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首页> 外文期刊>Infection and immunity >Listeria monocytogenes Phospholipase C-Dependent Calcium Signaling Modulates Bacterial Entry into J774 Macrophage-Like Cells
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Listeria monocytogenes Phospholipase C-Dependent Calcium Signaling Modulates Bacterial Entry into J774 Macrophage-Like Cells

机译：李斯特菌李斯特菌磷脂酶C依赖性钙信号调节细菌进入J774巨噬细胞样细胞。

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Listeria monocytogenes secretes several proteins that have been shown to contribute to virulence. Among these is listeriolysin O (LLO), a pore-forming hemolysin that is absolutely required for virulence. Two other virulence factors are phospholipases: a phosphatidylinositol-specific phospholipase C (PI-PLC [plcA]) and a broad-range PLC (plcB). Although mutations in plcA or plcB resulted in small increases in mouse 50% lethal dose (LD50), deletions in both genes resulted in a 500-fold increase in LD50. We have examined the role of these secreted proteins in host intracellular signaling in the J774 macrophage-like cell line. Measurements of cytosolic free calcium ([Ca2+]i) have revealed a rapid spike upon exposure of these cells to wild-typeL. monocytogenes. This is followed by a second peak at 5 min and a third prolonged peak with a maximal [Ca2+]i of 800 to 1,000 nM. The pattern of calcium changes was greatly altered by deletion of any of the three virulence factors. An LLO mutant produced none of these elevations in [Ca2+]i; however, a transient elevation was observed whenever these bacteria entered the cell. A PI-PLC mutant produced a diminished single elevation in [Ca2+]i at 15 to 30 min. A broad-range PLC mutant produced only the first calcium spike. Studies with inhibitors suggested that the first elevation arises from influx of calcium from the extracellular medium through plasma membrane channels and that the second and third elevations come from release of Ca2+ from intracellular stores. We observed that internalization of wild-type bacteria and the broad-range PLC mutant was delayed for 5 to 10 min, but the LLO and PI-PLC mutants were internalized rapidly upon infection. Inhibitors that affected calcium signaling changed the kinetics of association of wild-type bacteria with J774 cells, the kinetics of entry, and the efficiency of escape from the primary phagosome.
机译：单核细胞增生李斯特菌分泌几种蛋白质，这些蛋白质已被证明具有毒性。其中有李斯特菌溶血素O（LLO），这是一种形成孔的溶血素，绝对需要毒力。另外两个毒性因子是磷脂酶：磷脂酰肌醇特异性磷脂酶C（PI-PLC [ plcA ]）和大范围PLC（ plcB ）。尽管 plcA 或 plcB 的突变导致小鼠50％致死剂量（LD 50 ）的微小增加，但两个基因的缺失均导致500 LD 50 增长了两倍。我们已经检查了这些分泌蛋白在J774巨噬细胞样细胞系中宿主细胞内信号传导中的作用。胞浆游离钙（[Ca 2 + ] i ）的测量显示，这些细胞暴露于野生型 L时会迅速升高。单核细胞增生。随后是5分钟的第二个峰和最大[Ca 2 + ] i 为800至1,000 nM的第三个延长峰。钙变化的模式通过删除三种毒力因子中的任何一种而大大改变。 LLO突变体在[Ca 2 + ] i 中均不产生这些升高;但是，每当这些细菌进入细胞时，都会观察到瞬时升高。一个PI-PLC突变体在15至30分钟时使[Ca 2 + ] i 的单峰降低。一个宽范围的PLC突变体仅产生了第一个钙峰值。对抑制剂的研究表明，第一个升高是由于钙通过质膜通道从细胞外介质中流入而引起的，第二个和第三个升高是由于细胞内储存物中Ca 2 + 的释放。我们观察到野生型细菌和大范围PLC突变体的内在化被延迟了5到10分钟，但LLO和PI-PLC突变体在感染后迅速被内在化。影响钙信号传导的抑制剂改变了野生型细菌与J774细胞缔合的动力学，进入动力学以及从初级吞噬体逃逸的效率。

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