretion and maturation pathway of bacterial exotoxin

细菌外毒素分泌及成熟途径的研究

• 批准号: 14570237
• 负责人: OKAMOTO Keinosuke
• 金额: $ 2.24万
• 依托单位: OKAYAMA UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2002
• 资助国家: 日本
• 起止时间: 2002 至 2003
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14570237/
• 关键词: Enterotoxin; Escherichia coli; Aeromonas spp.; Secretion; Maturation; Membrane protein; Hemolysin; Diarrhea; 成熟化; 毒素; 細胞膜; 蛋白分解酵素; 菌体外毒素; 膜; グラム陰性菌; プロテアーゼ

Diarrhea caused by the infection of bacteria is usually induced, by the action of the enterotoxin released from the bacteria into outside of the bacteria. It means that the diarrhea is not induced by the bacteria which can not secrete the active toxin into the outside of the cell, even if the bacteria synthesized the toxin in cell. In order to emerge outside of the cell as the active toxin, the toxin synthesized in cell must cross two membranes and form active structure during the secretion. Without this event, the bacteria can not express the toxic activity. We investigated the event in the productions of heat-stable enterotoxin (ST) of Escherichia coli, and enterotoxin of Aeromonas spp. It becomes clear that ST crosses the outer membrane though the pore formed by ToIC and that both Leu-412 and Leu-3 play an important role in the transportation of ST. This suggests that the compound attaching these two residues might interfere the secretion of ST, resulting the suppression of the toxicity of the bacteria producing ST. Subsequently, we found that the enterotoxin of Aeromas spp. possesses hemolytic activity, too. Then, we examined the hemolytic activity of the isolates from patients. Some of strains did not show hemolytic activity but showed enterotoxic activity. To clear the relationship between two activities, we cloned the gene encoding the new hemolysin. The results showed that there are differences in the DNA sequence between the wild type hemolysin gene and the new hemolysin gene in the amino terminal region. Furthermore, we found the protease is involved in the maturation of the hemolysin and that the protease function in cell to survive the bacteria in severe conditions, too.

由细菌感染引起的腹泻通常是通过从细菌释放到细菌外部的肠毒素的作用引起的。这意味着即使细菌在细胞内合成了毒素，但细菌不能将活性毒素分泌到细胞外，因此腹泻不是由细菌引起的。细胞内合成的毒素在分泌过程中必须穿过两层膜形成活性结构，才能以活性毒素的形式出现在细胞外。如果没有这个事件，细菌就不能表达毒性活性。本研究对大肠杆菌产耐热肠毒素（ST）和气单胞菌产肠毒素进行了研究。结果表明，ST通过ToIC形成的孔穿过外膜，Leu-412和Leu-3在ST的转运中起重要作用，这表明连接这两个残基的化合物可能会干扰ST的分泌，从而抑制产ST细菌的毒性。也具有溶血活性。然后，我们检测了患者分离物的溶血活性。部分菌株无溶血活性，但有肠毒活性。为了明确两者之间的关系，我们克隆了编码新溶血素的基因。结果表明，野生型溶血素基因和新溶血素基因在氨基末端区域的DNA序列存在差异。此外，我们发现蛋白酶参与溶血素的成熟，并且蛋白酶在细胞中也发挥作用，使细菌在恶劣条件下生存。

项目成果

Ritsuko Yokoyama, Yoshio Fujii, Yoko Noguchi, Tomohiko, Nomura, Masahiko Akita, Kojun Setstu, Shigeo Yamamoto, Keinosuke Okamoto: "Physicochemical and Biological Properties of an Extracellular Serine Protease of Aeromonas sobria"Microbiology and Immunolog

Ritsuko Yokoyama、Yoshio Fujii、Yoko Noguchi、Tomohiko、Nomura、Masahiko Akita、Kojun Setstu、Shigeo Yamamoto、Keinosuke Okamoto：“气单胞菌胞外丝氨酸蛋白酶的物理化学和生物学特性”微生物学和免疫学

Hiroyasu Yamanaka, Tomohiko Nomura, Naoyuki Morisada, Sumio Shinoda, Keinosuke Okamoto: "Site-directed mutagenesis studies of the amino acid residue at position 412 of Escherichia coli TolC which is required for the activity"Microbial Pathogenesis. 33(2).

Hiroyasu Yamanaka、Tomohiko Nomura、Naoyuki Morisada、Sumio Shinoda、Keinosuke Okamoto：“大肠杆菌 TolC 412 位氨基酸残基的定点诱变研究，这是微生物发病机制活性所需的”。

Tomohiko Nomura, Yoshio Fujii, Hiroyasu Yamanaka, Hidetomo Kobayashi, Keinosuke Okamoto: "The protein encoded at the 3' end of the serine protease gene of Aeromonas sobria functions as a chaperone in the production of the protease"Journal of Bacteriology.

Tomohiko Nomura、Yoshio Fujii、Hiroyasu Yamanaka、Hidetomo Kobayashi、Keinosuke Okamoto：“在气单胞菌丝氨酸蛋白酶基因 3 端编码的蛋白质在蛋白酶的产生中充当伴侣”《细菌学杂志》。

Sakae Arimoto-Kobayashi: "Inhibitory effects of (-)-epigallocatechin gallate on the mutation, DNA strand cleavage and DNA-adduct formation by heterocyclic amines"J.Arg.Food Chem.. 51・17. 5150-5153 (2003)

Sakae Arimoto-Kobayashi：“(-)-表没食子儿茶素没食子酸酯对杂环胺的突变、DNA链断裂和DNA加合物形成的抑制作用”J.Arg.Food Chem.. 51・17 (2003)。

Hiroyasu Yamanaka et al.: "Site-directed mutagenesis studies of the amino acid residue at position 412 of Escherichia coli TolC which is required for the activity"Microbial Pathogenesis. 33. 81-89 (2002)

Hiroyasu Yamanaka 等人：“对大肠杆菌 TolC 412 位氨基酸残基进行定点诱变研究，这是微生物发病机制活性所必需的”。