CA++-MEDIATED ENHANCEMENT OF SALMONELLA INVASION

我们最近证明了沙门氏菌是随着 他们体内的生理钙离子水平显著提高 体外穿透HeLa细胞的能力。HeLa中的这种刺激 细胞穿透是剂量依赖的，需要细菌 新陈代谢和RNA合成，不能用其他 二价阳离子，是处理细胞的稳定性质。在这 建议，我们将研究钙离子的作用机制 促进沙门氏菌侵袭。钙离子介导的 变化是沙门氏菌内外的表现 膜多肽和脂多糖决定簇将 经生化和凝胶分离分析后测定 电泳法。寻找钙离子应答基因 并确定它们在沙门氏菌侵袭增强中的作用，我们 将使用lambda placMU载体构建操纵子基因融合。 这些融合菌株将被用来评估细胞内 钙离子应答基因的表达。钙离子介导的增强型 其他沙门氏菌毒力产物的表达(即细胞毒素 和肠毒素)也将被检测。为了研究体内的 钙离子促进沙门氏菌入侵的意义，我们将 钙强化沙门氏菌与对照沙门氏菌引出液体的比较 积聚、产生和炎症反应，引起组织 破坏兔结扎环，并从 肠道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~(++)的响应 增强沙门氏菌。上皮细胞的抵抗力 将检查干扰素治疗后的穿透性。CA++- 巨噬细胞中介导的增强摄取或存活也将是 考虑过了。我们还将寻找新的抗原决定簇和 非淋巴样细胞侵袭后产生干扰素的能力 钙离子增强型沙门氏菌。这些研究的结果将 对加深我们对……的理解 细菌侵袭上皮细胞的机制、作用 钙离子在兼性细胞内细菌病中的作用及丰富我们的 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.