Determination of the critical region of listeriolysin O (LLO) for the cytokine-inducing activity and application of LLO to vaccination with killed BCG.

李斯特菌溶血素O(Listeriolysin O，LLO)是单核细胞增多性李斯特氏菌的主要毒力因子，是胆固醇结合型细胞溶血素的成员之一。我们已经报道，LLO诱导内源性Th1型细胞因子的产生不依赖于膜的裂解活性。提示LLO在体内不发挥细胞溶解活性的情况下，可能具有促进保护性免疫生成的佐剂作用。因此，在本研究中，我们试图确定LLO仅对细胞因子诱导活性的临界区域，并通过给予脂质体包裹的LLO作为佐剂来评价其作为佐剂的有效性。第四结构域(LLO415)缺失的LLO对表达细胞因子活性具有重要的作用，显示了干扰素-γ的诱导活性。然而，重组的第四结构域不能诱导细胞因子的产生。当N-末端部分进一步缺失时，LLO415的活性受到相当大的抑制。这些数据表明，LLO的N端而不是C端的第四个结构域是细胞因子诱导活性的关键。在小鼠脾细胞中未观察到LLO的活性，并被抗CD14抗体阻断，提示LLO的信号转导可能是通过LPS的信号转导途径。此外，免疫沉淀显示LLO与J774.1细胞表面的两个分子结合，分子量约为50-60 kDa。我们认为这些分子参与了LLO的信号转导。为了确定LLO作为佐剂的效果，用灭活的卡介苗和脂质体包裹的LLO免疫C3H/HEN小鼠。LLO无细胞杀伤活性，并促进保护性T细胞的产生，提示LLO具有促进Th1型宿主抵抗的佐剂作用。另一方面，由于LLO415不能很好地被脂质体包裹，其佐剂活性还有待于阐明。

Listeriolysin O (LLO) is demonstrated to be a mojor virulence factor of Listeria monocytogenes and is one of the member of cholesterol-binding cytolysins. we have reported that LLO induces endogenous Th1-type cytokine productions independent of the membrane lytic activity. It suggests that LLO may play a role as an adjuvant to promote the generation of protective immunity if it does not exert the cytolytic activity in vivo. In the present study, therefore, we attempted to determine the critical region of LLO only for the cytokine-inducing activity and evaluated its effectiveness as an adjuvant by administration of LLO encapsulated with liposome containing cholesterol.LLO deleted for the fourth domain (LLO415), which is important for expression of the cytolytic activity, showed IFN-γ inducing activity. However, recombinant fourth domain did not induce the cytokine production. The activity of LLO415 was considerably suppressed when the N-terminal portion was further deleted. These data indicated that the N-terminus of LLO but not C-terminal fourth domain is critical for the cytokine-inducing activity. The activity of LLO was not observed in spleen cells of C3H/HeJ mice and was blocked by anti-CD14 antibody, suggesting that the signal of LLO may transduce via a signaling pathway of LPS.Moreover, immunoprecipitation revealed that LLO bound to two molecules on J774.1 cells, of which molecular weights are approximately 50-60kDa. We believe that these molecules are involved in the signal transduction of LLO.To determine the efficacy of LLO as an adjuvant, C3H/HeN mice were immunized with killed BCG along with LLO encapsulated in the liposome. The LLO showed no cytolytic activity and promoted generation of protective T cells, suggesting that LLO functions as the adjuvant to promote Th1-type host resistance. On the other hand, because LLO415 was not trapped in liposome well, the adjuvant activity was remained to be elucidated.