Analysis of the secretion system and the gene expression of Burkholderia cepacia virulence factors.

洋葱伯克霍尔德菌毒力因子分泌系统及基因表达分析

• 批准号: 10670259
• 负责人: ABE Mitsuko
• 金额: $ 1.79万
• 依托单位: Yamaguchi University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1998
• 资助国家: 日本
• 起止时间: 1998 至 1999
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10670259/
• 关键词: Burkholderia cepacia; protease; disulfide oxidoreductase; DsbA; hemolytic substance; TypeII secretion system; LysR family; 溶血物質; TypeII分泌機構; セパシア菌; 発現調節タンパク

To investigate the mechanisms of the production of protease and hemolytic substance, cepalycin, produced by Burkholderia cepacia, we analyzed Tn5-generated protease-negative mutant and cepalycine-negative mutant derived from strain KF1 and strain JN106.1. A protease-negative and lipase-positive mutant of KF1 was complemented by a clone having an open reading frame coding a 212-amino acid polypeptide which showed homology to Escherichia coli periplasmic disulfide bond oxidoreductase, DsbA.2. The DsbA mutant showed the following phenotypes. (1)defective in the production of extracellular protease and alkaline phosphatase, (2)repression of growth, (3)defective in motility, (4)increase in sensitivity to CdィイD12+ィエD1 and ZnィイD12+ィエD1, (5)increase in sensitivity to a variety of antibiotics including β-lactams and sodium dodecyl sulfite.3. Several protease- and lipase-negative mutants of KF1 were analyzed. The region complementing protease production of these mutants was located in a 9.9kb fragment. Analysis of the nucleotide sequence revealed gspD, -E, -F, -C, -G, -H, -I, -J, -K and gspL which encode for components involved in a type II secretion system, a Sec-dependent general secretory pathway of gram-negative bacteria. The gsp genes were presumed to be organized in three operons consisting of gspD-E-F, gspC(in opposite direction) and gspG-H-I-J-K-L.These results suggested that B. cepacia protease is secreted by a type II secretion system and requires the disulfide oxidoreduction system for production/secretion.4. A cepalycin- and protease-deficient mutant of JN106 had a mutation in an open reading frame for a 327-amino acid polypeptide belonging to LysR family proteins. A typical helix-turn-helix motif was found in the N-terminal region. Protease activity and cepalycin activity were restored when the lysR family gene termed cviR(B. cepacia virulence regulator) on a plasmid, suggesting that the cviR gene is involved in the production of both protease and cepalycin.

为了研究洋葱伯克霍尔德菌产生蛋白酶和溶血物质--头孢菌素的机制，我们分析了来自菌株KF 1和菌株JN106.1的Tn 5产生的蛋白酶阴性突变体和头孢菌素阴性突变体。蛋白酶阴性和脂肪酶阳性的突变体KF 1的克隆具有开放的阅读框编码212个氨基酸的多肽，显示同源性大肠杆菌周质二硫键氧化还原酶，DsbA.2的补充。DsbA突变体表现出以下表型。（1）细胞外蛋白酶和碱性磷酸酶的产生缺陷;（2）生长抑制;（3）运动缺陷;（4）对Cd（Ⅱ）D12+ Cd（Ⅱ）D1和Zn（Ⅱ）D12+ Zn（Ⅱ）D1的敏感性增加;（5）对多种抗生素包括β-内酰胺类和十二烷基亚硫酸钠的敏感性增加。几个蛋白酶和脂肪酶阴性突变体的KF 1进行了分析。这些突变体的蛋白酶生产互补区位于9.9kb的片段中。对核苷酸序列的分析揭示了gspD、-E、-F、-C、-G、-H、-I、-J、-K和gspL，它们编码参与II型分泌系统的组分，II型分泌系统是革兰氏阴性菌的Sec依赖性一般分泌途径。推测gsp基因由gspD-E-F、gspC（反向）和gspG-H-I-J-K-L三个操纵子组成。洋葱蛋白酶由II型分泌系统分泌，并且需要二硫化物氧化还原系统用于生产/分泌。JN 106的头孢菌素和蛋白酶缺陷型突变体在属于LysR家族蛋白的327个氨基酸多肽的开放阅读框中具有突变。在N-末端区域发现了典型的螺旋-转角-螺旋基序。当lysR家族基因命名为cviR（B. cviR基因与蛋白酶和cepalycin的产生有关。

项目成果

M. Abe: "Burkholderia cepacia"Infection Control. 7(11)(in Jaoanese). 1448-1453 (1998)

M. Abe：“洋葱伯克霍尔德菌”感染控制。

阿部光子: "Burkholderia cepaciaの低分子溶血物質cepalycinとプロテアーゼの産生機構"日本細菌学雑誌. 54(1). 148 (1999)

Mitsuko Abe：“洋葱伯克霍尔德菌中低分子量溶血物质头孢霉素和蛋白酶的产生机制”日本细菌学杂志 148（1999）。

M. Abe: "The Dsb(disulfide bond formation) system of Burkholderia cepacia and the maturation of protease and β-lactamase"Proceedings of The 33th Meeting of The Japan Pseudomonas aeruginosa Society. in Japanese. 103-107 (1998)

M. Abe：“洋葱伯克霍尔德氏菌的 Dsb（二硫键形成）系统以及蛋白酶和 β-内酰胺酶的成熟”日本铜绿假单胞菌学会第 33 届会议记录（1998 年）。

阿部光子: "Burkholderia cepacia(セパシア菌)" Infection Control. 7・11. 66-71 (1998)

阿部光子：“洋葱伯克霍尔德菌”感染控制7·11（1998）。

阿部光子: "Burkholderia cepaciaのDsb系とプロテアーゼおよびβ-ラクタマーゼの成熟化"第32回緑膿菌感染症研究会講演記録. 103-107 (1998)

阿部光子：《洋葱伯克霍尔德氏菌的Dsb系统与蛋白酶和β-内酰胺酶的成熟》第32届铜绿假单胞菌感染研究组讲座记录103-107（1998）。