Department of Biological Sciences, Purdue University, West Lafayette, Indiana, United States of America.
PLoS One. 2011 Apr 26;6(4):e19331. doi: 10.1371/journal.pone.0019331.
Enterohemorrhagic Escherichia coli (EHEC) O157:H7 causes hemorrhagic colitis and may result in potentially fatal hemolytic uremia syndrome in humans. EHEC colonize the intestinal mucosa and promote the formation of actin-rich pedestals via translocated type III effectors. Two EHEC type III secreted effectors, Tir and EspFu/TccP, are key players for pedestal formation. We discovered that an EHEC effector protein called Non-LEE-encoded Ligase (NleL) is an E3 ubiquitin ligase. In vitro, we showed that the NleL C753 residue is critical for its E3 ligase activity. Functionally, we demonstrated that NleL E3 ubiquitin ligase activity is involved in modulating Tir-mediated pedestal formation. Surprisingly, EHEC mutant strain deficient in the E3 ligase activity induced more pedestals than the wild-type strain. The canonical EPEC strain E2348/69 normally lacks the nleL gene, and the ectopic expression of the wild-type EHEC nleL, but not the catalytically-deficient nleL(C753A) mutant, in this strain resulted in fewer actin-rich pedestals. Furthermore, we showed that the C. rodentium NleL homolog is a E3 ubiquitin ligase and is required for efficient infection of murine colonic epithelial cells in vivo. In summary, our study demonstrated that EHEC utilizes NleL E3 ubiquitin ligase activity to modulate Tir-mediated pedestal formation.
肠出血性大肠杆菌（EHEC）O157：H7 可引起出血性结肠炎，并可能导致人类潜在致命性溶血尿毒综合征。EHEC 定植于肠道黏膜，并通过转位型 III 效应子促进富含肌动蛋白的足状突起的形成。两种 EHEC 型 III 分泌效应子 Tir 和 EspFu/TccP 是形成足状突起的关键因素。我们发现一种称为非 LEE 编码连接酶（NleL）的 EHEC 效应蛋白是一种 E3 泛素连接酶。在体外，我们表明 NleL C753 残基对其 E3 连接酶活性至关重要。在功能上，我们证明了 NleL E3 泛素连接酶活性参与调节 Tir 介导的足状突起的形成。令人惊讶的是，缺乏 E3 连接酶活性的 EHEC 突变株诱导的足状突起比野生型菌株更多。经典的 EPEC 菌株 E2348/69 通常缺乏 nleL 基因，而在该菌株中异位表达野生型 EHEC nleL，但不是催化缺陷型 nleL(C753A)突变体，导致富含肌动蛋白的足状突起减少。此外，我们表明 C. rodentium NleL 同源物是一种 E3 泛素连接酶，是体内有效感染鼠结肠上皮细胞所必需的。总之，我们的研究表明，EHEC 利用 NleL E3 泛素连接酶活性来调节 Tir 介导的足状突起的形成。
