宿主细胞磷脂酶D调控李斯特菌侵入上皮细胞过程的机制研究

发布时间：2018-07-07 16:35

本文选题：单核细胞增生李斯特菌 + vero细胞　；参考：《中国人民解放军军事医学科学院》2010年博士后论文

【摘要】：病原体单核细胞增生李斯特菌能够诱导自身侵入非吞噬细胞,侵入过程受到宿主细胞肌动蛋白骨架的紧密调控。我们在本研究中报道了这一侵入过程的一个新的调节因子,磷脂酶D (phospholipase D, PLD)。PLD可以特异性地催化水解细胞膜的重要组成成份-磷脂酰胆碱生成磷脂酸和胆碱,是细胞内的重要信号效应蛋白,参与调控许多重要细胞生理过程,尤其与细胞肌动蛋白骨架重排关系密切。我们发现李斯特菌在内化侵入vero上皮细胞过程中能够刺激宿主PLD的激活。两种PLD的化学抑制剂(1-丁醇,2,3-DPG)预处理细胞能够抑制李斯特菌诱导的PLD激活及李斯特菌的内化侵入。过表达酯酶缺陷型PLD蛋白或沉默内源PLD表达都能抑制李斯特菌的内化侵入。在一系列能够影响侵入能力的缺陷型李斯特菌中,只有内化素B (InlB)缺陷的菌株不能激活宿主细胞的PLD,而纯化的InlB蛋白能够直接通过其细胞表面受体Met来激活宿主细胞PLD活性并能挽救缺陷株对宿主细胞的侵染能力以及激活宿主PLD的能力。进一步,我们发现,李斯特菌和InlB都能诱导细胞内cofilin蛋白的短暂磷酸化。在细胞内表达不能磷酸化的cofilin S3A,或是表达cofilin的特异性磷酸水解酶slingshot 1L,不但显著抑制了李斯特菌的内化侵入,而且能够抑制InlB诱导的PLD激活。相反,在细胞内表达类磷酸化的cofilin (cofilin S3D)可显著抑制李斯特菌的内化侵入,并能挽救cofilin基因沉默细胞中的InlB诱导的PLD激活。最后,在vero细胞中表达能够与cofilin特异性结合的PLD的F-3片段,可抑制InlB诱导的PLD激活和李斯特菌的内化侵入。因此,我们的研究结果显示李斯特菌的InlB可能是一种新的PLD信号激动剂,其可通过cofilin-PLD信号级联调节李斯特菌的内化侵入。
[Abstract]:Listeria monocytogenes can induce self-invasion of non-phagocytes and the invasion process is closely regulated by the actin cytoskeleton of host cells. In this study, we report a new regulatory factor for the invasion process. Phospholipase D (phospholipase D, PLD). PLD can specifically catalyze the hydrolysis of phosphatidylcholine into phosphatidic acid and choline, an important component of cell membrane. It is an important signal effector protein in cells, which is involved in many important cellular physiological processes, especially the cytoskeleton rearrangement of actin. We found that Listeria can stimulate host vero activation during the process of internalization and invasion of vero epithelial cells. Pretreatment with two chemical inhibitors of PLD (1-butanol 2-butanol 3-DPG) could inhibit the PLD activation induced by Listeria sp. And the invasion of Listeria. Overexpression of esterase deficient PLD protein or silencing of endogenous PLD protein could inhibit the invasion of Listeria. In a series of defective Listeria that can affect invasiveness, The purified InlB protein can activate the PLD activity of the host cell directly through its cell surface receptor Met and can save the invading ability of the defective strain to the host cell. And the ability to activate the host PLD. Furthermore, we found that both Listeria and InlB could induce transient phosphorylation of cofilin protein in cells. The expression of cofilin S3A, which can not be phosphorylated in cells, or slingshot 1L, a specific phosphohydrolase expressing cofilin, not only inhibited the invasion of Listeria, but also inhibited the InlB-induced activation of cofilin. On the contrary, the expression of phosphorylated cofilin (cofilin S3D in cells significantly inhibited the invasion of Listeria and saved the InlB-induced activation of cofilin gene in cells. Finally, the expression of F-3 fragment of PLD specifically binding to cofilin in vero cells inhibited the InlB-induced activation of PLD and the internalization of Listeria sp. Therefore, our results suggest that Listeria sp. InlB may be a new PLD signal agonist, which can regulate the invasion of Listeria by cofilin-PLD signal cascade.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：博士后
【学位授予年份】：2010
【分类号】：R378

【共引文献】