Kinetics of CagA type IV secretion by Helicobacter pylori and the requirement for substrate unfolding
Kinetics of CagA type IV secretion by Helicobacter pylori and the requirement for substrate unfolding
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DOI：
10.1111/mmi.14772
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发表时间：
2021-06-28
影响因子：
3.6
通讯作者：
Fischer, Wolfgang
中科院分区：
文献类型：
--
作者：
Lettl, Clara;Haas, Rainer;Fischer, Wolfgang
IV型效应蛋白的分泌是人类病原体与其靶细胞相互作用的重要原理。相应的分泌系统可能运输大量的效应蛋白，这些效应蛋白必须在相应的时空背景下部署，或者仅运输单一的易位蛋白，如人类胃病原体幽门螺杆菌产生的CagA效应蛋白。为了更详细地分析幽门螺杆菌分泌CagA IV型的动力学和作用模式，我们在这里描述了一种新的，高度敏感的基于荧光素酶的分裂易位报告，它可以很容易地适应不同的终点或实时测量。利用这个报告基因，我们发现幽门螺杆菌细胞能够快速地将它们有限的CagA供应注入培养的胃上皮细胞。我们进一步使用报告系统来解决CagA是否必须在IV型分泌系统易位之前展开的问题。我们发现，与CagA共易位的蛋白结构域作为蛋白融合体比在其他分泌系统中更容易作为底物耐受，但也提供证据表明，效应蛋白的展开是其运输的先决条件。
Type IV secretion of effector proteins is an important principle for interaction of human pathogens with their target cells. The corresponding secretion systems may transport a multitude of effector proteins that have to be deployed in the respective spatiotemporal context, or only a single translocated protein, as in the case of the CagA effector protein produced by the human gastric pathogen Helicobacter pylori. For a more detailed analysis of the kinetics and mode of action of CagA type IV secretion by H. pylori, we describe here, a novel, highly sensitive split luciferase-based translocation reporter which can be easily adapted to different end-point or real-time measurements. Using this reporter, we showed that H. pylori cells are able to rapidly inject a limited amount of their CagA supply into cultured gastric epithelial cells. We have further employed the reporter system to address the question whether CagA has to be unfolded prior to translocation by the type IV secretion system. We showed that protein domains co-translocated with CagA as protein fusions are more readily tolerated as substrates than in other secretion systems, but also provide evidence that unfolding of effector proteins is a prerequisite for their transport.