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Kinetics of CagA type IV secretion by Helicobacter pylori and the requirement for substrate unfolding
Molecular Microbiology ( IF 3.6 ) Pub Date : 2021-06-14 , DOI: 10.1111/mmi.14772
Clara Lettl 1, 2 , Rainer Haas 1, 2 , Wolfgang Fischer 1, 2
Affiliation  

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.

中文翻译:

幽门螺杆菌分泌CagA IV型的动力学和底物展开的要求

效应蛋白的IV型分泌是人类病原体与其靶细胞相互作用的重要原理。相应的分泌系统可以运输必须在各自的时空环境中部署的大量效应蛋白,或者仅运输单个易位蛋白,如人类胃病原体幽门螺杆菌产生的 CagA 效应蛋白的情况。为了更详细地分析幽门螺杆菌分泌 CagA IV 型的动力学和作用方式,我们在此描述了一种新型的、高度灵敏的基于分裂荧光素酶的易位报告基因,它可以很容易地适应不同的终点或真实-时间测量。使用这个记者,我们展示了幽门螺杆菌细胞能够将有限量的 CagA 供应快速注入培养的胃上皮细胞。我们进一步采用报告系统来解决 CagA 是否必须在通过 IV 型分泌系统易位之前展开的问题。我们表明,与 CagA 作为蛋白质融合物共易位的蛋白质结构域比在其他分泌系统中更容易作为底物被耐受,但也提供了证据表明效应蛋白的展开是其运输的先决条件。
更新日期:2021-06-14
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