PLASMID-MEDIATED VIRULENCE IN SALMONELLA

这个项目的长期目标是确定质粒的作用- 系统性非伤寒沙门氏菌致病中的介导性基因 感染。都柏林沙门氏菌的整个8.2kb基本毒力区域 已对pSDL2进行了测序，并进行了突变分析 序列中所需的基因座，现在被称为SPV基因，保守于 所有的毒力质粒。SPV基因是根据生长情况而诱导的 在体外是有限的，并且表达取决于染色体 饥饿调节基因座Katf(Rpos)，导致假设 质粒毒力位点参与了生物体的适应。 对寄主的生长限制，很可能是在细胞内 巨噬细胞的环境。在这项提案中，基本的结构 质粒毒力位点的基因将由特定的位点定义 突变。毒力基因调控的分子机制 表达将通过生化和遗传学相结合的方式来阐明 接近。SPV基因在体内的作用部位将被确定 在受感染的小鼠的脾中，体外细胞培养系统将 以反映SPV基因座在体内的活性。这个 将确定每个SPV基因的重要性和作用。以下是 提出了具体的目标：L)确定每个SPV基因在 都柏林链霉菌野生型质粒编码的毒力。非极SPV 将构建并测试完整毒力质粒中的突变 在沙门氏菌病小鼠模型中。2)明确分子机制 通过SpvR调控转录激活SPV操纵子 蛋白质，使用体外DNA结合研究和体内研究相结合的方法 基因分析。3)确定调控的分子机制 通过染色体饥饿诱导的交替Sigma. 卡特夫系数(Rpos)。4)确定质粒介导的增殖是否 都柏林沙门氏菌在脾内的感染发生在巨噬细胞内。受感染的 采集脾细胞，FACS将细胞分散并分选成 不同的种群，每个细胞的活细菌数量将是 下定决心。5)建立脾细胞体外培养体系。 巨噬细胞检测SPV基因对细胞内生长的作用 在静息和细胞因子激活的巨噬细胞中。

The long-term goal of this project is to define the role of plasmid- mediated genes in the pathogenesis of systemic non-typhoid Salmonella infections. The entire 8.2 kb essential virulence region of the S. dublin plasmid pSDL2 has been sequenced, and mutation analysis has defined required loci within the sequence, now termed the spv genes, conserved in all virulence plasmids. The spv genes are induced in response to growth limitation in vitro, and expression is dependent on the chromosomal starvation regulatory locus katF (rpoS), leading to the hypothesis that the plasmid virulence locus is involved in the adaptation of the organism to growth limitation by the host, most likely in the intracellular environment of the macrophage. In this proposal, essential structural genes of the plasmid virulence locus will be defined by site-specific mutations. The molecular mechanism for the regulation of virulence gene expression will be elucidated by a combined biochemical and genetic approach. The site of action of the spv genes in vivo will be determined in the spleen of infected mice, and an in vitro cell culture system will be developed to reflect the activity of the spv locus in vivo. The importance and role of each spv gene will be identified. The following Specific Aims are proposed: l) to define the role of each spv gene in the virulence encoded by the wild-type plasmid in S. dublin. Non-polar spv mutations in the complete virulence plasmid will be constructed and tested in the mouse model of salmonellosis. 2) to define the molecular mechanism for transcriptional activation of the spv operon by the SpvR regulatory protein, using a combination of in vitro DNA binding studies and in vivo genetic analysis. 3) to determine the molecular mechanism for regulation of the spv genes by the chromosomal starvation-induced alternative sigma- factor KatF (RpoS). 4) to establish whether plasmid-mediated proliferation of S. dublin within the spleen takes place within macrophages. Infected spleens will be harvested, the cells dispersed and sorted by FACS into different populations, and the number of viable bacteria per cell will be determined. 5) to establish an in vitro cell culture system using splenic macrophages to examine the role of the spv genes on intracellular growth in resting and cytokine-activated macrophages.