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

关于李斯特菌溶血素表达生物活性的分子机制，利用重组蛋白和突变菌株获得了以下结果。(1)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仅依赖于n端结构域1-3。(2)在ILO中观察到的极低的细胞因子诱导活性可能是由于在n端部分缺乏pest样序列。(3)经LLO刺激后，巨噬细胞首先产生IL-12和IL-18，然后这些诱导IFN-γ的细胞因子刺激NK细胞产生大量IFN-γ。(4)TLRs，尤其是TLR2和TLR4都是巨噬细胞在LLO反应中产生细胞因子所必需的。(5)在不表达表面TLRs的上皮细胞中，细胞因子的反应可能是由于LLO引起的亚致死水平的膜损伤引起的ca内流引发的细胞因子诱导。(6)建立了编码LLO基因hly的火焰缺失突变体。通过重组菌株，证实了细菌从吞噬体逃逸到巨噬细胞的胞质空间和pest样序列的存在是细胞因子反应的必要条件。(7)单核增生李斯特菌感染巨噬细胞后，诱导hly和orfA基因表达显著上调。感染巨噬细胞产生的氧自由基似乎是导致毒力基因表达上调的原因。

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.