HOST-SPECIFIC INDUCTION OF BACTERIAL VIRULENCE GENES

这一建议是基于一种全新的方法来隔离 与致病有关的细菌毒力基因，称为IVET(In vivo 表达技术)，这允许细菌的正向选择 在宿主组织中特异诱导的基因。这种方法论将 被用来描述调节的遗传和环境因素 体内诱导基因(IVI)在人类病原体中的表达 鼠伤寒沙门氏菌。这些研究将为我们提供对 调控致病性的分子机制，这是 是了解传染病的基础。遗传分析 回答依维特选择的基因是因为基因 在转录上与报告基因lacZ融合，从而使 在大肠杆菌中结构性表达IVI基因的调控突变体的恢复 体外的(通常是抑制条件)。我们的努力将集中在 那些协调调节IVI基因的调节因子，因此 定义可确保多方面细菌的调节组件 对多方面的主持人挑战的回应。跨行为监管 突变将被用来描述阳性和阴性病毒的特征 体内激活IV1转录和抑制IV1转录的调控元件 体外转录。将对这些突变体进行筛选，以寻找那些 在动物模型中发现毒性缺陷，表明它们或一些 在它们调节的产物中，在发病机制中起着重要作用。 顺式作用突变将被用来定义ivi启动子区域 重视建立协调IVI基因的共识位点 表情。界定协调监管的性质提供了一种手段 为了了解复杂的信号转导机制，使 细菌在这样不同的环境条件下生长(例如， 实验室介质与宿主组织)。此外，艾维特的选拔将 被扩展到对存活时间限制为 特定的宿主组织并因此可能包含组织特定的IVI基因， 其限制性表达将通过遗传分析进行检查。这些 疫苗和抗菌剂的研究都有实际应用 发展。

This proposal is based on a fundamentally new approach to the isolation of bacterial virulence genes involved in pathogenesis, termed IVET (in vivo expression technology), that allows for the positive selection of bacterial genes that are specifically induced in host tissues. This methodology will be used to characterize the genetic and environmental factors that regulate the expression of in vivo induced genes (ivi) in the human pathogen, Salmonella typhimurium. Such studies will provide insights into the molecular mechanisms governing the regulation of pathogenicity, which is fundamental to the understanding of infectious disease. Genetic analysis of genes that answer the IVET selection is facilitated because the genes are transcriptionally fused to the reporter gene, lacZ, allowing the recovery of regulatory mutants that constitutively express ivi genes in vitro (normally repressing conditions). Our efforts will be focused on those regulatory factors that coordinately regulate ivi genes, thus defining the regulatory components that ensure a multifaceted bacterial response to a multifaceted host challenge. Trans-acting regulatory mutations will be used to characterize the positive and negative ivi regulatory elements that activate ivi transcription in vivo and repress ivi transcription in vitro. These mutants will be screened for those that confer virulence defects in an animal model, suggesting that they, or some of the products they regulate, play an important role in pathogenesis. Cis-acting mutations will be used to define the ivi promoter region with an emphasis on establishing a consensus site for coordinate ivi gene expression. Defining the nature of coordinate regulation provides a means to under stand the sophisticated signal transduction mechanisms that enable bacteria to grow under such different environmental conditions (e.g., laboratory media vs. host tissues). Additionally, the IVET selection will be extended to screening for ivi fusions whose survival is limited to a particular host tissue and thus may contain tissue-specific ivi genes, whose restricted expression will be examined by genetic analysis. These studies have practical applications for both vaccine and antimicrobial development.