The tail-specific protease (Tsp) of Chlamydiae spp.: Mechanistic and structural characterization of its interactions with host-cell proteins

衣原体是专性细胞内病原体，利用分泌的效应蛋白修饰真核宿主细胞的信号通路。据报道，在人类病原体沙眼衣原体中，丝氨酸蛋白酶CT441通过切割p65干扰宿主炎症反应的NF-kappaB途径。此外，CT441特异性靶向雌激素受体α的类固醇受体RNA激活因子1（甾体受体RNA激活因子1，SRA1）的蛋白组分SRAP1。有研究表明，这种相互作用将SRAP1保留在宿主细胞质中，从而阻止了许多雌二醇诱导的靶基因的表达，这些基因对宿主细胞内衣原体的生存和发育至关重要。大部分未被鉴定的CT441显示了与大肠杆菌中参与细菌蛋白质质量控制的尾部特异性蛋白酶（Tsps）的序列相似性。本项目的目的是为了解衣原体对宿主-细胞通路的调节作出贡献。我们将重点阐明衣原体Tsps与宿主细胞蛋白相互作用的详细机制。我们的目标是确定CT441及其同源物的三维结构。基于结构信息，位点定向诱变将用于进行详细的机制分析。此外，我们将在体外研究CT441与SRAP1的相互作用。利用CT441和SRAP1的全长蛋白或单个结构域进行生化相互作用分析，蛋白酶分析和x射线晶体学将有助于阐明相互作用的模式。最终，我们的目标是确定SRAP1和衣原体Tsp蛋白之间复合物的分子结构。我们将利用人上生殖道衣原体感染的输卵管组织模型研究CT441在生理环境下对宿主细胞SRAP1水平的影响。该项目有望揭示衣原体通过雌激素调节宿主细胞代谢和感染性的分子基础。

Chlamydiae are obligate intracellular pathogens that use secreted effector proteins to modify signaling pathways of eukaryotic host cells. In the human pathogen Chlamydia trachomatis, the serine protease CT441 has been reported to interfere with the NF-kappaB pathway of the host inflammatory response by cleaving p65. In addition, CT441 specifically targets SRAP1, the protein component of steroid-receptor RNA activator 1 (SRA1) of estrogen receptor alpha. It has been suggested that this interaction retains SRAP1 in the host-cell cytoplasm, thereby preventing the expression of many estradiol-induced target genes which is important for chlamydial survival and development within the host cell. The largely uncharacterized CT441 shows sequence similarities to tail-specific proteases (Tsps) involved in bacterial protein-quality control in E. coli. The goal of this project is to contribute to the understanding of the modulation of host-cell pathways by Chlamydiae. We will focus on elucidating the detailed mechanisms of interactions between chlamydial Tsps with host-cell proteins. We aim to determine three-dimensional structures for CT441 and homologues from related species. Based on structural information, site-directed mutagenesis will be used to conduct a detailed mechanistic analysis. In addition, the interaction between CT441 and SRAP1 will be studied in vitro. Using full-length proteins or individual domains of CT441 and SRAP1 in biochemical interaction analyses, protease assays and X-ray crystallography will help to clarify the mode of the interaction. Ultimately, we aim to determine the molecular structure of a complex between SRAP1 and a chlamydial Tsp protein. A human fallopian-tube tissue model of chlamydial infections of the upper genital tract will be used to study the influence of CT441 on host cell SRAP1 levels in a physiological environment. The project is expected to reveal the molecular basis for chlamydial modulation of host-cell metabolism and infectivity by estrogen.