Functional Analysis of Cu/Zn Superoxide Dismutase

描述（由申请人提供）：沙门氏菌在美国每年导致140万例肠胃炎和肠热病例，并且是导致死亡的所有其他食源性细菌病原体的主要原因。最严重的疾病是由鼠伤寒沙门氏菌在吞噬细胞中存活引起的，吞噬细胞通常通过产生包括超氧化物（O2-）在内的各种抗菌剂来杀死细菌。吞噬细胞02-破坏细菌细胞的机制是完全未知的。鼠伤寒沙门氏菌菌株14028具有两个质周超氧化物歧化酶。在Gifsy-2噬菌体上编码的SodCI通过保护抗吞噬细胞02-而对毒力有重要贡献。染色体编码的SodCII，我们已经证明在感染期间表达，即使在没有SodCI的情况下，也不会促进生存。这两种酶在体外的表现相似，但有一个明显的例外，即SodCI不会通过渗透休克释放，我们将这种现象称为“栓系”。这是质周蛋白的新特性。我们假设SodCII是产生的，但没有功能，可能是由于巨噬细胞吞噬体中的蛋白质水解降解，并且SodCI与一些质周成分的物理或空间关联解释了它在毒力中的优先作用。本建议的具体目的是：1。确定吞噬体中SodCI和SodCII的命运。我们将同时监测动物感染期间以及组织培养巨噬细胞中SodCI和SodCII蛋白的产生。2. 确定SodCI的结构和功能特征，使其能够抵抗吞噬性超氧化物。我们将利用SodCI和SodCII的活性差异。杂交蛋白将被构建和表征。来自其他致病菌和特定位点定向突变体的sodc也将测试其补充SodCI的能力。抗吞噬超氧化物的能力将与其他特性相关，包括通过渗透休克释放。3. 确定在体内的功能是否需要黏附于外周质，并了解黏附的生化性质。对不再系留的突变体进行基因筛选，以及对外质中SodCI与什么相互作用进行生化鉴定，将有助于了解系留及其在SodCI功能中的作用。这项研究解决了先天免疫的一个基本问题，并对对抗各种重要病原体具有重要意义。

DESCRIPTION (provided by applicant): Salmonella cause 1.4 million cases of gastroenteritis and enteric fever per year in the US and lead all other foodborne bacterial pathogens as a cause of death. The most serious disease results from S. typhimurium survival in phagocytes, which normally kill bacteria by producing a variety of antimicrobials including superoxide (O2-). The mechanism by which phagocytic 02- damages bacterial cells is completely unknown. S. typhimurium strain 14028 possesses two periplasmic superoxide dismutases. SodCI, encoded on the Gifsy-2 phage, contributes significantly to virulence by protecting against phagocytic 02-. The chromosomally encoded SodCII, which we have shown is expressed during infection, does not contribute to survival, even in the absence of SodCI. The two enzymes behave similarly in vitro with the notable exception that SodCI is not released by osmotic shock, a phenomenon we term "tethering." This is a novel property for a periplasmic protein. We hypothesize that SodCII is produced but is non-functional, probably due to proteolytic degradation in the macrophage phagosome, and that the physical or spatial association of SodCI with some periplasmic component accounts for its preferential role in virulence. The specific aims of this proposal are to: 1. Determine the fate of SodCI and SodCII in the phagosome. We will simultaneously monitor the production of both SodCI and SodCII protein during infection in an animal as well as in tissue culture macrophages. 2. Determine the structural and functional characteristics of SodCI that allow it to protect against phagocytic superoxide. We will exploit the differential activity of SodCI and SodCII. Hybrid proteins will be constructed and characterized. SodCs from other pathogenic bacteria and specific site directed mutants will also be tested for the ability to complement SodCI. The ability to combat phagocytic superoxide will be correlated with other characteristics including release by osmotic shock. 3. Determine if tethering to the periplasm is required for function in vivo and understand the biochemical nature of tethering. A genetic screen for mutants that are no longer tethered as well as a biochemical identification of what SodCI interacts with in the periplasm will lead to an understanding of tethering and its role in SodCI function. This research addresses a fundamental issue in innate immunity and has implications for combating a variety of important pathogens.