Analysis and role of the inhibition of apoptosis by the Chlamydia trachomatis OmpA protein

沙眼衣原体（Ctr）是一种专性细胞内细菌：它在被感染的人上皮细胞的液泡内复制。已知Ctr可以保护受感染的人类细胞免受许多实验性促凋亡刺激。细胞凋亡保护的分子机制存在争议。我们在感染的人细胞上使用bcl -2家族抑制剂观察到，Ctr可以通过蛋白酶敏感因子在促凋亡bcl -2家族效应蛋白Bak水平上阻止细胞凋亡。因此，抑制作用发生在细胞色素c释放的上游，而抗凋亡bcl -2样蛋白的下游。由于没有已知的人类蛋白经常参与凋亡信号传导的这一步，这强烈表明衣原体蛋白干扰细胞凋亡。我们发现衣原体主要外膜孔蛋白OmpA从细菌转移到线粒体，在那里它被发现插入膜并靠近Bak。我们将Ctr的抗凋亡活性映射到已知的Bak在细胞凋亡过程中的激活步骤上，并能够监测Bak的抑制作用。在未感染的细胞中OmpA的表达被cr -感染精确地表型化了bac抑制。Bak存在于与线粒体外膜孔蛋白VDAC2结合的静息细胞中，在细胞凋亡诱导下被释放。我们的研究结果表明，OmpA在Bak从VDAC2中释放时可以隔离和抑制Bak，并且我们已经从Simkania negevensis （SnOmpA）中鉴定出一个具有类似功能的相关蛋白。“类衣原体”，与原生动物相关的细菌，如副衣原体，诱导而不是抑制人类细胞的凋亡。我们发现，如果线粒体凋亡被阻断，副原体可以在人细胞中生长，而OmpA可以支持副原体的生长。在本项目中，我们将首先对Ctr和Sn OmpA进行比较研究，了解其抗凋亡机制的保守程度。在衣原体感染期间，OmpA如何转运到线粒体尚不清楚。我们假设它发生在细菌脱落的外膜囊泡中，并建议测试这种机制。最后，我们将从分子层面对OmpA的抗凋亡作用进行了解。我们将首先比较OmpA和VDAC2的抗凋亡活性。我们将进一步跟进我们的发现，OmpA可以降低线粒体Bax的浓度，很可能是通过增强Bax向细胞质的逆转录，这是一种已建立的抗凋亡机制。这些实验不仅有助于了解OmpA在细胞凋亡抑制中的作用，还有助于了解Bax和Bak的活化和活性。我们的数据表明，衣原体利用它们与线粒体的进化关系来阻止受感染的人类细胞的线粒体凋亡。我们期望这个项目的结果将揭示革兰氏阴性细菌与人类细胞相互作用的一个新的方面。

Chlamydia trachomatis (Ctr) is an obligate intracellular bacterium: it replicates within a vacuole in infected human epithelial cells. Ctr is known to protect infected human cells against numerous experimental pro-apoptotic stimuli. The molecular mechanism of apoptosis protection is contentious. We had observed, using Bcl-2-family inhibitors on infected human cells, that Ctr can block apoptosis through a protease-sensitive factor at the level of the pro-apoptotic Bcl-2-family effector protein Bak. Inhibition thus occurs upstream of cytochrome c-release but downstream of anti-apoptotic Bcl-2-like proteins. As no human protein is regularly known to act at this step of apoptotic signaling, this strongly suggested a chlamydial protein interfering with apoptosis. We found that the chlamydial major outer membrane porin, OmpA, translocates from the bacteria to mitochondria where it is found membrane-inserted and in the proximity of Bak. We mapped the anti-apoptotic activity of Ctr onto the known activation steps of Bak during apoptosis and were able to monitor Bak inhibition. Expression of OmpA in uninfected cells precisely phenocopied Bak-inhibition by Ctr-infection. Bak is in resting cells bound to a mitochondrial outer membrane porin, VDAC2, from where it is released upon apoptosis-induction. Our results suggest that OmpA sequesters and inhibits Bak upon its release from VDAC2, and we have identified a related protein from Simkania negevensis (SnOmpA) with functionally similar activity. ‘Chlamydia-like’, protozoa-associated bacteria such as Parachlamydia induce rather than inhibit apoptosis in human cells. We have found that Parachlamydia can grow in human cells if mitochondrial apoptosis is blocked, and that OmpA can support the growth of Parachlamydia. In this project we will first conduct a comparative study of Ctr and Sn OmpA to understand the degree of conservation of the anti-apoptotic mechanism. How OmpA translocates to mitochondria during chlamydial infection is unclear. We hypothesize that it occurs in outer membrane vesicles that are shed by the bacteria and propose to test this mechanism. Lastly, we aim at a molecular understanding of the anti-apoptotic effect of OmpA We will first compare the anti-apoptotic activity of OmpA and VDAC2. We will further follow up on our finding that OmpA can reduce mitochondrial concentration of Bax, very likely through enhancing the retro-translocation of Bax into the cytosol, an established anti-apoptotic mechanism. These experiments will help understand not only the function of OmpA in apoptosis inhibition but also provide information on activation and activity of Bax and Bak. Our data suggest that Chlamydiales exploit their evolutionary relationship to mitochondria to block mitochondrial apoptosis of an infected human cell. We expect that the results of this project will shed light on a novel facet of interaction of Gram-negative bacteria and human cells.