Mechanisms of Impaired Mycobacteria Sensing by TLR2 and TLR4 Polymorphic Variants

描述(申请人提供)：结核分枝杆菌(Mtb)感染了超过三分之一的人口，每年导致800万新肺结核病例，全球约250万人死亡。结核病与艾滋病毒的致命联系以及多重耐药菌株的出现，加剧了全球结核病的负担，导致世界卫生组织将结核病宣布为“全球卫生紧急情况”。新药、疫苗和更好的治疗方法的设计需要确定控制免疫反应的分子机制及其对分枝杆菌感染的调节。Toll样受体(Toll-like Receptor，TLR)2和TLR4是分枝杆菌重要的感受器，在激活天然宿主反应中发挥关键作用，这一点从缺乏这些受体的小鼠对分枝杆菌感染的易感性增加和细胞因子的产生减少可见一斑。在人类中，TLR2(例如R753Q)和TLR4(例如D299G)单核苷酸多态(SNPs)与结核分支杆菌感染和/或严重程度相关。然而，这些SNP影响受体信号转导的分子机制尚不清楚。根据我们的初步数据，我们提出了一个假设，即该基因的多态影响分枝杆菌的识别、TLR信号体的组装和激活激酶和转录因子、改变炎症介质的表达、吞噬和体内感染的易感性。这一假设将在以下具体目标中进行检验：1.定义 TLR2/4 SNPs对TLR的表达、定位、信号小体组装以及对分枝杆菌应答的关键激酶和转录因子的激活的影响；2.确定TLR2/4 SNPs对炎症介质的表达、分枝杆菌吞噬作用和细胞内存活的影响；以及3.确定TLR2/4 SNPs对表达人类突变同源基因的转基因小鼠体内分枝杆菌感染易感性的影响。我们的研究有望确定SNPs影响分枝杆菌识别、TLR2/4信号体组装、激酶和转录因子激活的机制，并将这些变化与下游对宿主防御分枝杆菌至关重要的反应联系起来，如炎性细胞因子的表达和吞噬作用。我们将设计新的转基因小鼠，表达人类R753Q TLR2或D299G TLR4 SNPs的同源基因，以确定这些突变对分枝杆菌感染的易感性和严重性的影响。阐明TLR2/4 SNPs抑制宿主对分枝杆菌的抵抗力的分子基础，以及鉴定功能受损的中间产物，将为开发新的方法和药物来改善TLR介导的Mtb传感和宿主防御铺平道路。这些进展将对改善美国结核病患者的公共健康至关重要。

DESCRIPTION (provided by applicant): Mycobacterium tuberculosis (Mtb) infects over one-third of the human population, causing 8 million new cases of pulmonary tuberculosis (TB) each year and ~2.5 million deaths worldwide. The global burden of tuberculosis has been compounded by its deadly association with HIV and by the emergence of multi-drug resistant strains, leading the World Health Organization to declare TB as "a global health emergency". The design of new drugs, vaccines and better therapeutics requires identification of the molecular mechanisms governing immune responses and their regulation upon mycobacterial infection. Toll-like receptors (TLR) 2 and TLR4 are mycobacterial important sensors that play a key role in activating innate host responses, as evidenced by an increased susceptibility to mycobacterial infections and impaired cytokine production in mice deficient for these receptors. In humans, the TLR2 (e.g., R753Q) and TLR4 (e.g., D299G) single nucleotide polymorphisms (SNPs) have been correlated with incidence and/or severity of Mtb infections and TB. However, the molecular mechanisms by which such SNPs affect receptor signaling are unknown. Based on our preliminary data, we formulated the hypothesis that the polymorphisms affect recognition of mycobacteria, TLR signalosome assembly and activation of kinases and transcription factors, altering expression of inflammatory mediators, phagocytosis and predisposing to infection in vivo. This hypothesis will be tested in the following Specific Aims to: 1. Define the impact of the TLR2/4 SNPs on TLR expression, localization, signalosome assembly and activation of key kinases and transcription factors in response to mycobacteria; 2. Determine the effect of the TLR2/4 SNPs on expression of inflammatory mediators, mycobacterial phagocytosis and intracellular survival; and 3. Identify the consequences of TLR2/4 SNPs on susceptibility to mycobacterial infections in vivo in transgenic mice expressing orthologs of human mutations. Our studies are expected to identify mechanisms by which the SNPs affect mycobacterial recognition, TLR2/4 signalosome assembly, activation of kinases and transcription factors, and to link these changes to downstream responses important for host defense against mycobacteria, such as expression of inflammatory cytokines and phagocytosis. We will engineer new transgenic mice expressing orthologs of human R753Q TLR2 or D299G TLR4 SNPs to identify the consequences of these mutations on susceptibility and severity of mycobacterial infections. Elucidation of the molecular basis by which TLR2/4 SNPs compromise host defense against mycobacteria and identification of intermediates with compromised functions will pave the way for the development of new approaches and drugs to improve TLR-mediated Mtb sensing and host defense. These advances would be of key importance for improving public health in tuberculosis patients in the U.S.