Department of Microbiology, School of Public Health, Southern Medical UniversityGuangzhou, China.
Key Laboratory of Tropical Disease Research of Guangdong Province, Southern Medical UniversityGuangzhou, China.
Front Cell Infect Microbiol. 2017 Sep 21;7:410. doi: 10.3389/fcimb.2017.00410. eCollection 2017.
Enterohemorrhagic (EHEC) O157:H7 EspF is an important multifunctional protein that destroys the tight junctions of intestinal epithelial cells and promotes host cell apoptosis. However, its molecular mechanism remains elusive. We knocked out the sequence (747 bp, Δ), N-terminal sequence (219 bp, Δ ), and C-terminal sequence (528 bp, Δ ) separately using the pKD46-mediated λ Red homologous recombination system. Then, we built the corresponding complementation strains, namely, Δ, Δ , and Δ by overlap PCR, which were used in infecting HT-29 cells and BALB/C mice. The level of reactive oxygen species, cell apoptosis, mitochondrial trans-membrane potential, inflammatory factors, transepithelial electrical resistance (TER), and animal mortality were evaluated by DCFH-DA, double staining of Annexin V-FITC/PI, JC-1 staining, ELISA kit, and a mouse assay. The wild-type (WT), Δ, Δ, Δ , Δ , Δ , and Δ groups exhibited apoptotic rates of 68.3, 27.9, 64.9, 65.7, 73.4, 41.3, and 35.3% respectively, and mean TNF-α expression levels of 428 pg/mL, 342, 466, 446, 381, 383, and 374 pg/mL, respectively. In addition, the apoptotic rates and TNF-α levels of the WT, Δ, and Δ were significantly higher than that of Δ, Δ , Δ , and Δ group ( < 0.05). The N-terminal of EspF resulted in an increase in the number of apoptotic cells, TNF-α secretion, ROS generation, mitochondria apoptosis, and pathogenicity in BalB/c mice. In conclusion, the N-terminal domain of the Enterohemorrhagic O157:H7 EspF more strongly promotes apoptosis and inflammation than the C-terminal domain.
肠出血性大肠杆菌（EHEC）O157：H7 EspF 是一种重要的多功能蛋白，可破坏肠上皮细胞的紧密连接并促进宿主细胞凋亡。然而，其分子机制仍不清楚。我们使用 pKD46 介导的 λ Red 同源重组系统分别敲除了序列（747 bp，Δ）、N 端序列（219 bp，Δ）和 C 端序列（528 bp，Δ）。然后，我们通过重叠 PCR 构建了相应的互补菌株，即Δ、Δ和Δ，用于感染 HT-29 细胞和 BALB/C 小鼠。通过 DCFH-DA、Annexin V-FITC/PI 双染、JC-1 染色、ELISA 试剂盒和小鼠试验评估活性氧（ROS）水平、细胞凋亡、线粒体跨膜电位、炎症因子、跨上皮电阻（TER）和动物死亡率。野生型（WT）、Δ、Δ、Δ、Δ、Δ和Δ组的凋亡率分别为 68.3%、27.9%、64.9%、65.7%、73.4%、41.3%和 35.3%，TNF-α 的平均表达水平分别为 428 pg/mL、342、466、446、381、383 和 374 pg/mL。此外，WT、Δ和Δ组的凋亡率和 TNF-α 水平明显高于Δ、Δ、Δ和Δ组（<0.05）。EspF 的 N 端导致凋亡细胞数量、TNF-α 分泌、ROS 生成、线粒体凋亡和 BalB/c 小鼠的致病性增加。总之，肠出血性大肠杆菌 O157：H7 EspF 的 N 端比 C 端更能促进细胞凋亡和炎症。
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