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Stringent response and programed cell death mechanism of periodontal pathogenic bacteria

牙周病原菌的严格反应和程序性细胞死亡机制

基本信息

批准号：
16591832
负责人：
KAMAGUCHI Arihide
金额：
$ 2.3万
依托单位：
Health Sciences University of Hokkaido
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2004
资助国家：
日本
起止时间：
2004 至 2006
项目状态：
已结题

项目摘要

Porphyromonas gingivalis is a putative major etiology agent of adult periodontitis. It is suppose that P. gingivalis may survive in the oral biofilm. However, P. gingivalis quickly autolyzes in the culture media. To examine the molecular components of the P gingivalis autolysate, two-dimensional gel electrophoresis was performed. Many protein spots varied both in number and volume. One of these spots included Rgp as determined by N-terminal amino acid sequencing. Mutant of N-acetylmuramoyl-L-alanine amidase like protein gene of P. gingivalis was constructed by alleic exchange. Autolysis rate of the autolysin mutant was the same as that of parent strain. These results indicate that many autolysin may act the autolysis of P. gingivalis.From consideration that inside of oral biofilm may be innutritious space, mimic condition of oral biofilm was prepared by low concentration of THM medium. Major component of THM medium was Trypyone which is composed from peptide. P gingivalis could growth with metabolizing not amino acid but peptide. In low concentration of THM medium, autolysis rate of P gingivalis was decreased and living cells was increased. Proteome analysis of P. gingivalis cells in the low concentration of THM medium indicated that expression of many proteins decreased. It is considered that low concentration of THM medium is stringent state for P. gingivalis. One of the stringent response gene (relA/spot) mutant of P gingivalis decreased autolysis rate than parent strain. It was also observed that many protein expression were differ from relA/spot mutant and parent strain.These results suggest that high peptide concentration induce autolysis (programmed cell death) and low peptide concentration in vitro induce the stringent response gene expression, consequently autolysis rate of P. gingivalis turned slow. This mechanism may be possible to explain the survival of P. gingival in biofilm.

牙龈卟啉单胞菌是公认的成人牙周炎的主要病原体。推测牙龈卟啉单胞菌可能在口腔生物膜中存活。然而，牙龈卟啉单胞菌在培养基中迅速自溶。为了研究牙龈卟啉单胞菌自溶物的分子组成，进行了二维凝胶电泳。许多蛋白质点在数量和体积上都有变化。这些斑点之一包括通过N-末端氨基酸测序确定的Rgp。采用丙氨酸交换法构建牙龈卟啉单胞菌N-乙酰胞壁酰-L-丙氨酸酰胺酶样蛋白基因突变体。自溶素突变株的自溶速率与出发菌株相同。考虑到口腔生物被膜内可能是无营养的空间，采用低浓度THM培养基制备口腔生物被膜模拟条件。THM培养基的主要成分是由肽组成的胰蛋白酶。牙龈卟啉单胞菌的生长代谢不是氨基酸，而是肽。在低浓度THM培养基中，牙龈卟啉单胞菌自溶率降低，活细胞数增加。蛋白质组学分析表明，在低浓度THM培养基中牙龈卟啉单胞菌细胞的许多蛋白质的表达下降。认为低浓度THM培养基对牙龈卟啉单胞菌是严格状态。其中一株严格反应基因（relA/spot）突变株的自溶率较亲本菌株降低。结果表明，在体外高浓度的肽诱导自溶（程序性细胞死亡），低浓度的肽诱导严格反应基因的表达，从而使自溶速率变慢。这可能是牙龈卟啉单胞菌在生物膜中存活的原因之一。