Goldfine H, Wadsworth S J, Johnston N C
Department of Microbiology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104-6076, USA.
Infect Immun. 2000 Oct;68(10):5735-41. doi: 10.1128/IAI.68.10.5735-5741.2000.
Infection of the J774 murine macrophage-derived cell line with Listeria monocytogenes results in several elevations of intracellular calcium during the first 15 min of infection. These appear to result from the actions of secreted bacterial proteins, including phosphatidylinositol-specific phospholipase C (PI-PLC), a broad-range phospholipase C, and listeriolysin O (LLO) (S. J. Wadsworth and H. Goldfine, Infect. Immun. 67:1770-1778, 1999). We have measured hydrolysis of host PI and the activation of host polyphosphoinositide-specific PLC and host phospholipase D (PLD) during infection with wild-type and mutant L. monocytogenes. Elevated hydrolysis of host PI occurred within the first 10 min of infection and was dependent on both bacterial PI-PLC and LLO, both of which were required for the earliest elevations of intracellular calcium in the host cell. A more rapid hydrolysis of host PI was observed at 30 min after infection, at the time when wild-type bacteria have been internalized. Activation of host PLC, also occurred in the first 10 min of infection but was not dependent on the presence of bacterial PI-PLC. Similar observations were made in murine bone marrow-derived macrophages. In J774 cells, activation of host PLD was observed after 20 min of infection and was dependent on bacterial LLO. Mutants in the bacterial phospholipases produced levels of PLD activation similar to those produced by the wild type. Phorbol myristate acetate (PMA) also activated host PLD, while long-term treatment with PMA resulted in loss of the ability of L. monocytogenes to activate host PLD, suggesting an involvement of protein kinase C (PKC) in the activation of PLD. Rottlerin, an inhibitor of PKC delta in J774 cells, also inhibited the activation of PLD, but hispidin, an inhibitor of PKC betaI and betaII, did not. Pretreatment of J774 cells with the PLD inhibitor, 2, 3-diphosphoglycerate partially inhibited escape of the bacteria from the primary phagocytic vacuole.
用单核细胞增生李斯特菌感染J774小鼠巨噬细胞系会在感染的前15分钟内导致细胞内钙水平多次升高。这些升高似乎是由分泌的细菌蛋白的作用引起的，包括磷脂酰肌醇特异性磷脂酶C（PI-PLC）、一种广谱磷脂酶C和李斯特菌溶血素O（LLO）（S. J. 沃兹沃思和H. 戈德芬，《感染与免疫》67:1770 - 1778，1999）。我们已经测量了野生型和突变型单核细胞增生李斯特菌感染期间宿主PI的水解以及宿主多磷酸肌醇特异性PLC和宿主磷脂酶D（PLD）的激活情况。宿主PI的水解在感染的前10分钟内升高，并且依赖于细菌PI-PLC和LLO，这两者都是宿主细胞内钙最早升高所必需的。在感染后30分钟观察到宿主PI的水解更快，此时野生型细菌已被内化。宿主PLC的激活也发生在感染的前10分钟，但不依赖于细菌PI-PLC的存在。在小鼠骨髓来源的巨噬细胞中也有类似的观察结果。在J774细胞中，感染20分钟后观察到宿主PLD的激活，并且依赖于细菌LLO。细菌磷脂酶突变体产生的PLD激活水平与野生型相似。佛波酯（PMA）也激活宿主PLD，而长期用PMA处理导致单核细胞增生李斯特菌激活宿主PLD的能力丧失，这表明蛋白激酶C（PKC）参与了PLD的激活。rottlerin是J774细胞中PKCδ的抑制剂，也抑制了PLD的激活，但hispidin是PKCβI和βII的抑制剂，却没有抑制作用。用PLD抑制剂2,3 - 二磷酸甘油酸预处理J774细胞可部分抑制细菌从初级吞噬泡中逸出。
