Modulation of Inflammasome Activation by Yersinia

描述(申请人提供)：这个项目的总体目标是了解先天免疫系统如何感知和响应细菌病原体的分子机制，以及细菌病原体为避免免疫检测而使用的逃避策略。特别是，我们以假结核耶尔森菌为模型，它表达的许多毒力因子与其近亲和鼠疫耶尔森菌相同，以了解宿主-病原体的界面，并发现宿主对革兰氏阴性细菌的天然免疫反应的共同特征。革兰氏阴性菌多药耐药性的快速扩大增加了开发新的抗菌治疗方法的重要性，了解先天性免疫系统最初是如何检测细菌病原体和相应的病原体逃避策略的有望识别新的抗菌素。III型分泌系统(T3SS)是一种广泛保守的毒力决定因子，对从大肠杆菌到鼠疫杆菌的许多革兰氏阴性细菌的毒力是必不可少的，它向宿主细胞注入毒力因子，扰乱细胞信号通路。然而，T3SS的活性也可以被NLR家族的胞浆模式识别受体感知并诱导宿主免疫反应。以往的研究表明，假结核杆菌和鼠疫杆菌T3SS可诱导依赖NLRP3的炎性小体的激活，导致细胞死亡和caspase-1依赖的细胞因子分泌。此外，研究表明，耶尔森氏菌的基本毒力因子YopK，它调节T3SS的成孔活性，并限制其他Yop效应蛋白的易位，防止炎性小体激活。炎症体激活在宿主防御耶尔森氏菌中发挥着重要作用，因为YopK缺陷细菌的毒力缺陷在缺乏炎症体成分的小鼠身上得到恢复。然而，细胞如何感知耶尔森氏菌的T3SS活性以及YopK如何阻止这种感知的分子基础尚不清楚。我们提出了三个具体目标来解决我们知识中的这一重要差距。首先，我们将检验这样一种假设，即将转运子成分输送到宿主细胞胞浆通过破坏细胞内的隔室来激活NLRP3炎症体。其次，我们将验证YopK通过与转运子结合并限制转运子成分注入细胞来阻止NLRP3炎症体激活的假设。第三，我们将检验TE假设，即体内炎症小体激活通过产生依赖于caspase-1的细胞因子来控制耶尔森菌感染，这些细胞因子诱导特定免疫细胞亚群的激活，促进细菌清除。

DESCRIPTION (provided by applicant): The overall goal of this project is to understand molecular mechanisms of how the innate immune system senses and responds to bacterial pathogens and the evasion strategies utilized by bacterial pathogens to avoid immune detection. In particular we use Yersinia pseudotuberculosis, which expresses many of the same virulence factors as its close relative and plague pathogen Yersinia pestis, as a model to understand the host- pathogen interface and discover shared features of host innate immune responses to Gram-negative bacterial pathogens. The rapid expansion of multi-drug resistance among Gram-negative bacteria has increased the importance of developing new approaches to antimicrobial therapeutics, and understanding how the innate immune system initially detects bacterial pathogens and the corresponding pathogen evasion strategies holds promise for identifying novel antimicrobials. The Type III secretion system (T3SS) is a broadly conserved virulence determinant that is essential for virulence of many Gram-negative bacterial pathogens from E. coli to Y. pestis, and injects virulence factors into host cells that disrupt cellular signling pathways. However, T3SS activities can also be sensed by cytosolic pattern recognition receptors of the Nod-like receptor (NLR) family and induce host immune responses. Previous studies demonstrated that the Y. pseudotuberculosis and Y. pestis T3SS induces activation of an NLRP3-dependent inflammasome, resulting in cell death and caspase-1 dependent cytokine secretion. Furthermore, studies demonstrated that the essential Yersinia virulence factor YopK, which modulates the pore-forming activity of the T3SS and limits translocation of other Yop effector proteins, prevents inflammasome activation. Inflammasome activation plays an important role in host defense against Yersinia, as the virulence defect of YopK-deficient bacteria is restored in mice that lack inflammasome components. However, the molecular basis for how cells sense the activity of Yersinia's T3SS and how YopK prevents this sensing is not known. We propose three Specific Aims to address this important gap in our knowledge. First we will test the hypothesis that delivery of translocon components into the host cell cytosol activates the NLRP3 inflammasome through disruption of intracellular compartments. Second we will test the hypothesis that YopK prevents NLRP3 inflammasome activation by binding to the translocon and limiting injection of translocon components into the cell. Third, we will test te hypothesis that inflammasome activation in vivo controls Yersinia infection via production of caspase-1 dependent cytokines that induce activation of specific immune cell subsets that promote bacterial clearance.