PLASMID-MEDIATED VIRULENCE IN SALMONELLA

该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基因 感染. 该菌株的8.2kb的基本毒力区是完整的。都柏林 质粒pSDL 2已测序，突变分析已确定 序列中所需的基因座，现在称为spv基因，在 所有的毒力质粒。 spv基因在生长的反应中被诱导 限制在体外，表达依赖于染色体 饥饿调节位点katF（rpoS），导致假设， 质粒毒力基因座参与生物体的适应 宿主的生长限制，最有可能在细胞内 巨噬细胞的环境。 在这一建议中，基本结构 质粒毒力基因座的基因将通过位点特异性 突变。 毒力基因调控的分子机制 表达将通过生物化学和遗传学的结合来阐明。 approach.将确定spv基因在体内的作用位点 在感染小鼠的脾脏中，体外细胞培养系统将 以反映体内SPV基因座的活性。 的 将鉴定每个SPV基因的重要性和作用。 以下 具体的目的是：1）确定每个spv基因在 在S.都柏林 非极性spv 将构建并检测完整毒力质粒中的突变 在沙门氏菌病的小鼠模型中。 2)来定义分子机制 对于通过SpvR调节基因转录激活spv操纵子， 蛋白质，使用体外DNA结合研究和体内 遗传分析 3)以确定调控的分子机制 的spv基因的染色体饥饿诱导的交替西格玛， 因子KatF（RpoS）。4)以确定质粒介导的增殖 色葡萄脾脏内的都柏林发生在巨噬细胞内。 感染 收获脾，将细胞分散并通过FACS分选成 不同的群体，每个细胞的活细菌数量将是 测定 5)建立脾细胞体外培养体系， 巨噬细胞，以检查SPV基因对细胞内生长的作用 在静息和精氨酸激活的巨噬细胞中。

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.