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"Reverse Evolution" Approach to Identify Coxiella burnetii Strategies of Intracellular Survival

“逆向进化”方法鉴定伯内氏立克次体的细胞内生存策略

基本信息

批准号：
10163127
负责人：
Edward I. Shaw
金额：
$ 7.22万
依托单位：
OKLAHOMA STATE UNIVERSITY STILLWATER
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-05-18 至 2023-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10163127
关键词：
Acute Address Animals Bacteria Bacterial Genes Bacterial Proteins Binding Biology Catalogs Categories Cell physiology Cells Cellular Stress Centers for Disease Control and Prevention (U.S.)Chromosomes Chronic Disease Citrates Communities Coxiella burnetii Culture Media Cysteine Cytolysis DNA DNA Sequence Alteration DNA sequencing Data Deletion Mutagenesis Detection Diagnostic Disease Environment Evolution Face Foundations Funding Future Gene Expression Genes Genetic Genetic Transcription Goals Growth Human Infection Insertional Mutagenesis Intervention Laboratories Length Libraries Life Methods Molecular Molecular Analysis Mutation Nature Organism Pathogenesis Pathway interactions Phagolysosome Phagosomes Phase Phase Transition Physiological Process Proteins Proteomics Q Fever Research Serial Passage Stress System Testing Time Type IV Secretion System Pathway Vacuole Vesicle Virulence Virulent Zoonoses base chicken egg comparative gene synthesis innovation mutant parasitism pathogen sound stressor therapeutic target transcriptome sequencing

项目摘要

Project Summary Coxiella burnetii (Cb) is an obligate intracellular bacterium in nature and the causative agent of human acute Q fever as well as chronic disease. Upon infection, Cb is trafficked along a bacterially modified endocytic pathway and establishes a parasitophorous vacuole (PV) that retains many of the features of mature phagolysosomes. Throughout the infectious cycle within a host cell, Cb face an array of physiological needs and stresses unique to life within an acidified intracellular vesicle. The pathogen manipulates a plethora of host cell pathways and processes from within the PV via a bacterial Type 4 Secretion System (T4BSS) in order to survive and replicate. However, axenic media supports Cb replication in an environment where the bacterial genes required for many of its adaptations to intracellular growth are no longer needed. Deletion and insertion mutants have demonstrated some of Cb host cell specific gene requirements, however, an understanding of the breadth of the bacteria’s genes, and/or their expression, that will change when cellular stresses are removed is still lacking. This is a fundamental gap in our understanding of how Cb subverts host cell processes during infection and will lead to a broader understanding of Cb biology and pathogenesis. The long-term goal of our research is to elucidate molecular mechanisms employed by Cb to orchestrate parasitism of the host cell during infection. Our hypotheses are 1- Cb changes its gene expression within the first few passages upon transition from intracellular to axenic media growth, and 2- Cb will progressively acquire and accumulate DNA mutations upon transition from intracellular to axenic media growth after repetitive passages since certain bacterial genes/proteins are no longer required for successful growth. We will test our hypotheses by 1- Identifying Cb gene expression changes that occur during early and long term passages after inoculating axenic growth media with host cell propagated Cb, and 2- Identifying Cb genetic mutations/changes that occur/accumulate over multiple passages after inoculating axenic growth media with host cell propagated Cb. Understanding the virulence mechanisms employed by this unique pathogen to survive within the harsh environment of the host cell phagosome and cause disease will enable us to develop countermeasures to this poorly understood bacteria.

项目摘要贝氏柯克斯体（Coxiellaburnetii，Cb）是自然界中的一种专性胞内细菌，也是人类感染的病原体急性Q热以及慢性疾病。感染后，Cb沿着细菌修饰的内吞蛋白转运，途径，并建立了一个寄生液泡（PV），保留了许多成熟的特征，吞噬溶酶体在宿主细胞内的整个感染周期中，Cb面临一系列生理需求并对酸化的胞内囊泡内的生命施加独特的压力。病原体操纵过多的宿主通过细菌4型分泌系统（T4 BSS）从PV内的细胞途径和过程，生存和复制。然而，无菌培养基在细菌生长的环境中支持Cb复制。它对细胞内生长的许多适应所需的基因不再需要。缺失和插入突变体已经证明了一些Cb宿主细胞特异性基因的要求，然而，细菌基因的宽度，和/或它们的表达，当细胞压力被去除时，仍然缺乏。这是我们理解Cb如何破坏宿主细胞过程中的一个根本差距。感染，并将导致更广泛的了解Cb的生物学和发病机制。我们的长期目标是研究的目的是阐明Cb在宿主细胞中的寄生作用的分子机制。感染我们的假设是1- Cb改变其基因表达的前几个通道后，从细胞内过渡到无菌培养基生长，2- Cb将逐渐获得，在重复培养后从细胞内向无菌培养基生长过渡时积累DNA突变因为成功生长不再需要某些细菌基因/蛋白质。我们将通过1-识别早期和长期发生的Cb基因表达变化来测试我们的假设在无菌生长培养基后用宿主细胞繁殖Cb进行传代，2-鉴定Cb 在无菌接种后的多次传代中发生/积累的遗传突变/变化具有宿主细胞繁殖的生长培养基Cb.了解这种病毒的毒力机制独特的病原体在宿主细胞吞噬体的恶劣环境下生存并引起疾病使我们能够开发出对抗这种知之甚少的细菌的对策。