ACTA AND THE ACTIN BASED MOBILITY OF LISTERIA

单核细胞增多性李斯特菌是一种食源性人类和动物病原体， 导致孕妇患重病，免疫功能受损 个人，包括艾滋病患者。毒力的关键决定因素 对于这种细胞内病原体来说，是它利用宿主细胞的能力 细胞内运动和细胞间扩散的肌动蛋白细胞骨架。 这项提案的长期目标是确定L. 单核细胞增多性表面蛋白ActA启动肌动蛋白的积累 细菌周围的细丝，引导这些聚合 肌动蛋白细丝转化为一种生产性、能动性的力量，并决定着 细丝伸长的速度，因此细菌移动的速度。 为此，我将首先分离错义等位基因和显性负值 ActA的等位基因，并分析体内产生的突变蛋白和 在体外研究它们成核肌动蛋白的能力，产生定向 运动，并与宿主蛋白相互作用。第二，我将测试 假设ACTA需要一个临界浓度才能 细菌通过改变水平在宿主细胞内启动运动 Acta Expression的。最后，因为最近的生化和基因 有证据表明Acta形成同源二聚体，我将分离Acta 在二聚化过程中有明显缺陷的突变体。这些变种人 将用于确定ActA是否需要形成二聚体 函数，如果是，Acta函数的哪个方面。

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.