Zeaiter Zaher, Cohen David, Müsch Anne, Bagnoli Fabio, Covacci Antonello, Stein Markus
Department of Medical Microbiology and Immunology, University of Alberta, Edmonton, Alberta, Canada, T6G 2H7.
Cell Microbiol. 2008 Mar;10(3):781-94. doi: 10.1111/j.1462-5822.2007.01084.x. Epub 2007 Nov 13.
Detergent-resistant membranes of eukaryotic cells are enriched in many important cellular signalling molecules and frequently targeted by bacterial pathogens. To learn more about pathogenic mechanisms of Helicobacter pylori and to elucidate novel effects on host epithelial cells, we investigated how bacterial co-cultivation changes the protein composition of detergent-resistant membranes of gastric adenocarcinoma (AGS) tissue culture cells. Using iTRAQ (isobaric tags for relative and absolute quantification) analysis we identified several cellular proteins, which are potentially related to H. pylori virulence. One of the proteins, which showed a significant infection-dependent increase in detergent resistance, was the polarity-associated serine/threonine kinase MARK2 (EMK1/Par-1b). We demonstrate that H. pylori causes the recruitment of MARK2 from the cytosol to the plasma membrane, where it colocalizes with the bacteria and interacts with CagA. Using Mardin Darby Canine Kidney (MDCK) monolayers and a three-dimensional MDCK tissue culture model we showed that association of CagA with MARK2 not only causes disruption of apical junctions, but also inhibition of tubulogenesis and cell differentiation.
真核细胞的抗去污剂膜富含许多重要的细胞信号分子，且常成为细菌病原体的攻击目标。为了更深入了解幽门螺杆菌的致病机制并阐明其对宿主上皮细胞的新作用，我们研究了细菌共培养如何改变胃腺癌（AGS）组织培养细胞抗去污剂膜的蛋白质组成。通过iTRAQ（相对和绝对定量的等压标签）分析，我们鉴定了几种可能与幽门螺杆菌毒力相关的细胞蛋白。其中一种蛋白，即极性相关丝氨酸/苏氨酸激酶MARK2（EMK1/Par-1b），在抗去污剂能力上表现出显著的感染依赖性增加。我们证明，幽门螺杆菌会导致MARK2从细胞质募集到质膜，在质膜上它与细菌共定位并与CagA相互作用。利用玛丁达比犬肾（MDCK）单层细胞和三维MDCK组织培养模型，我们发现CagA与MARK2的结合不仅会导致顶端连接的破坏，还会抑制肾小管形成和细胞分化。
