Interaction Between Endosymbiont Holospora and the Cell

内共生全孢菌与细胞之间的相互作用

• 批准号: 11694211
• 负责人: FUJISHIMA Masahiro
• 金额: $ 3.9万
• 依托单位: Yamaguchi University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B).
• 财政年份: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2000
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-11694211/
• 关键词: Endonuclear symbiont; Holospora; Stress responces; Paramecium; Endosymbiosis; Gene expressions; Targeting; Nuclear membrane; 核内共生細菌; ターゲッティング; 細胞内共生細菌; 原生動物

1. We found that nuclear envelope-recognition sensor substances of H.obtusa and H.undulata were lipo-polysaccharides (LPSs) on their outer membranes. Indirect immunofluorescence microscopy and immuno-electron microscopy with monoclonal antibodies for these LPSs showed that the antibodies reacted with their target nuclear envelopes.2. Holospora penetrates the target nuclear envelope with its specific tip. When this special tip was cut off from about 3,400 infectious forrns with a micro-dissection system and loaded on SDS-PAGE, 2 bands appeared with silver staining. These area of SDS-PAGE gels were cut and inside materials were electroeluted. Using these eluted substances as antigens, we succeeded to develop a monoclonal antibody specific for the special substance with indirect immunofluorescence microscopy.3. Eighteen spots were cut off from 2D-SDS-PAGE of H.obtusa and the micro-sequences of the purified proteins from their N-terminals were clarified and homology checks with known proteins were carried out. Also, their ORFs were searched from H.obtusa genomic DNA sequences so far clarified. As a the results, only 3 ORFs were found among 18 proteins, and 8 proteins showed high homologies with known proteins. The fact that only 3 ORF were detected shows that majority of the H.obtusa proteins may derived from the host cells.4. We found 12 host genes that were depressed and 3 host genes by the infection of H.obtusa by differential display (DD) method. Partial DNA sequences of 4 genes of the former ones were clarified. One of the 4 genes showed was an immobilization antigen A homologous gene of Paramecium tetraurelia.

1.结果表明，钝头海蜇和波纹海蜇的核膜识别感受物质均为外膜上的脂多糖。间接免疫荧光显微镜和单克隆抗体免疫电镜观察显示，这些LPS单克隆抗体与其靶核团发生反应.全孢子虫以其特有的尖端穿透靶核膜。用显微切割系统从约3,400个感染性组织中切下这种特殊的针尖，并将其上样于SDS-PAGE上，经银染后出现2条带。切割SDS-PAGE凝胶的这些区域，并将内部材料电洗脱。以这些洗脱物为抗原，通过间接免疫荧光显微镜技术成功制备了特异性的单克隆抗体.从2D-SDS-PAGE上切下18个斑点，从N-末端对纯化蛋白进行微序列分析，并与已知蛋白进行同源性比较。此外，从迄今为止澄清的钝头红球藻基因组DNA序列中搜索它们的ORF。结果表明，在18个蛋白质中，只有3个开放阅读框，其中8个蛋白质与已知蛋白质同源性较高。结果表明，该蛋白主要来源于宿主细胞.利用差异显示技术发现12个宿主基因被钝头血蜱抑制，3个宿主基因被钝头血蜱感染。对其中4个基因的部分DNA序列进行了测序。其中一个基因为草履虫的制动抗原A同源基因。

项目成果

Eriko Osakii and Masahiro Fujishima: "Purification of Germinal-Vesicle-Breakdown-inducing protein from the ciliate Tetrahymenapyrifomis"European Journal of Protistology. 36. 459-464 (2000)

Eriko Osakii 和 Masahiro Fujishima：“从纤毛虫四膜虫中纯化胚泡分解诱导蛋白”欧洲原生学杂志。

Maki Murata-Hori: "Effects of G1 arrest substances on cells in the G1, S and G2 phases of the cel cycle in Tetrahymena."European Journal of Protistology. 36. 313-318 (2000)

Maki Murata-Hori：“G1 阻滞物质对四膜虫细胞周期的 G1、S 和 G2 期细胞的影响。”《欧洲原生学杂志》。

Hideo Dohra: "Effects of antibiotics on early infection process of a macronuclear endosymbiotic bacterium Holospora obtusa of Parameclum caudatum."FEMS leteter. 179. 473-477 (1999)

Hideo Dohra：“抗生素对草履虫大核内共生细菌 Holospora obtusa 早期感染过程的影响。”FEMS 信件。

Miki Kawai: "Invasion of the macronucleus of Paramecium caudatum by the bacterium Holospora obtusa Fates of the bacteria and timings of invasion steps."European Journal of Protistology. 36. 46-52 (2000)

Miki Kawai：“Holospora obtusa 细菌对尾草履虫大核的入侵。细菌的命运和入侵步骤的时间。”欧洲原生学杂志。

Miki Kawai and Masahiro Fujishima: "Invasion of the macronucleus of Paramecium caudatum by the bacterium Holospora obtusa Fates of the bacteria and timings of invasion steps."European Journal of Protistology. 36. 46-52 (2000)

Miki Kawai 和 Masahiro Fujishima：“Holospora obtusa 细菌对尾草履虫大核的入侵。细菌的命运和入侵步骤的时间。”欧洲原生学杂志。