Manipulation of Host Innate Immunity by Listeria

微生物发病机制中的一个中心问题是免疫系统的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。单核细胞增多性李斯特菌作为一种高度易处理的细胞内病原体模型来解决这一问题。利用缺乏分泌孔形成溶血(LLO)的野生型和突变型细菌，发现了导致宿主基因表达的两条不同的途径：一条以Toll样受体信号为特征的空泡途径和一条以产生干扰素β(IFNb)为特征的胞质途径。IFNb的表达至少部分是由新近发现的SecA2途径分泌的单核细胞自溶素(P60和NAMA)介导的。这看起来就像是单核细胞增多性李斯特菌在操纵宿主系统。 通过产生一种特定的肽聚糖(PGN)裂解产物而产生的先天免疫。在目标I中，将使用野生型L.单核细胞基因和LLO阴性突变体与枯草杆菌或表达LLO的枯草杆菌结合来检测巨噬细胞对空泡和胞浆细菌的转录反应。 使用从缺乏IFNabR、MyD88或TLR2的小鼠身上分离的巨噬细胞，将建立一组代表空泡和胞质信号的基因。在AIM II中，影响巨噬细胞信号的细菌自溶素将通过引入框内缺失和从野生型和NOD1和NOD2基因敲除的小鼠和巨噬细胞中产生的突变体的特征来确定。在AIM III中，特定的PGN片段的贡献将用反相高压液相色谱和质谱仪结合将纯化的PGN片段导入细胞，并使用含有和不含LLO的脂质体来确定。在目标四中，激活的腹膜巨噬细胞将被用来检验这一假设，即胞浆途径实际上检测到来自空泡的物质(PGN) 吞噬小体降解产生的隔间。最后，将单核细胞增多性乳杆菌诱导的复合转录图谱与结核分枝杆菌、图拉氏杆菌和包膜螺旋体诱导的复合转录图谱进行比较。

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.