Inhibition of the host cell AIM2 inflammasome by Mycobacterium tuberculosis

结核分枝杆菌对宿主细胞 AIM2 炎性体的抑制

基本信息

批准号：
8720174
负责人：
VOLKER BRIKEN
金额：
$ 22.69万
依托单位：
UNIV OF MARYLAND, COLLEGE PARK
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-04-15 至 2016-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8720174
关键词：
AIM2 gene Apoptosis Apoptotic Binding Bone Marrow Cell Line Cells Cessation of life Complement Complex Cosmids Cytosol DNA DNA Sequence Analysis Dendritic Cells Drug Design Drug resistance in tuberculosis Francisella Future Generations Genes Genetic Screening Genome Genomics Human Immune Immune response Immunology In Vitro Individual Infection Interferons Interleukin-1 Knowledge Lead Libraries Listeria Measures Mediating Molecular Analysis Morbidity - disease rate Mus Mutagenesis Mutate Mycobacterium smegmatis Mycobacterium tuberculosis Phagocytes Pharmaceutical Preparations Phenotype Process Production Proteins Publications Recombinant Vaccines Reporting Research Project Grants Side Site Testing Time Tuberculosis Vaccine Design Vaccines Virulence base drug development ds-DNA gain of function improved in vivo interest killings loss of function macrophage mortality mutant novel novel therapeutics pathogen public health relevance repository tuberculosis drugs

项目摘要

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.

抽象的结核分枝杆菌 (Mtb) 是一种导致显着发病率和死亡率的人类病原体全球范围内越来越多的结核病病例是由多重耐药性和极端耐药性引起的。 Mtb 菌株强调了对新疗法的需求，宿主对 Mtb 感染的有效反应取决于。关于 IL-1 的生产它是由巨噬细胞和树突状细胞中的炎性体复合物介导的黑色素瘤中缺失 2 (AIM2) 蛋白是炎症小体成分之一，能够结合细胞。与细胞质中的弗朗西斯菌和李斯特菌等病原体的双链 DNA 结合，然后诱导炎症体复合物的组装和激活在这里，我们报告了这一新颖且令人惊讶的发现： Mtb 能够抑制宿主细胞 AIM2 炎症小体的激活。将能够识别介导这种新型免疫逃避策略的 Mtb 基因。通过比较宿主细胞 AIM2-炎症小体激活后的“功能获得”(GoF) 遗传筛选用现有 312 个耻垢分枝杆菌 (Msme) 克隆文库的单个克隆感染吞噬细胞用含有粘粒（约 40kbp 插入片段大小）的 Mtb-DNA 转染该文库已成功使用。我们在 GoF 筛选 Mtb 基因组中具有最强抗凋亡基因的 Msme 克隆。 IL-1 减少¿与野生型 Msme 感染细胞相比，分泌量将被选择，并且接下来，我们将使用三种不同的策略来确定粘粒的 Mtb-DNA 插入片段的序列。我们将鉴定介导 AIM2 炎症体抑制的粘粒之一中的 Mtb 基因。 Mtb 中的缺失突变体将覆盖已识别基因组区域的所有基因并分析突变体预期 AIM2 介导的炎症小体激活增加。其次，体外转座子。粘粒 DNA 的诱变和随后的 DNA 序列分析将生成一个有序的文库这些突变的粘粒将被转染到Msme中并降低AIM2-的能力。第三，将分析已识别基因的 Mtb 转座子突变体。从存储库（TARGET）订购并分析炎症小体激活的增加。结论是，拟议的研究将导致鉴定对于介导重要的 Mtb 基因抑制宿主细胞 AIM2 炎症小体激活，这些突变体将使未来的体内研究成为可能。为了确定这种新型免疫逃避策略对于 Mtb 的毒力有多重要。假定的毒力基因可能成为药物开发和改进重组疫苗的新靶点设计。