Reprogramming of host cell cholesterol homeostasis by Coxiella burnetii

 描述(申请人提供)：伯氏柯克斯体是人类Q热的病原体，Q热是一种新出现的传染病，也是培养阴性心内膜炎的主要原因。柯克斯体是一种专有的细胞内细菌病原体，通过气溶胶传播，在自然感染期间主要针对肺泡巨噬细胞。一旦进入细胞，柯克斯体就会操纵宿主细胞机制，促进一种名为寄生虫性液泡(PV)的特殊隔室的生物发生。虽然PV是柯克斯体致病的核心，但PV生物发生和维持背后的机制却鲜为人知。Coxiella PV富含类固醇，而扰乱宿主细胞胆固醇的药物抑制剂可以抑制PV的形成和随后的细菌生长。尽管越来越多的证据表明胆固醇在PV的形成和柯克斯体与宿主的相互作用中起着关键作用，但关于这两种情况的信息都很少 涉及细菌或宿主因素。这项应用的目的是识别和表征细菌驱动的宿主细胞胆固醇稳态的变化。这一提议将检验这样一种假设，即柯克斯体蛋白通过细菌专门的IV型分泌系统(T4SS)分泌到宿主细胞质中，操纵宿主胆固醇稳态，作为支持细胞内细菌生长和生存的机制。在第一个特定目标中，将通过对感染的肺泡巨噬细胞的转录组分析来确定以柯克斯体T4SS效应蛋白为靶点的宿主胆固醇稳态途径。这项研究将确定柯克斯体差异调控的关键宿主细胞基因。在鉴定后，宿主途径将被扰乱，并确定对柯克斯体存活的影响。在第二个目标中，将对操纵宿主胆固醇的柯克斯氏菌蛋白质进行表征。这将通过筛选已经失去改变宿主细胞胆固醇稳态能力的柯克斯体转座子突变体来实现。鉴定出的柯克斯体蛋白将通过分析分离的突变株的表型以及相应蛋白质的功能分析来鉴定。这项拟议的工作将确定柯克斯体感染期间参与胆固醇稳态的宿主和病原体蛋白，为治疗干预开辟新的途径。

 DESCRIPTION (provided by applicant): Coxiella burnetii is the causative agent of human Q fever, an emerging infectious disease and a leading cause of culture-negative endocarditis. An obligate intracellular bacterial pathogen spread through aerosols, Coxiella primarily targets alveolar macrophages during natural infection. Once inside the cell, Coxiella manipulates host cell machinery to promote the biogenesis of a specialized compartment called the parasitophorous vacuole (PV). While the PV is central to Coxiella pathogenesis, the mechanisms behind PV biogenesis and maintenance are poorly understood. The Coxiella PV is sterol-rich, and pharmaceutical inhibitors that perturb host cell cholesterol inhibit PV formation and subsequent bacterial growth. Despite increasing evidence that cholesterol plays a critical role in PV formation and Coxiella-host interactions, there is very little information on either the bacterial or host factors involved. The objective of this application is to identify and characterie bacterial-driven changes in host cell cholesterol homeostasis. This proposal will test the hypothesis that Coxiella proteins, secreted into the host cell cytoplasm through the bacteria's specialized Type IV Secretion System (T4SS), manipulate host cholesterol homeostasis as a mechanism to support intracellular bacterial growth and survival. In the first specific aim, host cholesterol homeostasis pathways targeted by Coxiella T4SS effector proteins will be identified through transcriptome analysis of infected alveolar macrophages. This study will identify key host cell genes differentially regulated by Coxiella. Following their identification, the host pathways will be disrupted and the effect on Coxiella survival determined. In the second aim, Coxiella proteins that manipulate host cholesterol will be characterized. This will be accomplished by screening for Coxiella transposon mutants that have lost the ability to alter host cell cholesterol homeostasis. The identified Coxiella proteins will be characterized by analyzing the phenotype of the isolated mutants, as well as functional analysis of the corresponding protein. The proposed work will identify both host and pathogen proteins involved in cholesterol homeostasis during Coxiella infection, opening new avenues for therapeutic intervention.