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。最著名的是二元ADP-核糖化蛋白毒素家族成员对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ADP-核糖化G-肌动蛋白位于Arg177。该修饰(I)抑制肌动蛋白聚合，以及(Ii)将肌动蛋白转变为覆盖蛋白，从而抑制未修饰肌动蛋白的聚合。最近有报道，肠沙门氏菌SpvB蛋白引起靶细胞细胞骨架的重新分布，并具有ADP-核糖基转移酶活性，从而导致肌动蛋白的修饰。与肌动蛋白-ADP-核糖化毒素不同，SpvB蛋白通过III型分泌直接从病原体输送到宿主细胞。这项建议的主要目的是在分子和细胞水平上阐明SpvB导致肌动蛋白细胞骨架重排的细胞毒作用和作用方式。为了实现这一目标，我们建议1)详细表征SpvB诱导的肌动蛋白ADP-核糖化，包括胞液因子对肌动蛋白ADP-核糖基转移酶活性的调节，2)鉴定SpvB对肌动蛋白的修饰，以及3)利用生化方法和成像技术以及完整细胞中的荧光显微镜，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相互作用的功能影响。

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.