Molecular analysis of actin-based motility of Burkholderia spp.

假麦氏伯克氏菌是一种引起类鼻疽病的细菌病原体，类鼻疽病是一种在亚热带地区流行的动物和人类的严重和新出现的疾病。与李斯特菌属、志贺氏菌属、立克次体属和分枝杆菌属的一些成员一样，假假杆菌进化出了一种非凡的能力，可以进入宿主细胞，并通过劫持组装肌动蛋白（细胞骨架的关键结构成分）的细胞机制，在细胞内部和细胞之间推进自己。在迄今为止研究的所有案例中，这些生物体通过肌动蛋白“火箭”向邻近细胞发射自己的能力是导致疾病的必要条件，可能是因为它有助于细菌在逃避宿主免疫监视的同时进行细胞间传播。李斯特菌和志贺氏菌肌动蛋白依赖运动的分子基础已经得到了广泛的研究，部分原因是为了了解生物体如何引起疾病，但也因为它提供了对细胞内肌动蛋白组装如何被精心安排和调节的见解。肌动蛋白是我们细胞中最丰富的蛋白质之一，对肌动蛋白组装的控制对许多过程至关重要，包括胚胎发育过程中的细胞迁移、伤口愈合和侵袭性癌症，以及免疫细胞抵抗感染的运动和能力。在BBSRC的资助下，我们最近发现了一种细菌因子，该细菌因子是假芽孢杆菌（BimA）基于肌动蛋白的运动所必需的，以及相关伯克霍尔德氏菌物种中类似的功能蛋白。虽然我们已经确定这些因子结合肌动蛋白并促进其在试管和宿主细胞内的组装，但它们这样做的机制尚不清楚。在本建议中，我们力求：确定肌动蛋白结合和组装所需的BimA蛋白区域。2. 确定BimA是否通过添加磷酸盐和/或糖基团发生在细胞内，并研究其功能后果。3. 检查BimA在细菌表面组装的复合体以及其中发生的蛋白质-蛋白质相互作用。4. 检测假芽孢杆菌诱导的细胞内肌动蛋白尾部的宿主细胞蛋白并确定其功能相关性。5. 确定来自不同伯克霍尔德菌种的BimA蛋白是否促进不同成分复合物的形成。综上所述，我们相信这些实验将解释关键微生物毒力因子的作用模式，并产生关于肌动蛋白动力学在我们细胞中控制的机制的重要新知识。

Burkholderia pseudomallei is a bacterial pathogen that causes melioidosis, a severe and emerging disease of animals and humans that is endemic in the subtropics. In common with some members of the genera Listeria, Shigella, Rickettsia and Mycobacterium, B. pseudomallei has evolved the remarkable ability to enter host cells and propel itself within and between cells by hijacking the cellular machinery that assembles actin, a key structural component of the cell skeleton. In all cases so far examined, the ability of these organisms to fire themselves into adjacent cells on actin 'rockets' is required to cause disease, presumably as it aids cell-to-cell spread of the bacteria while evading host immune surveillance. The molecular basis of the actin-dependent movement of Listeria and Shigella have been extensively studied, partly to understand how the organisms cause disease, but also because it provides insights into how actin assembly is orchestrated and regulated inside cells. Actin is one of the most abundant proteins in our cells and control of actin assembly is essential for a multitude of processes, including cell migration during embryo development, wound healing and invasive cancer, as well as the movement and ability of immune cells to fight infection. With BBSRC funding we recently identified a bacterial factor that is required for actin-based motility of B. pseudomallei (BimA), as well as similar functional proteins in related Burkholderia species. Though we have established that these factors bind actin and promote its assembly in the test tube and inside host cells, the mechanism by which they do this is not understood. In this proposal we seek to: 1. Identify regions of the BimA protein that are required for actin binding and assembly. 2. Determine whether modifications of BimA by addition of phosphate and/or sugar groups occur inside cells, and investigate their functional consequences. 3. Examine the complex assembled by BimA at the bacterial surface and the protein-protein interactions that occur within it. 4. Detect host cell proteins in B. pseudomallei-induced actin tails inside cells and determine their functional relevance. 5. Determine if BimA proteins from different Burkholderia species promote the formation of complexes with differing compositions. Taken together, we believe that these experiments will explain the mode of action of key microbial virulence factors and produce important new knowledge on the mechanisms by which actin dynamics are controlled in our cells.