Analysis of the impact of N. meningitidis on receptor tyrosine kinase activation in mammalian cells and on the cell cycle

脑膜炎奈瑟菌是人类特有的共生菌，但偶尔会穿透鼻咽粘膜进入血流，引起严重败血症和/或急性化脓性脑膜炎。像许多其他细菌病原体一样，脑膜炎奈瑟氏菌可以激活宿主细胞的信号转导途径来建立黏附和诱导摄取。我们的初步数据表明，宿主蛋白的酪氨酸磷酸化在脑膜炎球菌与哺乳动物细胞的相互作用中发挥着重要作用，从而导致细胞骨架重塑并随后吞噬病原体。我们可以证明，OPC介导的脑膜炎奈瑟菌与整合素的相互作用导致非受体酪氨酸激酶(RTKs)c-Src和粘着斑激酶的激活，这与脑膜炎奈瑟菌的摄取过程相偶联。除了非RTKs的激活，脑膜炎奈瑟氏菌还导致RTKs的磷酸化，包括ErbB家族的成员。有趣的是，一项对激活的RTK的调查显示，一些以前没有被描述过的激酶被激活，它们参与了脑膜炎奈瑟菌与宿主细胞的相互作用(例如，胰岛素受体、酪氨酸相关激酶B受体和ePhin受体(EphA3和EphB1)，我们的目标是在当前的提案中进行分析。特别是，将确定EPhin受体在(I)细菌黏附、侵袭和(Ii)调节内皮屏障完整性中的作用。在本应用程序的第二部分中，我们将重点介绍细胞周期的变化。最近的进一步数据表明，脑膜炎奈瑟氏菌也能够干扰宿主细胞周期，例如以不同的方式调节细胞周期蛋白的表达。我们将分析(I)主要周期蛋白丰度及其亚细胞定位的变化(Ii)脑膜炎奈瑟菌感染细胞中编码细胞周期调节分子的mRNA水平的转录组，(Iii)脑膜炎球菌感染引起的细胞周期停滞以及(Iv)细菌对某些细胞周期表型的黏附/侵袭/细胞内复制的影响。脑膜炎奈瑟菌与鼻咽粘膜上皮细胞的相互作用将作为研究该病原体对细胞周期改变的影响的模型，这种改变可能促进和促进感染或建立携带者状态。

Neisseria meningitidis is an obligate commensal in humans, but occasionally may penetrate the mucosal membrane of the nasopharynx to enter the bloodstream and cause severe septicemia and/or acute purulent meningitis. Like many other bacterial pathogens N. meningitidis can activate host cell signal transduction pathways to establish adhesion and induce uptake. Our preliminary data indicated an important function of tyrosine phosphorylation of host proteins during the interaction of meningococci with mammalian cells, which results in cytoskeletal remodelling with subsequent engulfment of the pathogen. We could show, that Opc-mediated interaction of N. meningitidis with integrins results in activation of the non-receptor tyrosine kinases (RTKs) c-Src and the focal adhesion kinase, which couple to the uptake process of N. meningitidis. Besides activation of non-RTKs N. meningitidis also leads to phosphorylation of RTKs, including members of the ErbB family. Interestingly, a survey of activated RTKs revealed that several kinases are activated that have not been described before to be involved in the context of the interaction of N. meningitidis with host cells (e.g. insulin receptor, tyrosine-related kinase B receptor and ephrin receptors (EphA3 and EphB1), which we aim to analyse within the current proposal. In particular, the role of Ephrin receptors in (i) bacterial adhesion, invasion and (ii) modulation of endothelial barrier integrity will be determined. In the second part of this application we will focus on alterations of the cell cycle. Further recent data showed that N. meningitidis is also capable to interfere with the host cell cycle, and for example differently regulate expression of cyclins. We will analyse (i) changes in the abundance of major cyclins and their subcellular localization (ii) the transcriptome of mRNA levels encoding cell cycle-regulatory molecules in N. meningitidis-infected cells, and (iii) cell cycle phase arrest in response to meningococcal infection as well as the (IV) influence on bacterial adhesion/invasion/intracellular replication to certain cell cycle phenotypes. Interaction of N. meningitidis with epithelial cells of the nasopharyngeal mucosa will serve as a model to study the influence of this pathogen on cell cycle alteration that might either facilitate and promote infection or establish a carrier state.