Inhibition of the host cell AIM2 inflammasome by Mycobacterium tuberculosis

摘要 结核分枝杆菌（Mycobacterium tuberculosis，Mtb）是一种引起严重发病和死亡的人类病原体 国际吧越来越多的结核病病例是由多重耐药和极端耐药引起的。 结核分枝杆菌菌株强调需要新的治疗方法。宿主对结核分枝杆菌感染的有效反应取决于 对巨噬细胞和树突状细胞中炎性体复合物介导的IL-1产生的影响 细胞黑色素瘤中的缺失2（AIM 2）蛋白是炎性体组分之一，并且它能够结合 双链DNA的病原体，如弗朗西斯和李斯特菌在细胞胞质溶胶，然后诱导 炎性小体复合物的组装和活化。在这里，我们报告了一个新奇而令人惊讶的发现， Mtb能够抑制宿主细胞AIM 2-炎性体的活化。我们假设基因筛查 将能够鉴定介导这种新型免疫逃避策略的Mtb基因。我们建议使用 “功能获得性”（GoF）遗传筛选，通过比较施用后的宿主细胞AIM 2-炎性小体活化来进行。 用312 M的现有文库的单个克隆感染吞噬细胞。smeglobulin（Msme）克隆 用含有Cosmos的Mtb-DNA（~ 40 kbp插入大小）转染。此库已成功用于 我们在一个GoF筛选抗细胞凋亡基因的结核分枝杆菌基因组。Msme克隆出了最强的 将选择与野生型Msme感染细胞相比IL-1分泌减少的细胞， 将测定粘粒的Mtb-DNA插入片段的序列。接下来，我们将使用三种不同的策略， 鉴定介导AIM 2-炎性小体抑制的一种补体中的Mtb基因。的中国梦作出 Mtb中的缺失突变体，其将覆盖所鉴定的基因组区域的所有基因，并分析Mtb中的缺失突变体。 突变体的AIM 2介导的炎性小体活化的预期增加。二、体外转座子 粘粒DNA的诱变和随后的DNA序列分析将产生有序的 转座子突变体将这些突变的cosmetic转染到Msme中，并将其降低AIM 2- 将分析炎性小体活化。第三，将鉴定的基因的Mtb转座子突变体 从储存库（TARGET）订购，并分析增加的炎性小体激活。在 结论，拟议的研究将导致Mtb基因的鉴定，重要的介导 抑制宿主细胞AIM 2-炎性体活化。这些突变体将使未来的体内研究， 为了确定这种新的免疫逃避策略对Mtb毒力的重要性。另外这些 推测的毒力基因可能是药物开发和改进重组疫苗的新靶点 设计

Abstract Mycobacterium tuberculosis (Mtb) is a human pathogen that causes significant morbidity and mortality worldwide. An increasing number of tuberculosis cases are caused by multi-drug and extreme-drug resistant Mtb strains underscoring the need for novel therapeutics. The efficient host response to Mtb infection depends on the production of IL-1¿ which is mediated by the inflammasome complex in macrophages and dendritic cells. The Absent In Melanoma 2 (AIM2) protein is one of the inflammasome components and it is able to bind to double stranded DNA of pathogens such as Francisella and Listeria in the cell cytosol and then induces assembly and activation of the inflammasome complex. Here we report the novel and surprising finding that Mtb is able to inhibit the activation of the host cell AIM2-inflammasome. We hypothesize that genetic screens will be able to identify the Mtb gene(s) mediating this novel immune evasion strategy. We propose to use a "Gain-of-Function" (GoF) genetic screen by comparing the host cell AIM2-inflammasome activation after infection of phagocytes with individual clones of an existing library of 312 M. smegmatis (Msme) clones transfected with Mtb-DNA containing cosmids (~40kbp insert size). This library has successfully been used by us during a GoF screen for anti-apoptosis genes in the Mtb genome. The Msme clones with the strongest reduction in IL-1¿ secretion when compared to wild-type Msme infected cells will be selected and the sequence of the Mtb-DNA insert of the cosmid will be determined. Next, we will use three different strategies to identify the Mtb gene(s) in one of the cosmids mediating the AIM2-inflammasome inhibition. We will make deletion mutants in Mtb which will cover all of the genes of the identified genomic region and analyze the mutants for an expected increase in AIM2-mediated inflammasome activation. Second, in vitro transposon mutagenesis of the cosmid DNA and subsequent DNA sequence analysis will generate an ordered library of transposon mutants. These mutated cosmids will be transfected into Msme and the capacity of reducing AIM2- inflammasome activation will be analyzed. Third, Mtb transposon mutants of the identified genes will be ordered from a repository library (TARGET) and analyzed for increased inflammasome activation. In conclusion, the proposed studies will lead to the identification of Mtb gene(s) important for mediating suppression of host cell AIM2-inflammasome activation. These mutants will enable future in vivo studies in order to determine how important this novel immune evasion strategy is for virulence of Mtb. In addition, these putative virulence genes could be novel targets for drug development and improving recombinant vaccine design.