GENETIC AND MOLECULAR BASIS OF BACTERIAL INVASTION

描述(改编自申请人的摘要)：我们的目标是 研究是为了更好地了解糖尿病的遗传和分子基础 细菌入侵。特别是，我们建议将重点放在 鼠伤寒沙门氏菌的鉴定及特性研究 在受感染的宿主细胞和受感染的宿主细胞内表达的序列 动物。我们开发了一种新的方法，称为差示荧光法 诱导，它利用绿色荧光蛋白和荧光 激活的排序器(FACS)用于识别表达的细菌基因 仅在动物细胞内或在感染期间。我们将重点关注 巨噬细胞可诱导启动子序列(Mig)和动物诱导基因(Aig) 通过对鼠伤寒沙门氏菌SL1344进行细胞分选(FACS)，从 受感染动物的组织或受感染的培养细胞。四个MIG 到目前为止，我们鉴定的启动子序列代表了不同的一般 与细胞内生长和持久性相关的基因类别。 我们提出了几种策略来确定这些潜在函数 遗传序列的多参数流式细胞仪分析方法和 共聚焦显微镜分析细胞内运输。最后，我们 建议分离mig和aig基因的变种以遵循 受感染动物组织中的野生型和无毒突变生物。 因此，我们的目标是系统地研究 巨噬细胞和使用我们创造的突变菌株GFT感染的动物 我们构建的基因融合和FACS细胞分选的分辨率 发现感染发病机制所必需的基因。我们相信 这些原则可以广泛地应用于研究一些 传染病。

DESCRIPTION (Adapted from the applicant's abstract): The goal of our research is to understand better the genetic and molecular basis of bacterial invasion. In particular, we propose to focus on the identification and characterization of Salmonella typhimurium genetic sequences that are expressed within infected host cells and within infected animals. We have developed a new method termed differential fluorescence induction, which utilizes green fluorescent protein and a fluorescent activated sorter (FACS) to identify bacterial genes that are expressed exclusively within animal cells or during infection. We will focus on macrophage-inducible promoter sequences (mig) and animal-induced genes (aig) isolated from S. Typhimurium SL1344 by sorting cells (by FACS) from the tissue of infected animals or from infected cultured cells. Four mig promoter sequences we have identified thus far represent distinct general categories of genes associated with intracellular growth and persistence. We propose several strategies to determine the potential function of these genetic sequences using the methods of multi-parametric FACS analysis and confocal microscopy to analyze intracellular trafficking. Finally, we propose to isolate variants of the mig and aig genes to follow the fate of wildtype and avirulent mutant organisms in the tissues of infected animals. Thus, our aim is to systematically examine gene expression within macrophages and infected animals using the mutant strains we create, the GFT genetic fusions we construct, and the resolving power of FACS cell sorting to discover genes essential for the pathogenesis of infection. We believe that these principles can be broadly applied to the study of a number of infectious diseases.