HOST-SPECIFIC INDUCTION OF BACTERIAL VIRULENCE GENES

这项建议是基于一种全新的隔离方法， 细菌毒力基因参与发病机制，称为IVET（体内 表达技术），其允许细菌的阳性选择， 在宿主组织中特异性诱导的基因。 这种方法将 用于表征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。 这些研究将提供深入了解 控制致病性调节的分子机制， 对理解传染病至关重要。 遗传分析 的基因，回答IVET选择是便利的，因为基因， 与报告基因lacZ转录融合， 恢复组成型表达IVI基因的调节突变体， 体外（正常抑制条件）。 我们的努力将集中在 这些调节因子协调调节ivi基因，因此 定义了确保多方面细菌 应对多方面的东道国挑战。 反式调节 突变将用于表征阳性和阴性IVI 在体内激活IVI转录并抑制IVI的调节元件 体外转录。 这些突变体将被筛选出那些 在动物模型中赋予毒力缺陷，这表明它们或一些 它们调节的产物在发病机制中起重要作用。 顺式作用突变将被用于定义IVI启动子区域， 重点建立协调ivi基因一致位点 表情 界定协调监管的性质提供了一种手段 去理解复杂的信号传导机制， 细菌在这种不同的环境条件下生长（例如， 实验室培养基与宿主组织）。此外，IVET选择将 可以扩展到筛选存活率仅限于 特定的宿主组织并因此可能含有组织特异性IVI基因， 其限制性表达将通过遗传分析来检测。 这些 这些研究在疫苗和抗菌剂方面都有实际应用 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.