Functional Analysis of Cu/Zn Superoxide Dismutase

描述（由申请方提供）：沙门氏菌每年在美国引起140万例胃肠炎和肠热病，并导致所有其他食源性细菌病原体死亡。最严重的疾病是由S.鼠伤寒杆菌在吞噬细胞中存活，吞噬细胞通常通过产生包括超氧化物（O2-）在内的多种抗菌剂来杀死细菌。吞噬性O2-损伤细菌细胞的机制是完全未知的。S.鼠伤寒沙门氏菌14028株具有两种周质超氧化物歧化酶。 SodCI，在Gifsy-2噬菌体上编码，通过保护免受吞噬性02-而显著地有助于毒力。染色体编码的SodCII，我们已经表明，在感染过程中表达，不有助于生存，即使在没有SodCI。这两种酶在体外的行为相似，但值得注意的是，SodCI不会通过渗透压休克释放，这种现象我们称之为“束缚”。“这是周质蛋白的一个新特性。我们推测，SodCII的产生，但无功能，可能是由于在巨噬细胞吞噬体的蛋白水解降解，和SodCI与一些周质组分的物理或空间关联占其优先作用的毒力。这项建议的具体目标是：1.确定SodCI和SodCII在吞噬体中的命运。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，使其能够防止吞噬超氧化物。我们将利用SodCI和SodCII的不同活性。杂交蛋白质将被构建和表征。还将测试来自其他病原性细菌和特定位点定向突变体的SodC补充SodCI的能力。对抗吞噬超氧化物的能力将与其他特征相关，包括通过渗透压休克释放。3.确定在体内是否需要与周质的连接来发挥功能，并了解连接的生化性质。不再拴系的突变体的遗传筛选以及SodCI在周质中相互作用的生物化学鉴定将导致对拴系及其在SodCI功能中的作用的理解。这项研究解决了先天免疫中的一个基本问题，并对对抗各种重要病原体有影响。

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.