Elucidation of the Role of Chlamydial ClpX During Development and Differentiation

项目摘要 沙眼衣原体是国内细菌性性传播感染的主要原因， 在全球范围内，每10万人中的病例率每年都在增加。然而，感染者往往不能 寻求治疗主要是因为约70%的感染无症状。未经检查，慢性 感染可导致许多后遗症，包括异位妊娠和输卵管因素不孕症的妇女， 男性附睾炎和不育症，那些接受治疗的人通常被处方广谱 抗生素鉴于感染病例率稳步上升，慢性问题的风险以及有限的选择， 对于抗生素管理，更全面地了解衣原体生物学， 有针对性的治疗至关重要。 尽管衣原体的基因组高度简化，但它经历了复杂的发育周期， 细菌在功能和形态上有两种不同的形式：感染性的，非感染性的， 复制性初级体（EB）和非感染性复制性网状体（RB）。EB启动感染 通过与宿主细胞结合并诱导宿主细胞摄取。在细胞内，EB经历初级分化， 一个RB，然后将产生一个RB群。在一个未知的信号中期发育周期，新 形成的RB将经历从RB到EB的二次分化。EB和RB之间的转换不是 由重新分配细胞内蛋白质的分裂事件介导。相反，主要（EB至RB）和 二次分化（RB到EB）可能需要蛋白质周转。因此，我们假设ClpX 在衣原体分化过程中通过靶向蛋白降解发挥关键作用。为了验证这一 假设，我们产生了许多构建体，用于过表达各种突变ClpX同种型， 衣原体该提案的总体目标是描绘ClpX介导的细胞因子降解的机制。 通过适配器依赖性或非依赖性手段，其可以揭示靶的网络， 有助于衣原体的分化。来自其他实验室的这些突变的先前体外表征 研究其他细菌ClpX旁系同源物使我们能够对ClpX功能进行更有针对性的评估， 比标准的丙氨酸扫描要好目的1将通过以下方式确定ClpX在衣原体生物学的哪些方面发挥作用： 观察这些突变蛋白对衣原体发育、分化状态的影响， 形态学目标2的拟议实验将确定衣原体ClpX的靶点，这将提供 重要的洞察其基板识别和处理。我们开发了一种化学交联 与衣原体蛋白的Click-iT标记相结合的方法，特别是当 进行ClpX及其衔接子和底物的亲和纯化。总的来说，这些实验 揭示ClpX在衣原体中的作用，这反过来将为衣原体提供机制性的见解 差异化，并将打开大门，有针对性的治疗策略。

PROJECT SUMMARY Chlamydia trachomatis is the leading cause of bacterial sexually transmitted infection both domestically and globally, with the rate of cases per 100,000 increasing every year. However, infected individuals often fail to seek treatment largely due to the asymptomatic nature of approximately 70% of infections. Unchecked, chronic infection can lead to numerous sequelae, including ectopic pregnancy and tubal factor infertility in women or epididymitis and sterility in men, and those who do receive treatment are typically prescribed broad spectrum antibiotics. Given the steadily increasing case rate of infections, the risk of chronic issues, and the limited options for antibiotic stewardship, a more comprehensive understanding of chlamydial biology to produce a more targeted therapeutic treatment is critical. Despite having a highly reduced genome, Chlamydia undergoes a complex developmental cycle in which the bacteria differentiate between two functionally and morphologically distinct forms: the infectious, non- replicative elementary body (EB) and the non-infectious, replicative reticulate body (RB). An EB initiates infection by binding to and inducing uptake into a host cell. Within the cell, the EB undergoes primary differentiation into an RB, which will then give rise to a population of RBs. At an unknown signal mid-developmental cycle, newly formed RBs will undergo secondary differentiation from RB to EB. The transitions between EBs and RBs are not mediated by division events that re-distribute intracellular proteins. Rather, both primary (EB to RB) and secondary (RB to EB) differentiation likely require protein turnover. As such, we hypothesize that ClpX plays a critical role during chlamydial differentiation through targeted protein degradation. To test this hypothesis, we generated numerous constructs for overexpression of various mutant ClpX isoforms in Chlamydia. The overarching goal of this proposal is to delineate a mechanism of ClpX-mediated degradation of substrates through adaptor dependent or independent means, which may uncover a network of targets that contribute to chlamydial differentiation. Previous in vitro characterization of these mutations from other labs studying other bacterial ClpX paralogs allows us to take a more targeted assessment of ClpX function rather than standard alanine scanning. Aim 1 will determine in which facets of chlamydial biology ClpX plays a role by observing the effects that these mutant proteins exert on chlamydial development, differentiation state, and morphology. The proposed experiments of Aim 2 will identify targets of chlamydial ClpX, which will provide significant insight into its substrate recognition and processing. We have developed a chemical crosslinking approach combined with Click-iT labeling of chlamydial proteins, specifically, to maintain complex stability when performing affinity purification of ClpX and its adaptors and substrates. Collectively, these experiments will uncover the role that ClpX serves in Chlamydia, which in turn will provide mechanistic insight into chlamydial differentiation and will open the door to targeted therapeutic strategies.