Development of rapid diagnostic system for periodontal disease using cell division related genes form P.gingivalis

利用牙龈卟啉单胞菌细胞分裂相关基因开发牙周病快速诊断系统

• 批准号: 13672164
• 负责人: ANSAI Toshihiro
• 金额: $ 2.3万
• 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 (C)
• 财政年份: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2003
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13672164/
• 关键词: FtsZ; Periodontal disease; P.gingivalis; cell division; Multiplex PCR; P. gingivalis; ペプチドグリカン; cell division

We have already isolated and sequenced a gene encoding the MurC protein from Porphyromonas guigivalis (PgMurC gene), an oral anaerobic rod-shaped bacterium implicated in progressive 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 and we found that the three ORFs had a significant similarity with FtsQ (16%), FtsA (33%), and FtsZ (54%) in F.coli respectively. The FtsZ from P.giugivalis (PgFtsZ) possessed the clear motifs for GTP binding and hydrolysis, and the purified PgFtsZ protein exhibited GTPase activity with the following properties 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. A series of mutants deleted from the C-teminus of PgFtsZ were … More 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. Sequence comparison of the known prokaryotic FtsZs revealed that this region contained a highly conserved domain including 10 amino acids, designated A-domain, in which Ala320 and Gly322 of PgFtsZ was conserved throughout a broad variety of species. Therefore, we analyzed the role of Ala320 and GIy322 by site-directed mutagenesis. Consequently, we found that overexpression of ZA320H and ZA320R resulted in the normal phenotype, unlike the wild type. Similarly, overexpresson of ZG322P and ZG322H resulted in the normal phenotype. These results suggested that Ala320 and Gly322 are highly conserved and are crucial for cell division. The A-domain was also conserved among other periodontopathogens including A.actinomycetemcomitans, T.denticola, P.intermedia. We are currently planning to develop a novel system for differentiating these periodontopathic bacteria using the Multiplex PCR. Less

我们已经分离并测序了吉氏卟啉单胞菌MurC蛋白的编码基因(PgMurC基因)，PgMurC基因是一种与进行性牙周病有关的口腔厌氧杆状细菌。MurC蛋白在肽聚糖的合成中起作用，并催化细胞壁肽聚糖的生物合成的第一步。PgMurC基因与大肠杆菌中的MRA区高度相似，我们发现这三个ORF分别与FtsQ(16%)、FTSA(33%)和FtsZ(54%)有显著的相似性。从P.giugivalis获得的FtsZ蛋白(PgFtsZ)具有明确的GTP结合和水解模体，纯化的PgFtsZ蛋白具有不同于其他已知FtsZ蛋白的以下特性：1)Na+和K+离子对其GTP酶活性有抑制作用。2)PgFtsZ在无镁离子存在的情况下仍表现出其GTP酶活性，并在EDTA作用下完全保持其活性。从pgFtsZ的C-teminus中缺失的一系列突变体为…更多的产物，观察其形态的变化。我们发现Delta C-177突变体在C末端缺失了177个氨基酸残基，变成了正常细胞。这些结果表明，T281到E330的氨基酸残基可能对PgFtsZ的功能起重要作用。已知的原核生物FtsZ的序列比较表明，该区域含有一个高度保守的结构域，包含10个氨基酸，命名为A区，其中PgFtsZ的Ala320和Gly322在广泛的物种中都是保守的。因此，我们用定点突变的方法分析了Ala320和GIy322的作用。因此，我们发现ZA320H和ZA320R的过表达导致了正常的表型，与野生型不同。同样，ZG322P和ZG322H过表达导致正常表型。这些结果表明，Ala320和Gly322是高度保守的，对细胞分裂至关重要。A区在其他牙周病病原体中也是保守的，包括伴生放线菌、齿状毛滴虫、中间毛滴虫。我们目前正计划开发一种新的系统，使用多重聚合酶链式反应来区分这些牙周病细菌。较少

项目成果

Yh, W., Ansai, T.et al.: "A conserved Ala320 in the FtsZ of Porphyromonas gingivalis is important for Cell division."Curr.Microbiol.. 45. 355-361 (2002)

Yh, W., Ansai, T.et al.：“牙龈卟啉单胞菌 FtsZ 中保守的 Ala320 对于细胞分裂很重要。”Curr.Microbiol.. 45. 355-361 (2002)

Akifusa, S., Ansai, T.et al.: "Characterization of the Porphyromonas gingivalis FtsZ containing a novel GTPase activity"Curr. Microbiol.. 44. 267-272 (2002)

Akifusa, S., Ansai, T.等人：“含有新型 GTP 酶活性的牙龈卟啉单胞菌 FtsZ 的表征”Curr。

Kusaba, A., Ansai, T., et al.: "Cloning and sequencing of a hemK-family gene in Porphyromonas gingivalis"DNA seq.. (in press).

Kusaba, A.、Ansai, T. 等人：“牙龈卟啉单胞菌中 hemK 家族基因的克隆和测序”DNA seq..（正在出版）。

Ansai, T. et al.: "Construction of a pepO gene-deficient mutant of Porphyromonas gingivalis."Oral Microbiol. Immunol.. 18. 398-400 (2003)

Ansai, T. 等人：“牙龈卟啉单胞菌 pepO 基因缺陷突变体的构建。”口腔微生物。

Ansai, T. et al.: "Phosphatases coutaining phosphotyrosyl phosphatase activity from Prevotella intermedia : structure, function, and evolution"Recent Res. Devel. Microbiol.. 4. 569-584 (2000)

Ansai, T. 等人：“中间普雷沃氏菌中具有磷酸酪氨酰磷酸酶活性的磷酸酶：结构、功能和进化”最近的研究。