Development of antisense therapy for periodontal diseases targeted bacterial cell-division related genes

针对细菌细胞分裂相关基因的牙周病反义疗法的开发

• 批准号: 10557199
• 负责人: TAKEHARA Tadamichi
• 金额: $ 2.5万
• 依托单位: Kyushu Dental College
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B).
• 财政年份: 1998
• 资助国家: 日本
• 起止时间: 1998 至 2000
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-10557199/
• 关键词: Peptidoglycan; Peridontal diseases; P.gingivalis; Cell division

Peptidoglycan is known to be a heteropolymer with a unique chemical structure and biological activity, and be essential for cell viability of bacteria. We have already isolated and sequenced a gene encoding the MurC protein from Porphyromonas gingivalis (PgMurC gene), an oral anaerobic rod-shaped bacterium implicated in progressiove periodontal disease. The MurC protein functions in peptidoglycan synthesis and catalyzes the first step in the biosynthesis of cell wall peptidoglycan. The region including PgMurC gene appeared to be highly similar with mra region in E.coli, which contains genes concerned with peptidoglycan synthesis, and have been shown to be tightly clustered forming an operon. Then, we have sequenced the neibouring region of PgMurC gene, and we found that the three ORFs had a significant similarity with FtsQ (16%), FtsA (33%), and FtsZ (54%) in E.coli, respectively. The predicted FtsA from P.gingivalis (PgFtsA) had five motifs for ATPase domain, belonging to the actin fa … More mily, as in the FtsA from E.coli. When PgFtsA was overexpressed in E.coli, cell division was inhibited and its morphology changed to long filamentous cells. Electron micrographs of these cells revealed that the formation of aggregated structures existed in the E.coli cytoplasm. On the other hand, the FtsZ from P.gingivalis (PgFtsZ) possessed the clear motifs for GTP binding and hydrolysis, and the purified PgFtsZ protein exhibited GTPase activity with the following propeties different from other known FtsZ proteins ; 1) Na^+ and K^+ ions inhibited its GTPase activity. 2) PgFtsZ exhibited its GTPase activity even without Mg^<2+>, and completely retained its activity with EDTA.Very recently, a series of mutants deleted from the C-teminus of PgFtsZ were generated, and the change of their morphology were observed. We found that the delta C-177 mutant, deleted 177 amino acid residues from C-terminus, changed to the normal cells. These results suggest that amino acid residues from T281 to E330 may be important for the functional role in PgFtsZ.In order to identify amino acid residues in the corresponding region, mutant PgFtsZ proteins are currently generated through the use of site-directed mutagenesis. Less

肽聚糖是一种具有独特化学结构和生物活性的杂聚物，是细菌细胞生存所必需的。我们已经从牙龈卟啉单胞菌（Porphyromonas gingivalis，PgMurC基因）中分离并测序了MurC蛋白的编码基因，牙龈卟啉单胞菌是一种与进行性牙周病有关的口腔厌氧杆状细菌。MurC蛋白在肽聚糖合成中起作用，并催化细胞壁肽聚糖生物合成的第一步。包括PgMurC基因的区域似乎与大肠杆菌中的mra区域高度相似，其包含与肽聚糖合成有关的基因，并且已经显示紧密聚集形成操纵子。对PgMurC基因的邻近区域进行测序，发现这3个ORF与大肠杆菌中的FtsQ（16%）、FtsA（33%）和FtsZ（54%）的同源性较高。预测的牙龈卟啉单胞菌FtsA（PgFtsA）具有5个ATP酶结构域基序，属于肌动蛋白结构域， ...更多信息 mily，如大肠杆菌中的FtsA。当PgFtsA在大肠杆菌中过表达时，细胞分裂受到抑制，其形态变为长丝状细胞。电镜观察发现，大肠杆菌细胞质内存在聚集结构。另一方面，牙龈卟啉单胞菌FtsZ（PgFtsZ）具有清晰的GTP结合和水解基序，纯化后的PgFtsZ蛋白具有与其他已知FtsZ蛋白不同的性质：1）Na^+和K^+抑制其GTP酶活性。2)PgFtsZ在无Mg^<2+>存在下仍具有GT3活性，在EDTA存在下仍保持活性。我们发现Δ C-177突变体从C-末端缺失177个氨基酸残基，改变为正常细胞。这些结果表明，从T281到E330的氨基酸残基可能是重要的功能作用，在PgFtsZ。为了确定相应区域的氨基酸残基，突变PgFtsZ蛋白目前通过使用定点诱变产生。少

项目成果

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