Molecular analysis of apoptosis inhibition by Chlamydia trachomatis

沙眼衣原体（Ctr）是一种专性细胞内细菌，在人上皮细胞的细胞质液泡中发育。在医学上，Ctr是重要的，特别是作为眼睛和女性生殖道的病原体。从科学上讲，宿主细胞和细菌的相互作用是有趣的，而且在很大程度上还不为人所知。Ctr操纵宿主细胞的一个重要方式是抑制细胞凋亡：受感染的细胞对诱导未感染细胞线粒体凋亡的刺激具有强烈的抵抗力。这种抑制作用令人印象深刻，但却不为人所理解；过去已经提出了三种分子模型，但彼此不相容。当前项目的工作重点是对这三个假设进行严格的实验测试，并应用对细胞凋亡装置的最新研究进展。令人惊讶的是，所有三个假设都被证明是错误的，包括我们之前提出的假设。我们继续绘制抗凋亡活性图。通过完整细胞和体外重建线粒体凋亡系统的实验，我们发现抗凋亡活性涉及直接抑制线粒体凋亡的两种效应物Bax和Bak。这种抑制作用可能是化学修饰或结合伙伴的附着的结果。在这个项目的延续中，我们将追求两个目标：首先确定与这种抑制相关的Bax和Bak上的分子改变，其次确定这两种蛋白质的修饰或结合伙伴。在项目的第一部分，我们将使用缺乏Bax的HeLa细胞（因此最初专注于Bak），通过有限的蛋白水解、天然凝胶电泳和交联研究来绘制Bak改变图。在Bak激活过程中发生的分子内变化已被报道，因此这些研究将确定被c -感染阻断Bak激活的步骤。在项目的第二部分，我们将尝试明确Bak的修改和/或结合伙伴。通过对免疫沉淀的Bak进行蛋白质组学分析，或通过检测因与Bak接近而生物素化的蛋白质，我们将努力识别潜在的结合伙伴。通过包括蛋白质组学分析在内的方法，我们将寻找Bak的化学修饰。用Bak获得的结果将在Bax上进行测试。已确定的细菌或宿主细胞起源的潜在机制将被特别针对，以测试它们与抑制细胞凋亡和Ctr发展的相关性。我们相信，通过这些方法，我们有机会了解一种重要的细菌病原体如何操纵人类细胞，并描绘线粒体凋亡核心效应物Bax和Bak活化的新细节。

Chlamydia trachomatis (Ctr) is an obligate intracellular bacterium that develops in a cytoplasmic vacuole in human epithelial cells. Medically, Ctr is important especially as a pathogen of the eye and of the female genital tract. Scientifically, the interplay of host cell and bacterium is intriguing and in large parts not understood. One prominent way how Ctr manipulates the host cell is the inhibition of apoptosis: infected cells are strongly resistant to stimuli that would induce mitochondrial apoptosis in uninfected cells. This inhibition is very impressive but not understood; three molecular models have been put forward in the past but are incompatible with each other. The work in the current project has focused on a stringent experimental testing of these three hypotheses, applying recent advancements in the understanding of the apoptosis apparatus. Surprisingly, all three hypotheses turned out to be incorrect, including the one we had proposed ourselves some time ago. We moved on to map the anti-apoptotic activity. Using assays with intact cells and with in vitro reconstituted mitochondrial apoptosis systems we found evidence that the anti-apoptotic activity involves the direct inhibition of the two effectors of mitochondrial apoptosis, Bax and Bak. This inhibition may be the result of chemical modification or of the attachment of a binding partner. In this continuation of the project we will pursue the two goals of first identifying the molecular alterations on Bax and Bak associated with this inhibition and secondly identifying the modifications or binding partner of the two proteins. Using HeLa cells deficient for Bax (and thus initially focusing on Bak) we will, in the first part of the project, map Bak-alterations by limited proteolysis, native gel electrophoresis and cross-linking studies. Intramolecular changes in Bak that occur during its activation have been reported, and these studies will therefore pin down the step where Bak-activation is blocked by Ctr-infection. In the second part of the project we will attempt specifically to identify modifications and/or binding partners of Bak. Through proteomic analysis of immuno-precipitated Bak or through the detection of proteins biotinylated because of their proximity to Bak we will endeavour to identify potential binding partners. Through approaches also encompassing proteomic analyses we will search for chemical modifications of Bak. Results obtained with Bak will be tested on Bax. Identified underlying mechanisms of bacterial or host cell origin will be specifically targeted to test their relevance for both inhibition of apoptosis and development of Ctr. We believe that with these approaches we have the chance of understanding how an important bacterial pathogen manipulates human cells as well as delineating new details of the activation of the core effectors of mitochondrial apoptosis, Bax and Bak.