Molecular mechanism of biological activity expressed by hemolysins derived from Listeriae inside macrophages.

巨噬细胞内李斯特菌溶血素表达的生物活性的分子机制。

基本信息

批准号：
14370092
负责人：
MITSUYAMA Masao
金额：
$ 8.06万
依托单位：
Kyoto University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2002
资助国家：
日本
起止时间：
2002 至 2004
项目状态：
已结题

项目摘要

On the molecular mechanisms of biological activity expressed by hemolysins derived from various Listeria spp., the following results have been obtained by using recombinant proteins and mutant strains. (1)All the hemolysin proteins, LLO, ILO and LSO, consist of 4 domains and the known hemolytic activity is highly dependent on domain 4, while the newly found cytokine-inducing activity is exclusively dependent on domain 1-3 at he N-terminus. (2)The very low cytokine-inducing activity observed in ILO appeared to be due to the absence of PEST-like sequence at the N-terminus portion. (3)Upon stimulation with LLO, macrophage first produce IL-12 and IL-18, then these IFN-γ-inducing cytokines stimulate NK cells to produce a massive amount of IFN-γ. (4)TLRs, especially TLR2 and TLR4 are both required for the cytokine production by macrophages in response to LLO. (5)The cytokine response in epithelial cells that do not express surface TLRs appeared to be due to the cytokine induction triggered by the Ca-influx caused by sublethal level of membrane damage by LLO. (6)The in-flame deletion mutant of hly (a gene encoding LLO) has been established. By using recombinant strains, it was confirmed that both escape of bacteria from phagosome into cytosolic space in macrophages and the presence of PEST-like sequence are required for cytokine response. (7)Upon infection of macrophages by Listeria monocytogenes, a significant level of upregulation was induced in hly and orfA gene expression. Oxygen radicals produced by infected macrophages appeared to be responsible for this upregulation of virulence gene expression.

利用重组蛋白和突变菌株对多种李斯特菌溶血素表达的生物活性的分子机制进行了研究，得到了以下结果： (1)所有溶血素蛋白LLO、ILO和LSO均由4个结构域组成。已知的溶血活性高度依赖于结构域4，而新发现的细胞因子诱导活性完全依赖于N端的结构域1-3 (2)细胞因子诱导性非常低。在ILO中观察到的活性似乎是由于在N末端部分不产生PEST样序列(3)在用LLO刺激时，巨噬细胞首先产生IL-12和IL-18，然后产生这些IFN-γ诱导细胞因子。刺激NK细胞产生大量的IFN-γ (4)TLRs，特别是TLR2和TLR4都是巨噬细胞响应LLO产生细胞因子所必需的。不表达表面 TLR 的上皮细胞似乎是由于 LLO 亚致死水平的膜损伤引起的 Ca 内流触发细胞因子诱导 (6)hly（编码 LLO 的基因）的火焰缺失突变体。通过使用重组菌株，已证实细菌从吞噬体逃逸到巨噬细胞的胞质空间中以及 PEST 样序列的存在都是细胞因子反应所必需的。受感染的巨噬细胞产生的氧自由基似乎是毒力基因表达上调的原因。