Visualizing the Translocation and Localization of Bacterial Type III Effector Proteins by Using a Genetically Encoded Reporter System.
Visualizing the Translocation and Localization of Bacterial Type III Effector Proteins by Using a Genetically Encoded Reporter System.
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DOI：
10.1128/aem.03418-15
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发表时间：
2016-05
影响因子：
4.4
通讯作者：
Roe AJ
中科院分区：
文献类型：
--
作者：
Gawthorne JA;Audry L;McQuitty C;Dean P;Christie JM;Enninga J;Roe AJ
细菌III型分泌系统（T3SS）效应蛋白是许多动植物病原体感染的关键决定因素。然而，监测这些重要的毒力决定因素的易位和传递已被证明是技术上的挑战。在这里，我们使用基因工程LOV（光氧电压）传感域衍生物来监测细菌T3SS效应子的表达，易位和定位。我们发现大肠杆菌O157：H7细菌效应子融合体Tir-LOV在其易位后是功能性的，并且定位于离散病灶中的宿主细胞膜，这表明基于LOV的报告子可以用于可视化效应子易位，具有最小的操作和干扰。通过将LOV融合到福氏志贺菌效应子IpaB的C末端，证明了报告基因的多功能性的进一步证据。IpaB-LOV在易位前优先定位于细菌极。我们观察到IpaB-LOV以T3SS依赖的方式快速易位到宿主细胞中，在那里它定位在膜皱褶内的细菌进入位点。
Bacterial type III secretion system (T3SS) effector proteins are critical determinants of infection for many animal and plant pathogens. However, monitoring of the translocation and delivery of these important virulence determinants has proved to be technically challenging. Here, we used a genetically engineered LOV (light-oxygen-voltage) sensing domain derivative to monitor the expression, translocation, and localization of bacterial T3SS effectors. We found the Escherichia coli O157:H7 bacterial effector fusion Tir-LOV was functional following its translocation and localized to the host cell membrane in discrete foci, demonstrating that LOV-based reporters can be used to visualize the effector translocation with minimal manipulation and interference. Further evidence for the versatility of the reporter was demonstrated by fusing LOV to the C terminus of the Shigella flexneri effector IpaB. IpaB-LOV localized preferentially at bacterial poles before translocation. We observed the rapid translocation of IpaB-LOV in a T3SS-dependent manner into host cells, where it localized at the bacterial entry site within membrane ruffles.