ACTA AND THE ACTIN BASED MOBILITY OF LISTERIA

单核细胞增生李斯特菌是一种食源性人类和动物病原体， 导致孕妇严重疾病和免疫功能低下 包括艾滋病患者。 毒力的一个关键决定因素 这种细胞内的病原体利用宿主细胞的能力 肌动蛋白细胞骨架用于细胞内运动和细胞间扩散。 本提案的长期目标是确定L。 单核细胞增多症表面蛋白ActA启动肌动蛋白的积累 细菌周围的纤维，引导这些聚合物 肌动蛋白丝转化为生产力，运动力，并决定了速度 纤维的伸长和细菌运动的速度。 为此，我将首先分离错义等位基因和显性阴性 actA的等位基因，并在体内分析所得的突变蛋白， 在体外，它们能够使肌动蛋白成核，产生定向 运动，并与宿主蛋白质相互作用。 第二，我将测试 假设需要ActA的临界浓度， 通过改变细菌在宿主细胞内的水平， 一种表达。 最后，因为最近的生物化学和遗传学 有证据表明ActA形成同源二聚体，我将分离actA 在二聚化中特异性缺陷的突变体。这些突变体 将用于确定ActA是否需要形成二聚体 功能，如果是这样，对于ActA功能的哪个方面。

Listeria monocytogenes is a food-borne human and animal pathogen that causes serious illness in pregnant women and immunocompromised individuals, including AIDS patients. A key determinant of virulence for this intracellular pathogen is its ability to exploit the host cell actin cytoskeleton for intracellular movement and intercellular spread. The long-term objective of this proposal is to determine how the L. monocytogenes surface protein ActA initiates the accumulation of actin filaments around the bacterium, channels the polymerization of these actin filaments into a productive, motile force, and determines the rate of filament elongation and consequently the rate of bacterial movement. To this end, I will first isolate missense alleles and dominant negative alleles of actA and analyze the resulting mutant proteins in vivo and in vitro for their ability to nucleate actin, generate directional movement, and interact with host proteins. Second, I will test the hypothesis that a critical concentration of ActA is needed for the bacteria to initiate movement within host cells by altering the levels of actA expression. And finally, because recent biochemical and genetic evidence indicate that ActA forms homodimers, I will isolate actA mutants that are specifically defective in dimerization. These mutants will be used to determine whether dimer formation is necessary for ActA function, and if so, for what aspect of ActA function.