Stringent response and programed cell death mechanism of periodontal pathogenic bacteria

牙龈卟啉单胞菌是成人牙周炎的主要病原菌。推测牙龈假单胞菌可能在口腔生物膜中存活。然而，牙龈假单胞菌很快就会在培养基中自溶。采用双向凝胶电泳法对自溶P菌的分子组成进行了研究。许多蛋白质斑点在数量和体积上都不同。通过N-末端氨基酸序列测定，其中一个点含有RGP。采用等位交换的方法构建了牙龈假单胞菌N-乙酰-L-丙氨酸氨基转移酶基因突变体。自溶素突变体的自溶速率与亲本菌株相同。考虑到口腔生物膜内部可能存在营养不良的空间，采用低浓度的THM介质制备模拟口腔生物膜的条件。THM培养液的主要成分是由多肽组成的台盼酮。牙龈假单胞菌生长时代谢的不是氨基酸，而是多肽。在低浓度THM条件下，牙龈假单胞菌的自溶率降低，活细胞数增加。对低浓度THM培养的牙龈假单胞菌细胞的蛋白质组学分析表明，许多蛋白质的表达均下降。认为低浓度的THM是牙龈假单胞菌生长的严酷状态。牙龈假单胞菌的一个严格反应基因(relA/Spot)突变体比亲本菌株的自溶率降低。研究还发现，许多蛋白质的表达与relA/Spot突变体和亲本菌株不同，这些结果表明，高浓度的多肽在体外诱导自溶(细胞程序性死亡)，而低浓度的多肽在体外诱导严格的反应基因表达，从而使牙龈假单胞菌的自溶速度变慢。这一机制可能解释牙龈假单胞菌在生物膜中的存活。

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.