Institute of Medical Microbiology and Hygiene, Medical Center - University of Freiburg, Faculty of Medicine, 79104, Freiburg, Germany.
Veterinary Physiological Chemical Institute, Faculty of Veterinary Medicine, University of Leipzig, 04103, Leipzig, Germany.
Cell Death Differ. 2022 Oct;29(10):2046-2059. doi: 10.1038/s41418-022-00995-0. Epub 2022 Apr 9.
Apoptosis acts in defense against microbial infection, and many infectious agents have developed strategies to inhibit host cell apoptosis. The human pathogen Chlamydia trachomatis (Ctr) is an obligate intracellular bacterium that strongly inhibits mitochondrial apoptosis of its human host cell but there is no agreement how the bacteria achieve this. We here provide a molecular analysis of chlamydial apoptosis-inhibition in infected human cells and demonstrate that the block of apoptosis occurs during the activation of the effectors of mitochondrial apoptosis, Bak and Bax. We use small-molecule Bcl-2-family inhibitors and gene targeting to show that previous models cannot explain the anti-apoptotic effect of chlamydial infection. Although the anti-apoptotic Bcl-2-family protein Mcl-1 was strongly upregulated upon infection, Mcl-1-deficient cells and cells where Mcl-1 was pharmacologically inactivated were still protected. Ctr-infection could inhibit both Bax- and Bak-induced apoptosis. Apoptotic Bax-oligomerization and association with the outer mitochondrial membrane was reduced upon chlamydial infection. Infection further inhibited apoptosis induced conformational changes of Bak, as evidenced by changes to protease sensitivity, oligomerization and release from the mitochondrial porin VDAC2. Mitochondria isolated from Ctr-infected cells were protected against the pro-apoptotic Bcl-2-family proteins Bim and tBid but this protection was lost upon protease digestion. However, the protective effect of Ctr-infection was reduced in cells lacking the Bax/Bak-regulator VDAC2. We further found that OmpA, a porin of the outer membrane of Ctr, associated upon experimental expression with mitochondria and inhibited apoptosis, phenocopying the effect of the infection. These results identify a novel way of apoptosis inhibition, involving only the most downstream modulator of mitochondrial apoptosis and suggest that Chlamydia has a protein dedicated to the inhibition of apoptosis to secure its survival in human cells.
细胞凋亡在抵御微生物感染中起防御作用，许多传染性病原体已进化出抑制宿主细胞凋亡的策略。人类病原体沙眼衣原体（Ctr）是一种严格的细胞内细菌，强烈抑制其人类宿主细胞的线粒体凋亡，但对于细菌如何实现这一目标尚无共识。我们在此对感染人类细胞中的衣原体凋亡抑制进行了分子分析，并证明凋亡的阻断发生在线粒体凋亡效应子 Bak 和 Bax 的激活过程中。我们使用小分子 Bcl-2 家族抑制剂和基因靶向来表明，先前的模型无法解释衣原体感染的抗凋亡作用。尽管感染后抗凋亡的 Bcl-2 家族蛋白 Mcl-1 强烈上调，但 Mcl-1 缺陷细胞和 Mcl-1 被药物失活的细胞仍受到保护。Ctr 感染可以抑制 Bax 和 Bak 诱导的凋亡。凋亡的 Bax 寡聚化并与外线粒体膜结合在衣原体感染时减少。感染进一步抑制了 Bak 诱导的凋亡构象变化，这表现在蛋白酶敏感性、寡聚化和从线粒体 porin VDAC2 的释放方面的变化。从 Ctr 感染的细胞中分离出的线粒体对促凋亡的 Bcl-2 家族蛋白 Bim 和 tBid 具有保护作用，但这种保护在蛋白酶消化后丢失。然而，在缺乏 Bax/Bak 调节剂 VDAC2 的细胞中，Ctr 感染的保护作用降低。我们进一步发现，外膜的孔蛋白 OmpA，在实验表达时与线粒体结合并抑制凋亡，模拟了感染的效果。这些结果确定了一种新的凋亡抑制方式，仅涉及线粒体凋亡的最下游调节剂，并表明衣原体有一种专门用于抑制凋亡的蛋白，以确保其在人类细胞中的存活。
