Gene expression in stage-specific populations of Rickettsia prowazekii

描述（由申请人提供）：流行性斑疹伤寒是影响人类最严重的细菌性疾病之一，是由虱子传播的病原体，普氏立克次体引起的。由于该病死亡率高，缺乏有效的疫苗，并有可能通过气溶胶传播，因此普拉兹克氏弧菌被指定为一种特定病原体，有可能对公众健康和安全构成严重威胁。proproazekii是一种专性细胞内寄生细菌，只生长在宿主细胞的细胞质内，不受液泡膜的限制。这种利用细胞内环境并引起人类疾病的能力为我们的研究提供了基础，旨在阐明细胞内专性生长和普拉兹氏孢子虫毒力的机制。这一建议解决了关键阶段特异性基因的鉴定，当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立克次体时，这些基因受到调控。这一领域的一个主要障碍是

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.