Elucidation of the Role of Chlamydial ClpX During Development and Differentiation

阐明衣原体 ClpX 在发育和分化过程中的作用

基本信息

批准号：
10313291
负责人：
Nicholas A Wood
金额：
$ 3.63万
依托单位：
UNIVERSITY OF NEBRASKA MEDICAL CENTER
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-07-21 至 2023-07-20
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10313291
关键词：
ATP phosphohydrolase Adaptor Signaling Protein Affinity Affinity Chromatography Alanine Antibiotics Attenuated Vaccines Bacteria Binding Biochemical Biological Assay Biology Blindness Cells Characteristics Chemicals Chemistry Chlamydia Chlamydia Infections Chlamydia trachomatis Chronic Clinical Treatment ClpX protein Complex Confocal Microscopy Crosslinker Dependence Development Ectopic Pregnancy Epididymitis Evaluation Event Female infertility Genome Goals Immunity In Vitro Individual Infection Investigation Kinetics Knowledge Label Lead Lighting Mediating Microbe Microscopic Microscopy Mitochondria Modeling Molecular Morphology Mutation N-terminal Nature Organism Orthologous Gene Pathogenicity Peptide Hydrolases Physical condensation Physiological Play Population Process Protein Isoforms Proteins Proteomics Role Scanning Secondary to Sexually Transmitted Diseases Shapes Signal Transduction Sterility Structure System Testing Transmission Electron Microscopy Two-Hybrid System Techniques Variant Work Zinc cecal ligation puncture chronic infection crosslink endopeptidase Clp experimental study grasp in vivo infection rate infection risk insight men mutant novel overexpression paralogous gene protein degradation targeted treatment therapeutic development therapeutic target tubal infertility unfoldase uptake

项目摘要

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.

项目概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 万人中的病例数每年都在增加。然而，感染者往往无法寻求治疗主要是因为约 70% 的感染无症状。不受控制的、慢性的感染可导致许多后遗症，包括宫外孕和女性输卵管因素不孕或男性附睾炎和不育症，接受治疗的患者通常会接受广谱治疗抗生素。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，以产生更多针对性治疗至关重要。尽管基因组大大减少，但衣原体经历了一个复杂的发育周期，其中细菌区分两种功能和形态上不同的形式：感染性细菌、非感染性细菌复制基本体（EB）和非感染性复制网状体（RB）。 EB 引发感染通过结合宿主细胞并诱导其摄取。在细胞内，EB 初级分化为一个 RB，然后会产生 RB 群体。在发育周期中期的未知信号中，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 到 RB）和二次分化（RB 到 EB）可能需要蛋白质更新。因此，我们假设 ClpX 扮演通过靶向蛋白质降解在衣原体分化过程中发挥关键作用。为了测试这个假设，我们生成了许多用于过表达各种突变 ClpX 同种型的构建体衣原体。该提案的总体目标是描述 ClpX 介导的降解机制通过适配器依赖或独立的方式识别底物，这可能会揭示靶标网络有助于衣原体分化。其他实验室先前对这些突变的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丙氨酸扫描。目标 1 将确定 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、分化状态和形态学。目标 2 的拟议实验将识别衣原体 ClpX 的靶标，这将提供对其底物识别和处理的重要见解。我们开发了一种化学交联该方法与衣原体蛋白的 Click-iT 标记相结合，特别是在对 ClpX 及其接头和底物进行亲和纯化。总的来说，这些实验将揭示 ClpX 在衣原体中的作用，这反过来将为衣原体提供机制见解差异化将为靶向治疗策略打开大门。