CA++-MEDIATED ENHANCEMENT OF SALMONELLA INVASION

我们最近证明，沙门氏菌生长与 生理水平的Ca++显着提高， 在体外穿透HeLa细胞的能力。 HeLa细胞中的这种刺激 细胞渗透是剂量依赖性的，需要细菌 代谢和RNA合成，不能显示与其他 二价阳离子，并且是经处理的细胞的稳定性质。 在这 我们将探讨Ca ~（++）对细胞增殖的影响机制， 介导沙门氏菌入侵增强。 Ca++介导 改变是沙门氏菌的表达内部和外部 膜多肽和脂多糖决定簇将 通过生物化学和凝胶分离和分析后确定 电泳技术 为了鉴定Ca++响应基因， 并确定它们在沙门氏菌入侵增强中的作用，我们 将使用λ placMU载体构建操纵子基因融合体。 这些融合菌株将用于评估细胞内的 Ca++反应基因的表达。 Ca++介导的增强 其他沙门氏菌毒力产物的表达（即，细胞毒素 及肠毒素）亦会进行检验。 为了研究体内 Ca++增强沙门氏菌入侵的意义，我们将 比较Ca++-增强和对照沙门氏菌： 积累，产生炎症反应，引起组织 破坏兔结扎环，并从传播 进入血液或其他器官。 我们还将确定 不同寄主防御机制对Ca ~（++-）的反应 强化沙门氏菌 上皮细胞抵抗 将检查IFN处理后的渗透。 钙离子 巨噬细胞中介导的增强的摄取或存活也将是 考虑了 我们还将寻找新的抗原决定簇， 非淋巴样细胞在受侵袭后产生IFN的能力 CA++-增强沙门氏菌。 这些研究的结果将 为我们进一步了解 细菌侵入上皮细胞的机制， Ca++在兼性细胞内细菌疾病中的作用， 了解细菌宿主细胞生理学。

We have recently demonstrated that Salmonella grown with physiological levels of Ca++ are significantly enhanced in their ability to penetrate HeLa cells in vitro. This stimulation in HeLa cell penetration was dose dependent, required bacterial metabolism and RNA synthesis, could not be shown with other divalent cations and was a stable property of treated cells. In this proposal, we will investigate the mechanism by whch Ca++ mediates Salmonella invasion enhancement. Ca++-mediated alterations is the expression of Salmonella inner and outer membrane polypeptides and lipopolysaccharide determinants will be determined after isolation and analyses by biochemical and gel electrophoretic techniques. To identify Ca++-responsive genes and determine their role in Salmonella invasion enhancement, we will construct operon gene fusions using lambda placMU vectors. These fusion strains will be used to evaluate the intracellular expression of Ca++-responsive genes. Ca++-mediated enhanced expression of other Salmonella virulence products (ie. cytotoxin and enterotoxin) will also be examined. To investigate the in vivo significance of Ca++-enhanced Salmonella invasion, we will compare Ca++-enhanced and control Salmonella: to elicit fluid accumulation, produce and inflammatory reaction, cause tissue destruction in rabbit ligated loops, and to disseminate from the intestine into the blood or other organs. We will also determine the response of different host defense mechanisms to Ca++- enhanced Salmonella. The ability of epithelial cells to resist penetration after IFN treatment will be examined. Ca++- mediated enhanced uptake or survival in macrophages will also be considered. We will also look for new antigenic determinants and the ability of nonlymphoid cells to produce IFN after invasion by CA++-enhanced Salmonella. Findings from these studies will significantly contribute toward furthering our understanding of the mechanism of bacterial invasion in epithelial cells, the role of Ca++ in facultative intracellular bacterial disease and enrich our understanding of bacterial host cell physiology.