Modification of actin by bacterial protein toxins and effectors

各种细菌蛋白毒素和效应物直接修饰真核靶细胞的肌动蛋白。最为人所知的是由二元ADP-核糖基化蛋白毒素家族的成员对G-肌动蛋白的修饰。该毒素家族的原型是肉毒梭菌C2毒素。这些毒素在Arg 177处ADP-核糖基化G-肌动蛋白。修饰（i）抑制肌动蛋白聚合和（ii）将肌动蛋白转变成加帽蛋白，其抑制未修饰的肌动蛋白的聚合。最近，据报道，来自肠道沙门氏菌的SpvB蛋白引起靶细胞的细胞骨架的重新分布，并且具有导致肌动蛋白修饰的ADP-核糖基转移酶活性。与二元肌动蛋白-ADP-核糖基化毒素相反，SpvB蛋白通过III型分泌从病原体直接递送到宿主细胞中。本提案的主要目的是在分子和细胞水平上阐明导致肌动蛋白细胞骨架重排的SpvB的细胞毒性作用和作用模式。为了实现这一目标，我们提出1）详细表征SpvB诱导的肌动蛋白的ADP-核糖基化，包括胞质因子对SpvB的ADP-核糖基转移酶活性的调节，2）鉴定SpvB对肌动蛋白的修饰，和3）确定SpvB介导的肌动蛋白修饰对肌动蛋白成核、聚合、以及与各种肌动蛋白结合蛋白的相互作用。

Various bacterial protein toxins and effectors directly modify actin of eukaryotic target cells. Best known is the modification of G-actin by members of the family of binary ADP-ribosylating protein toxins. The prototype of this toxin family is Clostridium botulinum C2 toxin. These toxins ADP-ribosylate G-actin at Arg177. The modification (i) inhibits actin polymerization and (ii) turns actin into a capping protein, which inhibits the polymerization of unmodified actin. Recently, it was reported that the SpvB protein from Salmonella enterica causes redistribution of the cytoskeleton of target cells, and possesses ADP-ribosyltransferase activity that results in modification of actin. In contrast to binary actin-ADP-ribosylating toxins, the SpvB protein is directly delivered from the pathogen into host cells via type-III-secretion. The main objective of this proposal is to elucidate at the molecular and cellular level the cytotoxic effects and mode of action of SpvB that results in the rearrangement of the actin cytoskeleton. To achieve this aim, we propose 1) to characterize in detail the SpvB-induced ADP-ribosylation of actin, including regulation of the ADP-ribosyltransferase activity of the of SpvB by cytosolic factors, 2) to identify the modification of actin by SpvB, and 3) to determine the functional consequences of the SpvB-mediated actin modification on the kinetics of actin nucleation, polymerization, and interaction with various actin-binding proteins, using biochemical methods and imaging techniques and fluorescence microcopy in intact cells.