Mikrobiologisches Institut, Universitätsklinikum Erlangen, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany.
Lehrstuhl für Biotechnik, Department Biologie, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany.
PLoS Pathog. 2022 Feb 8;18(2):e1010266. doi: 10.1371/journal.ppat.1010266. eCollection 2022 Feb.
Inhibition of host cell apoptosis is crucial for survival and replication of several intracellular bacterial pathogens. To interfere with apoptotic pathways, some pathogens use specialized secretion systems to inject bacterial effector proteins into the host cell cytosol. One of these pathogens is the obligate intracellular bacterium Coxiella burnetii, the etiological agent of the zoonotic disease Q fever. In this study, we analyzed the molecular activity of the anti-apoptotic T4SS effector protein AnkG (CBU0781) to understand how C. burnetii manipulates host cell viability. We demonstrate by co- and RNA-immunoprecipitation that AnkG binds to the host cell DExD box RNA helicase 21 (DDX21) as well as to the host cell 7SK small nuclear ribonucleoprotein (7SK snRNP) complex, an important regulator of the positive transcription elongation factor b (P-TEFb). The co-immunoprecipitation of AnkG with DDX21 is probably mediated by salt bridges and is independent of AnkG-7SK snRNP binding, and vice versa. It is known that DDX21 facilitates the release of P-TEFb from the 7SK snRNP complex. Consistent with the documented function of released P-TEFb in RNA Pol II pause release, RNA sequencing experiments confirmed AnkG-mediated transcriptional reprogramming and showed that expression of genes involved in apoptosis, trafficking, and transcription are influenced by AnkG. Importantly, DDX21 and P-TEFb are both essential for AnkG-mediated inhibition of host cell apoptosis, emphasizing the significance of the interaction of AnkG with both, the DDX21 protein and the 7SK RNA. In line with a critical function of AnkG in pathogenesis, the AnkG deletion C. burnetii strain was severely affected in its ability to inhibit host cell apoptosis and to generate a replicative C. burnetii-containing vacuole. In conclusion, the interference with the activity of regulatory host cell RNAs mediated by a bacterial effector protein represent a novel mechanism through which C. burnetii modulates host cell transcription, thereby enhancing permissiveness to bacterial infection.
宿主细胞凋亡的抑制对于几种细胞内细菌病原体的存活和复制至关重要。为了干扰凋亡途径，一些病原体利用专门的分泌系统将细菌效应蛋白注入宿主细胞质。其中一种病原体是严格的细胞内细菌贝氏柯克斯体，这是动物传染病 Q 热的病原体。在这项研究中，我们分析了抗凋亡 T4SS 效应蛋白 AnkG（CBU0781）的分子活性，以了解贝氏柯克斯体如何操纵宿主细胞活力。我们通过共沉淀和 RNA 免疫沉淀证明，AnkG 与宿主细胞 DExD 框 RNA 解旋酶 21（DDX21）以及宿主细胞 7SK 小核核糖核蛋白（7SK snRNP）复合物结合，后者是正向转录伸长因子 b（P-TEFb）的重要调节剂。AnkG 与 DDX21 的共沉淀可能是通过盐桥介导的，并且独立于 AnkG-7SK snRNP 结合，反之亦然。已知 DDX21 促进 P-TEFb 从 7SK snRNP 复合物中释放。与文献中记录的释放的 P-TEFb 在 RNA Pol II 暂停释放中的作用一致，RNA 测序实验证实了 AnkG 介导的转录重编程，并表明参与凋亡、运输和转录的基因的表达受 AnkG 影响。重要的是，DDX21 和 P-TEFb 对于 AnkG 介导的宿主细胞凋亡抑制都是必不可少的，这强调了 AnkG 与 DDX21 蛋白和 7SK RNA 的相互作用的重要性。与 AnkG 在发病机制中的关键作用一致，AnkG 缺失的贝氏柯克斯体菌株在抑制宿主细胞凋亡和产生含有复制贝氏柯克斯体的复制空泡的能力方面受到严重影响。总之，细菌效应蛋白介导的对调节宿主细胞 RNA 活性的干扰代表了一种新的机制，通过该机制，贝氏柯克斯体调节宿主细胞转录，从而增强对细菌感染的易感性。
