Manipulation of Host Innate Immunity by Listeria

李斯特菌对宿主先天免疫的操纵

• 批准号: 6880450
• 负责人: DANIEL A PORTNOY
• 金额: $ 11.64万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-12-01 至 2009-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6880450
• 关键词: Listeria; Listeria infections; bacteria infection mechanism; genetic transcription; hemolysin; high performance liquid chromatography; immunity; interferon beta; laboratory mouse; liposomes; macrophage; mass spectrometry; peptidoglycan; toll like receptor

A central problem in microbial pathogenesis is how cells of the immune system integrate multiple signals to induce an appropriate response and how pathogens avoid and/or manipulate the host response. Listeria monocytogenes as a highly tractable model intracellular pathogen with which to approach this problem. Using wild-type and mutant bacteria lacking a secreted pore-forming hemolyisn (LLO), two separate pathways were discovered leading to host gene expression: a vacuolar pathway characterized by Toll-like Receptor signaling and a cytosolic pathway characterized by Interferon beta (IFNb) production. IFNb expression was mediated, at least in part, by L. monocyogenes autolysins (p60 and NamA) secreted by the recently discovered SecA2 pathway. It appeas as though L. monocytogenes is manipulating a host system of innate immunity by the production of a specific peptidoglycan (PGN) cleavage product. In Aim I, the macrophage transcriptional response to vacuolar and cytosolic bacteria will be determned using wildtype L. mononcytogenes and LLO-minus mutants in combination with B. subtilis or B. subtilis expressing LLO. Using macrophages isolated from mice lacking the IFNabR, MyD88, or TLR2, a panel of genes representing the vacuolar and cytosolic signaling will be established. In Aim II, the bacterial autolysins affecting macrophage signaling will be determined by introduction of in-frame deletions and characteization of the resulting mutants in mice and macrophages from wild-type and NOD1 and NOD2 knockouts. In Aim III, the contribution of specific PGN fragments will be determined using reverse phase high pressure liquid chromatography and mass spectrometry combined with introduction of purified PGN fragments into cells using liposomes with and without LLO. In Aim IV, activated peritoneal macrophages will be used to test the hypothesis will be tested that the cytosolic pathway actually detects material (PGN) derived from a vacuolar compartment resulting from degradation in a phagosome. In the final aim, the composite transcriptional 3rogram induced by L. monocytogenes will be compared with that induced by M. tuberculosis, F. tularensis and H. capsulatum.

微生物发病机制的一个核心问题是免疫系统细胞如何整合多种信号以诱导适当的反应，以及病原体如何避免和/或操纵宿主反应。单核细胞增生李斯特菌作为一个高度易处理的模型细胞内病原体与解决这一问题。使用缺乏分泌的成孔溶血素（LLO）的野生型和突变体细菌，发现导致宿主基因表达的两个单独的途径：以Toll样受体信号传导为特征的液泡途径和以干扰素β（IFNb）产生为特征的胞质途径。IFNb的表达至少部分由L.单核细胞自溶素（p60和NamA）由最近发现的SecA 2途径分泌。它看起来像L。单核细胞增多症在操纵宿主系统 通过产生特异性肽聚糖（PGN）裂解产物来产生先天免疫。在目的I中，巨噬细胞对空泡和胞质细菌的转录反应将使用野生型L.单核细胞基因和LLO-阴性突变体与B的组合。枯草芽孢杆菌或B.枯草芽孢杆菌表达LLO。 使用从缺乏IFNabR、MyD 88或TLR 2的小鼠分离的巨噬细胞，将建立代表空泡和胞质信号传导的一组基因。在目的II中，将通过引入框内缺失并表征小鼠和巨噬细胞中来自野生型和NOD 1和NOD 2敲除的所得突变体来确定影响巨噬细胞信号传导的细菌自溶素。在目的III中，将使用反相高压液相色谱法和质谱法结合使用具有和不具有LLO的脂质体将纯化的PGN片段引入细胞中来确定特异性PGN片段的贡献。在目的IV中，将使用活化的腹膜巨噬细胞来检验假设，即细胞溶质途径实际上检测到来自空泡细胞的物质（PGN）。 在吞噬体中降解而形成的隔室。最后，对L.单核细胞增多症将与M. tuberculosis，F. tularensis和H.荚膜