Molecular analysis of apoptosis inhibition by Chlamydia trachomatis

沙眼衣原体（Ctr）是一种专性细胞内细菌，在人类上皮细胞的胞质空泡中发育。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没有被理解。Ctr如何操纵宿主细胞的一个突出方式是抑制细胞凋亡：感染的细胞对将在未感染的细胞中诱导线粒体凋亡的刺激具有强烈的抗性。这种抑制是非常令人印象深刻的，但不理解;三个分子模型已经提出了在过去，但彼此不相容。在目前的项目中的工作集中在这三个假设的严格的实验测试，应用在细胞凋亡装置的理解的最新进展。令人惊讶的是，这三个假设都被证明是错误的，包括我们不久前提出的那个假设。我们继续绘制抗凋亡活性。使用完整细胞和体外重建线粒体凋亡系统的测定，我们发现证据表明，抗凋亡活性涉及直接抑制线粒体凋亡的两个效应子Bax和巴克。这种抑制可能是化学修饰或结合配偶体附着的结果。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和巴克的分子改变，其次确定这两种蛋白质的修饰或结合伴侣。使用Bax缺陷的HeLa细胞（因此最初关注巴克），我们将在项目的第一部分，通过有限的蛋白水解，天然凝胶电泳和交联研究绘制巴克改变。巴克在其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巴克激活被Ctr感染阻断的步骤。在项目的第二部分中，我们将特别尝试识别巴克的修饰和/或结合伙伴。通过免疫沉淀巴克的蛋白质组学分析或通过检测由于与巴克接近而生物素化的蛋白质，我们将努力鉴定潜在的结合伴侣。通过包括蛋白质组学分析的方法，我们将寻找巴克的化学修饰。使用巴克获得的结果将在Bax上进行检测。将特异性靶向细菌或宿主细胞来源的鉴定的潜在机制，以测试它们与细胞凋亡抑制和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，以及描绘线粒体凋亡的核心效应子Bax和巴克的激活的新细节。

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.