M. tuberculosis lipoprotein-TLR2 interactions

我们的目标是确定结核分枝杆菌结合的生化和结构基础 (MTB)脂蛋白到Toll样受体(TLR)-2以及由此产生的激动剂活性。结核分枝杆菌的TLR2识别 脂蛋白启动先天免疫，影响对结核分枝杆菌的获得性免疫。尽管这一关键角色 在结核病中，TLR2识别结核分枝杆菌脂蛋白的结构基础仍然很差 明白了。此外，TLR2还具有识别其他致病物种的功能，但其结构 TLR2激动剂活性的决定因素在很大程度上尚不清楚。已知脂蛋白的酰基结构 影响TLR2的识别，但蛋白质结构对TLR2结合的影响 未知。我们鉴定了三种不同的MTB脂蛋白，它们通过TLR2：LpqH(19-kDa)发出信号 脂蛋白)、LprG和LprA。这些脂蛋白都是TLR2激动剂，但在效力和表观上有所不同 它们活动的结构性决定因素。我们的数据表明，结核分枝杆菌的脂肪和蛋白质成分 脂蛋白可以影响TLR2激动剂的活性。我们正在构建重组标记脂蛋白和 可溶性TLR2融合蛋白在分析TLR2与配体相互作用中的结构与功能关系 来自MTB的这些具有重要病理生理意义的TLR2激动剂。目标1将使用细胞细胞因子分泌 His标记的重组结核分枝杆菌脂蛋白活性及其受体依赖性研究读数 (在与TLR2的异源二聚体中使用TLR1或TLR6作为共同受体，以及使用辅助受体， CD14和CD36)。目标2将确定影响结核分枝杆菌脂蛋白相互作用的结构特征 巨噬细胞和树突状细胞表达TLR2、TLR1、TLR6及其辅助受体(CD14和CD36) 来自三种受体基因缺陷的小鼠及未检测到的结核分枝杆菌脂蛋白变异的分析 酰化和/或带有蛋白质序列中的缺失、截断或突变(或使用最低活性 表达为重组蛋白或合成多肽的构建体)。AIM 3将使用直接 生化结合实验研究标记的重组可溶性TLR与脂蛋白分子的结合。 我们将测量不同结核分枝杆菌脂蛋白及其结构变体对TLR2的亲和力，以进一步 了解激动剂与TLR2结合的结构决定因素。总体而言，我们将确定结构 结核分枝杆菌脂蛋白与TLR2结合的基础，包括脂质和蛋白质组分的贡献。 相关性：这些研究将为TLR2的机制提供独特和新颖的见解 识别MTB。TLR2是参与识别结核分枝杆菌的关键免疫系统受体。更大 了解它的功能将有助于揭示导致宿主抗性的重要免疫机制 和/或在结核杆菌慢性感染期间逃避免疫。这可能有助于开发更好的治疗方法 肺结核。它还可以帮助设计更好的免疫佐剂，用于广泛的治疗用途。

Our goal is to determine the biochemical and structural basis for binding of Mycobacterium tuberculosis (MTB) lipoproteins to Toll-like receptor (TLR)-2 and resulting agonist activity. TLR2 recognition of MTB lipoproteins initiates innate immunity and influences adaptive immunity to MTB. Despite this critical role for TLR2 in tuberculosis, the structural basis for TLR2 recognition of MTB lipoproteins remains poorly understood. In addition, TLR2 functions in recognition of other pathogenic species, yet the structural determinants of TLR2 agonist activity are largely unexplored. It is known that acyl structures of lipoproteins influence their recognition by TLR2, but the influence of protein structures on TLR2 binding is essentially unknown. We have characterized three distinct MTB lipoproteins that signal through TLR2: LpqH (19-kDa lipoprotein), LprG and LprA. These lipoproteins are all TLR2 agonists but differ in potency and apparent structural determinants of their activity. Our data indicate that both lipid and protein components of MTB lipoproteins can influence TLR2 agonist activity. We are constructing recombinant tagged lipoproteins and soluble TLR2 fusion proteins to dissect structure-function relationships in TLR2-ligand interactions relevant to these pathophysiologically important TLR2 agonists from MTB. Aim 1 will use cellular cytokine secretion readouts to study the activity of His-tagged recombinant MTB lipoproteins and their receptor dependence (use of TLR1 or TLR6 as co-receptors in heterodimers with TLR2, as well as use of accessory receptors, CD14 and CD36). Aim 2 will determine structural features of MTB lipoproteins that affect interations with TLR2, TLR1, TLR6 and accessory receptors (CD14 and CD36) by use of macrophages and dendritic cells from mice that are genetically deficient in there receptors and analyses of MTB lipoprotein variantswithout acylation and/or with deletions, truncations or mutations in the protein sequence (or use of minimal active constructs expressed as recombinant proteins or made as synthetic peptides). Aim 3 will use direct biochemical binding assays to study binding of tagged recombinant soluble TLR and lipoprotein molecules. We will measure the affinities of different MTB lipoproteins and structural variants thereof for TLR2 to further understand the structural determinants of agonist binding to TLR2. Overall we will determine the structural basis for binding of MTB lipoproteins to TLR2, including contributions of lipid and protein components. RELEVANCE: These studies will provide unique and novel insights into the mechanisms by which TLR2 recognizes MTB. TLR2 is a key immune system receptor involved in recognition of MTB. Greater understanding of its function will help reveal important mechanisms in immunity that lead to host resistance and/or evasion of immunity during chronic infection by MTB. This may help develop better treatments for tuberculosis. It may also aid in design of better immune adjuvants for a wide array of therapeutic uses.