Lee Byung Cheol, Choi Sang Ho, Kim Tae Sung
School of Life Sciences and Biotechnology, Korea University, Anam-dong, Seongbuk-Gu, Seoul 136-701, Republic of Korea.
Microbes Infect. 2008 Nov-Dec;10(14-15):1504-13. doi: 10.1016/j.micinf.2008.09.006. Epub 2008 Sep 25.
During Vibrio vulnificus infection, V. vulnificus reaches the intestine and then invades the bloodstream by crossing the intestinal mucosal barrier of the host, which results in systemic septicemia. Previously, we reported that the RtxA toxin secreted through the RtxE transporter contributes to the cytotoxicity of V. vulnificus against intestinal epithelial cells. Here, we used gene mutants of rtxE and rtxA to determine the role that V. vulnificus RtxA toxin plays in the apoptotic death of human intestinal epithelial cells. The levels of DNA fragmentation were lower in human epithelial cells infected with an rtxE mutant of V. vulnificus than in those that were infected with the wild type. In addition, the rtxE mutant was found to induce lower levels of TUNEL positive cells and cell cycle arrest at the subG(1) than the wild type V. vulnificus. Furthermore, the decreased levels of DNA fragmentation, TUNEL positive cells and subG(1) arrest by the rtxE gene mutation were restored by the complementation of an rtxE gene into the rtxE mutant V. vulnificus. Finally, the rtxA mutant induced significantly lower levels of apoptotic cell death than the wild type. The levels of the PARP, cytochrome c, caspase-3, and mitochondrial membrane depolarization were lower in human epithelial cells infected with the rtxE and rtxA mutants, compared with the wild type and rtxE gene-complemented strains of V. vulnificus. Taken together, these results indicate that V. vulnificus RtxA toxin induces the apoptotic death through a mitochondria-dependent pathway in human intestinal epithelial cells exposed to V. vulnificus.
在创伤弧菌感染过程中，创伤弧菌到达肠道，然后通过穿越宿主的肠道黏膜屏障侵入血液，从而导致全身性败血症。此前，我们报道过通过RtxE转运蛋白分泌的RtxA毒素有助于创伤弧菌对肠道上皮细胞的细胞毒性。在此，我们使用rtxE和rtxA的基因突变体来确定创伤弧菌RtxA毒素在人肠道上皮细胞凋亡死亡中所起的作用。感染创伤弧菌rtxE突变体的人上皮细胞中的DNA片段化水平低于感染野生型的细胞。此外还发现，rtxE突变体诱导的TUNEL阳性细胞水平和细胞周期在亚G1期的停滞低于野生型创伤弧菌。此外，通过将rtxE基因互补到rtxE突变体创伤弧菌中，可恢复因rtxE基因突变导致的DNA片段化、TUNEL阳性细胞和亚G1期停滞水平的降低。最后，rtxA突变体诱导的凋亡细胞死亡水平明显低于野生型。与野生型和rtxE基因互补菌株相比，感染rtxE和rtxA突变体的人上皮细胞中PARP、细胞色素c、半胱天冬酶-3的水平以及线粒体膜去极化程度较低。综上所述，这些结果表明，创伤弧菌RtxA毒素在暴露于创伤弧菌的人肠道上皮细胞中通过线粒体依赖性途径诱导凋亡死亡。
