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

在各种李斯特菌溶血素表达生物活性的分子机制方面，利用重组蛋白和突变菌株获得了以下结果。(1)所有溶血素蛋白LLO、IIO和LSO都由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端的结构域1-3。(2)在iLO中观察到的很低的细胞因子诱导活性似乎是由于在N末端部分没有PEST类序列所致。(3)巨噬细胞在LLO刺激下，首先产生IL-12和IL-18，然后这些干扰素-γ诱导的细胞因子刺激NK细胞产生大量的干扰素-γ。(4)巨噬细胞产生细胞因子需要TLR2和TLR4受体，尤其是TLR2和TLR4。(5)不表达表面TLRs的上皮细胞的细胞因子反应可能是由于LLO亚致死性膜损伤引起的钙内流所触发的细胞因子诱导所致。(6)建立了HLY(编码LLO的基因)的火焰中缺失突变体。通过使用重组菌株，证实了细菌从吞噬小体逃逸到巨噬细胞的胞浆空间和存在类PEST序列都是细胞因子反应所必需的。(7)单核细胞增生性李斯特菌感染巨噬细胞后，HLY和OrfA基因表达显著上调。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。

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.