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HOST-SPECIFIC INDUCTION OF BACTERIAL VIRULENCE GENES

细菌毒力基因的宿主特异性诱导

基本信息

批准号：
2672367
负责人：
MICHAEL J MAHAN
金额：
$ 10.82万
依托单位：
UNIVERSITY OF CALIFORNIA SANTA BARBARA
依托单位国家：
美国
项目类别：
财政年份：
1994
资助国家：
美国
起止时间：
1994-08-01 至 2000-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/2672367
关键词：
Salmonella typhimurium bacterial genetics biological signal transduction gene induction /repression genetic regulation host organism interaction laboratory mouse molecular pathology nucleic acid sequence transcription factor transposon /insertion element virulence

项目摘要

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.

该提案基于一种全新的隔离方法参与发病机制的细菌毒力基因，称为IVET（体内表达技术），允许细菌的正选择在宿主组织中特异性诱导的基因。该方法将用于表征调节的遗传和环境因素人类病原体体内诱导基因（ivi）的表达，鼠伤寒沙门氏菌。此类研究将提供有关以下方面的见解：控制致病性调节的分子机制，是了解传染病的基础。遗传分析回答IVET选择的基因是有利于的，因为基因转录融合到报告基因 lacZ，从而允许恢复组成型表达 ivi 基因的调节突变体体外（通常是抑制条件）。我们的努力将集中于这些调节因子协调调节 ivi 基因，从而定义确保多方面细菌的监管成分应对多方面的主机挑战。交易监管突变将用于表征阳性和阴性 ivi 激活体内 ivi 转录并抑制 ivi 的调控元件体外转录。这些突变体将被筛选出那些在动物模型中赋予毒力缺陷，表明它们或某些它们调节的产物在发病机制中发挥着重要作用。顺式作用突变将用于定义 ivi 启动子区域强调建立协调ivi基因的共识位点表达。定义协调监管的性质提供了一种手段了解复杂的信号转导机制，使细菌在如此不同的环境条件下生长（例如，实验室介质与宿主组织）。此外，IVET 选择将扩展到筛选存活率有限的 ivi 融合体特定的宿主组织，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进行检查。这些研究对于疫苗和抗菌药物都有实际应用发展。