Expression and regulation of the Escherichia coli O157:H7 effector proteins NleH1 and NleH2.
Expression and regulation of the Escherichia coli O157:H7 effector proteins NleH1 and NleH2.
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DOI：
10.1371/journal.pone.0033408
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发表时间：
2012
期刊：
影响因子：
3.7
通讯作者：
Roe AJ
中科院分区：
文献类型：
--
作者：
Holmes A;Lindestam Arlehamn CS;Wang D;Mitchell TJ;Evans TJ;Roe AJ
E. coliO 157携带两个编码效应蛋白NleH 1和NleH 2的基因，这两个基因的同源性为87%。尽管蛋白质之间的相似性，启动子区上游的基因编码的效应器是更发散的，这表明基因的实际表达可能是差异调节。这是通过创建报告融合和检查它们在不同的遗传背景，媒体和与宿主细胞接触的表达进行测试。还在转染到宿主细胞中后测试蛋白质的功能。当在刺激三型分泌系统（T3 SS）表达的条件下培养时，NleH 1和NleH 2的表达增强，并且受到调节剂Ler和GrlA的影响。NleH 1的最大表达需要531 bp的上游非翻译区，而NleH 2只需要113 bp。有趣的是，与宿主细胞接触强烈抑制NleH 1和NleH 2的表达。转染后，当使用NF-κB荧光素酶报告基因测定进行评估时，两种蛋白质对NF-κB活化仅产生微小影响，该结果与最近的报告一致，该报告证明NF-κB的NleH 1调节依赖于RPS 3。这项研究证明了在研究细菌效应蛋白时考虑基因调控的重要性。尽管它们的序列相似性，NleH 1和NleH 2的表达差异，因此，可能会在感染过程中的不同时间易位。
E. coli O157 carries two genes encoding the effector proteins NleH1 and NleH2 which are 87% identical. Despite the similarity between the proteins, the promoter regions upstream of the genes encoding the effectors are more divergent suggesting that the actual expression of the genes may be differentially regulated. This was tested by creating reporter fusions and examining their expression in different genetic backgrounds, media and on contact with host cells. The function of the proteins was also tested following transfection into host cells. Expression of both NleH1 and NleH2 was enhanced when cultured under conditions that stimulated expression of the Type Three Secretion System (T3SS) and was influenced by the regulators Ler and GrlA. Maximal expression of NleH1 required 531 bp of the upstream untranslated region but NleH2 required only 113 bp. Interestingly, contact with host cells strongly repressed expression of both NleH1 and NleH2. Following transfection, both proteins produced only minor effects on NF-κB activation when assessed using a NF-κB luciferase reporter assay, a result that is consistent with the recent report demonstrating the dependence on RPS3 for NleH1 modulation of NF-κB. This study demonstrates the importance of considering gene regulation when studying bacterial effector proteins. Despite their sequence similarity, NleH1 and NleH2 are expressed differentially and may, therefore, be translocated at distinct times during an infection.
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DOI：
10.1073/pnas.0911609106
发表时间：
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影响因子：
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作者：
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