Gene expression in stage-specific populations of Rickettsia prowazekii

普瓦泽基立克次体特定阶段群体的基因表达

• 批准号: 8425189
• 负责人: DAVID WOOD
• 金额: $ 22.28万
• 依托单位: UNIVERSITY OF SOUTH ALABAMA
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-01-18 至 2014-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8425189
• 关键词: Address; Aerosols; Affect; Arthropods; Bacteria; Bacterial Infections; Cell Line; Cell Separation; Cells; Characteristics; Codon Nucleotides; Coupled; Cytolysis; Cytosol; Data; Disease; Environment; Epidemic; Epidemic Louse-Borne Typhus; Evaluation; Exhibits; Fluorescence; Fluorescence-Activated Cell Sorting; Foundations; Gene Expression; Gene Expression Profile; Genes; Goals; Growth; Harvest; Heterogeneity; Human; Infection; Lice; Membrane; Molecular; Pathogenicity; Play; Population; Protein Array; Proteins; Proteomics; Public Health; Quantitative Microscopy; Reporting; Research Design; Rickettsia; Rickettsia prowazekii; Safety; Severity of illness; Staging; System; Technology; Time; Typhus; Vaccines; Virulence; Virulent; War; base; combat; design; environmental change; human disease; intracellular parasitism; mortality; novel; novel therapeutics; pathogen; public health relevance; transmission process; vector

DESCRIPTION (provided by applicant): Epidemic typhus, one of the most serious bacterial diseases affecting humans, is caused by the louseborne pathogen, Rickettsia prowazekii. Due to the high mortality rate of this disease, the lack of an effective vaccine, and the possibility o aerosol dissemination, R. prowazekii is designated as a select agent and has the potential to pose a severe threat to public health and safety. R. prowazekii is an obligate intracellular, parasitic bacterium that grows only within the cytosol of the host cell, unbounded by a vacuolar membrane. This ability to exploit the intracellular environment and cause human disease provides the basis for our studies designed to elucidate the mechanisms underlying obligate intracellular growth and R. prowazekii virulence. This proposal addresses the identification of critical stage-specific genes that are regulated as a rickettsial infection proceeds from a few rickettsiae per cell to hundreds per cell. A major hurdle in this field is the heterogeneity in the bacterial load within an infected population and the inability to generate synchronous rickettsial cultures. To tackle this problem, we propose to use a fluorescence activated cell sorting (FACS) approach to separate R. prowazekii-infected host cells, based on bacterial load, into distinct stage-specific populations. Using genetically-modified GFP-expressing R. prowazekii, we have demonstrated that FACS can be used to separate R. prowazekii-infected host cells into distinct populations harboring uniform numbers of rickettsiae per cell (population gating). We hypothesize that FACS-based separation of rickettsiae-infected cells will facilitate identification of rickettsial genes that are regulated at specific growth stages and that play key roles in optimizing the growth, virulence, and subsequent spread of this intracellular pathogen. We will address this hypothesis through the following specific aims: In Aim 1 we will identify and separate R. prowazekii-infected host cells into distinct populations according to bacterial load. Rickettsial numbers in isolated populations will be evaluated by microscopy and quantitative PCR. Optimization will include the evaluation of a codon-adapted fluorescent protein. We will evaluate different host cell lines (e.g. vertebrate and arthropod) and a virulent rickettsial strain. The effect of initial host cell infection levels and harvest times wll be examined. In Aim 2 we will identify, at both the transcriptional (gene arrays) and protein level (differential proteomics), genes that are differentially expressed as the rickettsiae transition frm exponential growth to lysis stage. Completion of this project will significantly advance the field, providing an accurate assessment of gene expression at defined stages in the R. prowazekii intracellular growth cycle. This data will lay a foundation for further delineation of molecular mechanisms underlying the infection, intracellular growth, and virulence of R. prowazekii and may reveal new targets for design of novel therapeutics to combat this resourceful and dangerous pathogen.

描述(申请人提供)：流行性斑疹伤寒是影响人类的最严重的细菌疾病之一，由呼吸道传播的病原体普氏立克次体引起。由于这种疾病的高死亡率，缺乏有效的疫苗，以及气雾剂传播的可能性，普罗瓦泽克杆菌被指定为选择性制剂，并有可能对公众健康和安全构成严重威胁。普罗瓦泽克杆菌是一种专性胞内寄生细菌，只生长在宿主细胞的胞浆内，不受液泡膜的限制。这种利用细胞内环境并导致人类疾病的能力为我们的研究提供了基础，这些研究旨在阐明专性细胞内生长和普罗瓦泽克杆菌毒力的机制。这项建议涉及识别关键的阶段特定基因，这些基因随着立克次体感染从每个细胞几个立克次体发展到每个细胞数百个而受到调控。这一领域的一个主要障碍是 感染人群中的细菌载量以及无法同步产生立克次体培养。为了解决这个问题，我们建议使用荧光激活细胞分类(FACS)方法，根据细菌负载将感染Prowazekii的宿主细胞分离到不同的阶段特定种群。使用转基因的表达GFP的普氏立克次体，我们证明了FACS可以用于将感染普氏立克次体的宿主细胞分离到不同的群体中，每个细胞拥有统一数量的立克次体(群体门控)。我们推测，基于流式细胞仪的立克次体感染细胞的分离将有助于识别在特定生长阶段调节的立克次体基因，这些基因在优化这种细胞内病原体的生长、毒力和随后的传播中发挥关键作用。我们将通过以下具体目标来解决这一假设：在目标1中，我们将根据细菌载量鉴定和分离普罗瓦泽克杆菌感染的宿主细胞。隔离人群中立克次体数量将通过显微镜和定量聚合酶链式反应进行评估。优化将包括评估一种密码子适应的荧光蛋白。我们将评估不同的宿主细胞系(例如脊椎动物和节肢动物)和 一种致命的立克次体菌株。将检验初始寄主细胞感染水平和收获时间的影响。在目标2中，我们将在转录(基因阵列)和蛋白质水平(差异蛋白质组学)鉴定差异表达的基因，这些基因是立克次体从指数生长到裂解阶段的过渡阶段。该项目的完成将大大推进这一领域的发展， 提供对普罗瓦泽克乳杆菌细胞内生长周期中特定阶段的基因表达的准确评估。这些数据将为进一步描述普罗瓦泽克杆菌感染、细胞内生长和毒力的分子机制奠定基础，并可能为设计新的治疗方法来对抗这种足智多谋和危险的病原体提供新的靶点。