Functional Analysis of Cu/Zn Superoxide Dismutase

描述(申请人提供)：沙门氏菌在美国每年导致140万例胃肠炎和肠热病，并导致所有其他食源性细菌致死。最严重的疾病是鼠伤寒沙门氏菌在吞噬细胞中存活，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完全未知。鼠伤寒沙门氏菌14028菌株具有两种周质超氧化物歧化酶。编码在Gifsy-2噬菌体上的SodCI通过保护吞噬细胞02-而显著促进毒力。我们已经证明，染色体编码的SodCII在感染过程中表达，即使在没有SodCI的情况下，也不会对生存做出贡献。这两种酶在体外的表现相似，但值得注意的不同之处在于，SodCI不是通过渗透休克释放的，这种现象我们称之为“系留”。对于周质蛋白来说，这是一个新的性质。我们推测，SodCII是产生的，但不起作用，可能是由于巨噬细胞吞噬小体中的蛋白降解所致，并且SodCI与某些周质组分的物理或空间联系解释了其在毒力中的优先作用。这项建议的具体目的是：1.确定SodCI和SodCII在噬菌体中的命运。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，使其能够保护其免受吞噬细胞超氧化物的伤害。我们将利用SodCI和SodCII的不同活性。杂交蛋白将被构建和鉴定。来自其他病原菌和特定定点突变体的SoDC也将接受补充SodCI的能力测试。对抗吞噬细胞超氧化物的能力将与其他特征相关，包括通过渗透休克释放。3.确定是否需要系留到周质才能在体内发挥作用，并了解系留的生化性质。对不再拴系的突变体进行遗传筛选，以及对SodCI周质中与之相互作用的内容进行生化鉴定，将有助于理解拴系及其在SodCI功能中的作用。这项研究解决了先天免疫中的一个基本问题，并对抗击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.