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Abstract：
BACKGROUND: Enteropathogenic E. coli (EPEC) causes acute infantile diarrhea accounting for significant morbidity and mortality in developing countries. EPEC uses a type three secretion system to translocate more than twenty effectors into the host intestinal cells. At least four of these effectors, namely EspF, Map, EspG1/G2 and NleA, are reported to disrupt the intestinal tight junction barrier. We have reported earlier that the expression of EspF and Map in MDCK cells causes the depletion of the TJ membrane proteins and compromises the integrity of the intestinal barrier. In the present study, we have examined the role of the proline-rich repeats (PRRs) within the C-terminus of EspF in the depletion of the tight junction membrane proteins and identified key endocytosis markers that interact with EspF via these repeats.
RESULTS: We generated mutant EspF proteins which lacked one or more proline-rich repeats (PRRs) from the N-terminus of EspF and examined the effect of their expression on the cellular localization of tight junction membrane proteins. In lysates derived from cells expressing the mutant EspF proteins, we found that the C-terminal PRRs of EspF are sufficient to cause the depletion of TJ membrane proteins. Pull-down assays revealed that the PRRs mediate interactions with the TJ adaptor proteins ZO-1 and ZO-2 as well as with the proteins involved in endocytosis such as caveolin-1, Rab5A and Rab11.
CONCLUSIONS: Our study demonstrates the direct role of the proline-rich repeats of EspF in the depletion of the TJ membrane proteins and a possible involvement of the PRRs in the endocytosis of host proteins. New therapeutic strategies can target these PRR domains to prevent intestinal barrier dysfunction in EPEC infections.
RESULTS: We generated mutant EspF proteins which lacked one or more proline-rich repeats (PRRs) from the N-terminus of EspF and examined the effect of their expression on the cellular localization of tight junction membrane proteins. In lysates derived from cells expressing the mutant EspF proteins, we found that the C-terminal PRRs of EspF are sufficient to cause the depletion of TJ membrane proteins. Pull-down assays revealed that the PRRs mediate interactions with the TJ adaptor proteins ZO-1 and ZO-2 as well as with the proteins involved in endocytosis such as caveolin-1, Rab5A and Rab11.
CONCLUSIONS: Our study demonstrates the direct role of the proline-rich repeats of EspF in the depletion of the TJ membrane proteins and a possible involvement of the PRRs in the endocytosis of host proteins. New therapeutic strategies can target these PRR domains to prevent intestinal barrier dysfunction in EPEC infections.
摘要：
背景：肠致病性大肠杆菌（EPEC）引起急性婴儿腹泻，在发展中国家占显著的发病率和死亡率。EPEC使用3型分泌系统将超过20个效应子转移到宿主肠细胞中。这些效应物中至少有四个,即ESPF,地图,EspG1/G2和NleA，据报道会破坏肠道紧密连接屏障。我们先前报道了EspF和Map在MDCK细胞中的表达导致TJ膜蛋白的消耗并损害肠屏障的完整性。在本研究中,我们研究了EspFC末端富含脯氨酸的重复序列(PRRs)在紧密连接膜蛋白消耗中的作用,并确定了通过这些重复序列与EspF相互作用的关键胞吞标记.
结果：我们产生了突变的EspF蛋白，该蛋白从EspF的N端缺乏一个或多个富含脯氨酸的重复序列（PRR），并检查了它们的表达对紧密连接膜蛋白的细胞定位的影响。在来自表达突变EspF蛋白的细胞的裂解物中，我们发现EspF的C端PRRs足以引起TJ膜蛋白的消耗。下拉分析显示，PRR介导与TJ衔接蛋白ZO-1和ZO-2以及与内吞作用有关的蛋白质（例如caveolin-1，Rab5A和Rab11）的相互作用。
结论：我们的研究证明了富含脯氨酸的EspF重复序列在TJ膜蛋白消耗中的直接作用，以及PRRs可能参与宿主蛋白的内吞作用。新的治疗策略可以靶向这些PRR结构域以防止EPEC感染中的肠屏障功能障碍。
结果：我们产生了突变的EspF蛋白，该蛋白从EspF的N端缺乏一个或多个富含脯氨酸的重复序列（PRR），并检查了它们的表达对紧密连接膜蛋白的细胞定位的影响。在来自表达突变EspF蛋白的细胞的裂解物中，我们发现EspF的C端PRRs足以引起TJ膜蛋白的消耗。下拉分析显示，PRR介导与TJ衔接蛋白ZO-1和ZO-2以及与内吞作用有关的蛋白质（例如caveolin-1，Rab5A和Rab11）的相互作用。
结论：我们的研究证明了富含脯氨酸的EspF重复序列在TJ膜蛋白消耗中的直接作用，以及PRRs可能参与宿主蛋白的内吞作用。新的治疗策略可以靶向这些PRR结构域以防止EPEC感染中的肠屏障功能障碍。
