Role for gingipains in Porphyromonas gingivalis traffic to phagolysosomes and survival in human aortic endothelial cells
Role for gingipains in Porphyromonas gingivalis traffic to phagolysosomes and survival in human aortic endothelial cells
复制标题
DOI：
10.1128/iai.01013-06
复制
发表时间：
2007-05-01
影响因子：
3.1
通讯作者：
Yamamoto, Kenji
中科院分区：
文献类型：
--
作者：
Yamatake, Kumiko;Maeda, Maki;Yamamoto, Kenji
牙龈蛋白酶是负责牙龈卟啉单胞菌毒力的半胱氨酸蛋白酶。最近的研究表明，牙龈卟啉单胞菌被困在受感染细胞的自噬区室中，在那里它促进存活。在这项研究中，我们研究了牙龈卟啉菌蛋白酶在人主动脉内皮细胞中的细胞内运输和这种细菌的存活中的作用，以及这些酶在自噬途径中的任何可能的参与。尽管感染野生型（WT）牙龈卟啉单胞菌菌株可增强自噬作用，（ATCC 33277、381和W83）或缺乏牙龈卟啉菌蛋白酶的ATCC 33277突变体（KDP 136），我们发现超过90%的细胞内WT和KDP 136与组织蛋白酶B（一种溶酶体标志物）共定位，并且只有少数内化细胞与LC 3共定位，一种自噬体标记物，在感染后0.5至4小时内。免疫金电子显微镜分析进一步证实了这一点，从而表明牙龈卟啉单胞菌避开了自噬途径，而是直接进入溶酶体的内吞途径。在感染后的后期阶段，吞噬溶酶体中的WT菌株保留了它们的双膜结构。然而，这些隔室中的KDP 136失去了其双膜结构，这代表了其易破裂的特征。与超微结构观察一起，我们发现细胞内活WT细胞的数量比KDP 136细胞减少得更慢，从而表明牙龈卟啉菌蛋白酶有助于细菌存活，但不有助于感染细胞内的运输。
Gingipains are cysteine proteinases that are responsible for the virulence of Porphyromonas gingivalis. Recent studies have shown that P. gingivalis is trapped within autophagic compartments of infected cells, where it promotes survival. In this study we investigated the role of gingipains in the intracellular trafficking and survival of this bacterium in human aortic endothelial cells and any possible involvement of these enzymes in the autophagic pathway. Although autophagic events were enhanced by infection with either wild-type (WT) P. gingivalis strains (ATCC 33277, 381, and W83) or an ATCC 33277 mutant lacking gingipains (KDP136), we have found that more than 90% of intracellular WT and KDP136 colocalized with cathepsin B, a lysosome marker, and only a few of the internalized cells colocalized with LC3, an autophagosome marker, during the 0.5- to 4-h postinfection period. This was further substantiated by immunogold electron microscopic analyses, thus implying that P. gingivalis evades the autophagic pathway and instead directly traffics to the endocytic pathway to lysosomes. At the late stages after infection, WT strains in phagolysosomes retained their double-membrane structures. KDP136 in these compartments, however, lost its double-membrane structures, representing a characteristic feature of its vulnerability to rupture. Together with the ultrastructural observations, we found that the number of intracellular viable WT cells decreased more slowly than that of KDP136 cells, thus suggesting that gingipains contribute to bacterial survival, but not to trafficking, within the infected cells.