Newton Patrice, Latomanski Eleanor A, Newton Hayley J
Department of Microbiology and Immunology, Peter Doherty Institute for Infection and Immunity, University of Melbourne.
Department of Microbiology and Immunology, Peter Doherty Institute for Infection and Immunity, University of Melbourne;
J Vis Exp. 2016 Jul 6(113):54210. doi: 10.3791/54210.
Coxiella burnetii, the causative agent of Q fever, is an intracellular pathogen that relies on a Type IV Dot/Icm Secretion System to establish a replicative niche. A cohort of effectors are translocated through this system into the host cell to manipulate host processes and allow the establishment of a unique lysosome-derived vacuole for replication. The method presented here involves the combination of two well-established techniques: specific gene silencing using siRNA and measurement of effector translocation using a FRET-based substrate that relies on β-lactamase activity. Applying these two approaches, we can begin to understand the role of host factors in bacterial secretion system function and effector translocation. In this study we examined the role of Rab5A and Rab7A, both important regulators of the endocytic trafficking pathway. We demonstrate that silencing the expression of either protein results in a decrease in effector translocation efficiency. These methods can be easily modified to examine other intracellular and extracellular pathogens that also utilize secretion systems. In this way, a global picture of host factors involved in bacterial effector translocation may be revealed.
伯氏考克斯体是Q热的病原体，它是一种细胞内病原体，依赖IV型Dot/Icm分泌系统来建立复制龛。一组效应蛋白通过该系统转运到宿主细胞中，以操纵宿主过程，并为复制建立一个独特的源自溶酶体的液泡。这里介绍的方法涉及两种成熟技术的结合：使用小干扰RNA（siRNA）进行特定基因沉默，以及使用基于荧光共振能量转移（FRET）的底物测量效应蛋白转运，该底物依赖于β-内酰胺酶活性。应用这两种方法，我们可以开始了解宿主因子在细菌分泌系统功能和效应蛋白转运中的作用。在本研究中，我们研究了Rab5A和Rab7A的作用，它们都是内吞运输途径的重要调节因子。我们证明，沉默这两种蛋白中任何一种的表达都会导致效应蛋白转运效率降低。这些方法可以很容易地进行修改，以检测其他也利用分泌系统的细胞内和细胞外病原体。通过这种方式，可能揭示参与细菌效应蛋白转运的宿主因子的全貌。
