TIR domain containing protein from Brucella melitensis

描述（由申请方提供）：布鲁氏菌是一种高度传染性的细胞内病原体，在多种哺乳动物中发现，可引起流产、不育和波状热。与其他胞内病原体不同，布鲁氏菌属（Brucella spp.）不编码经典的毒性因子。然而，宿主细胞中的慢性细胞内持久性表明布鲁氏菌效应子可能与未鉴定的真核靶点相互作用，以改变细菌存活的细胞环境。关于布鲁氏菌蛋白质的信息很少，这些蛋白质有助于细菌的毒力，这表明迫切需要探索布鲁氏菌的发病机制。该提案旨在表征一种新鉴定的布鲁氏菌蛋白的贡献，称为TcpB，由BMEI 1674编码，其包含TIR结构域并模拟Toll样受体衔接蛋白TIRAP的特性。TcpB有效地阻断TLR 2和TLR 4介导的NF-？B活化并在细菌的毒力中起重要作用。我们的研究表明，TcpB与磷脂酰肌醇（PtDIns）相互作用，并与质膜和细胞骨架网络共定位。我们假设TcpB-PtDIns相互作用为TcpB提供TLR特异性，但TcpB-微管相互作用的意义，由TcpB的BB环基序介导的是未知的。TcpB介导NF-？B的抑制作用尚不清楚，也没有关于布鲁氏菌分泌Tcp B和Tcp B从布鲁氏菌空泡中逃逸的信息。基于我们的初步研究，TcpB除了抑制先天免疫应答外还发挥更广泛的作用。因此，本提案将详细描述TcpB，以阐明作用和细菌分泌的机制。本提案的具体目标是： 目标1。探讨TcpB介导NF-？B抑制。大规模的酵母双杂交筛选将分析TcpB与未鉴定的哺乳动物蛋白质的相互作用。阳性相互作用将通过共免疫沉淀和共亚细胞定位实验来证实。 目标2.分析羊种布氏杆菌分泌TcpB的情况及TcpB的膜穿透性。将在培养基和宿主细胞中分析羊种布鲁氏菌的TcpB分泌。将对浓缩的培养物上清液进行Western分析，以检测TcpB的分泌。为了分析细胞内TcpB的分泌，将使用基于肽标签（GSK标签）的报告系统过表达TcpB-GSK。为了分析细胞渗透性，将纯化的TcpB蛋白与哺乳动物细胞一起孵育，并通过蛋白质印迹法测定细胞内定位的TcpB。细胞因子ELISA将证实TcpB可以进入宿主细胞，并证明细胞内TcpB蛋白对细胞因子的抑制。 目的3：分析TcpB对微管蛋白聚合及TcpB介导的小G蛋白酶激活的影响。为了分析TcpB对微管蛋白组装动力学的影响，将在纯化的TcpB或含有介导微管相互作用的TcpB的BB环基序的肽的存在下进行基于荧光的微管蛋白聚合测定。完整微管在TcpB介导的NF-？在存在微管去稳定剂诺考达唑的情况下，通过报告基因测定分析B抑制。为了研究TcpB介导的小GTP酶的活化，将用TcpB转染的细胞裂解物进行GTP酶下拉测定。将进行吞噬测定以确定TcpB介导的膜皱褶和板状伪足突起是否增强吞噬作用。 我们的初步研究已经确定了一个B。Melitensis TIR结构域包含蛋白，称为TcpB，其具有改变宿主细胞中的信号传导事件和破坏先天免疫机制的能力。从这些特定目标中获得的关键信息将开始弥合我们对B的了解之间的差距。Melitensis毒力和毒力是如何调节的。

DESCRIPTION (provided by applicant): Brucellae are highly infectious intracellular pathogens found in a wide range of mammals causing abortion, infertility and undulant fever. Unlike other intracellular pathogens, Brucella spp. do not encode classical virulent factors. However, chronic intracellular persistence in host cells indicates Brucella effectors likely interact with unidentified eukaryotic targets to modify the cellular environment for bacterial survival. Minimal information is available on Brucella proteins that contribute to bacterial virulence indicating an immediate need to explore the mechanisms of Brucella pathogenesis. This proposal aims to characterize the contribution of a newly identified Brucella protein, termed TcpB encoded by BMEI1674 that contains a TIR domain and mimics properties of the Toll-like receptor adaptor protein TIRAP. TcpB efficiently blocks TLR2 and TLR4 mediated NF-?B activation and plays a significant role in the virulence of the bacterium. Our studies indicated that TcpB interacts with phosphatidylinositols (PtDIns) and co-localizes with the plasma membrane and cytoskeleton network. We hypothesize that TcpB-PtDIns interaction provides TLR specificity for TcpB, but the significance of TcpB-microtubule interaction, mediated by the BB-loop motif of TcpB is unknown. The exact mechanism of TcpB mediated NF-?B suppression is not yet understood, and no information is available on the secretion of TcpB by Brucella and the escape of TcpB from Brucella containing vacuoles. Based on our preliminary studies, TcpB plays a broader role in addition to the suppression of innate immune response. Therefore the present proposal will characterize TcpB in detail to unravel the mechanisms of action and bacterial secretion. The specific aims of the present proposal are: Aim 1. To explore the detailed mechanism of TcpB mediated NF-?B suppression. Large-scale yeast-two hybrid screening will analyze TcpB interaction with unidentified mammalian proteins. Positive interactions will be confirmed by co-immunuprecipitation and co-sub-cellular localization experiments. Aim 2. To analyze the secretion of TcpB by Brucella melitensis and membrane penetration of TcpB. Secretion of TcpB by Brucella.melitensis will be analyzed in culture medium as well as in host cells. Western analysis of concentrated culture supernatant will be performed to test the secretion of TcpB. To analyze the secretion of TcpB inside the cells, a peptide tag-based (GSK-tag) reporter system over expressing TcpB-GSK will be used. To analyze the cell permeability purified TcpB protein will be incubated with mammalian cells and intracellular localized TcpB will be determined by western blotting. A cytokine ELISA will confirm that TcpB can enter host cells as well as demonstrate the suppression of cytokine by intracellular TcpB protein. Aim 3: To analyze the effect of TcpB on tubulin polymerization and TcpB mediated activation of small GTPases. To analyze the effect of TcpB on kinetics of tubulin assembly, fluorescent-based tubulin polymerization assays will be performed in the presence of purified TcpB or peptide harboring the BB-loop motif of TcpB that mediates microtubule interaction. The role of intact microtubules for TcpB mediated NF-?B suppression will be analyzed by reporter assays in the presence of the microtubule destabilizing agent nocodazole. To investigate the TcpB mediated activation of small GTPases, GTPase pull-down assays will be performed with the cell lysate transfected with TcpB. Phagocytosis assays will be performed to address whether TcpB mediated membrane ruffles and lamellipodia protrusions enhances phagocytosis. Our Preliminary Studies have identified a B. melitensis TIR domain containing protein, termed TcpB that possesses the ability to alter signaling events in host cells and disrupt innate immune mechanisms. Critical information gained from these specific aims will begin to bridge the gap between what we know about B. melitensis virulence and how virulence is regulated.