Saha A K, Dowling J N, Pasculle A W, Glew R H
Department of Microbiology, Biochemistry and Molecular Biology, University of Pittsburgh School of Medicine, Pennsylvania 15261.
Arch Biochem Biophys. 1988 Aug 15;265(1):94-104. doi: 10.1016/0003-9861(88)90375-x.
The legionellae are facultative intracellular bacterial pathogens which multiply in host phagocytes. Legionella micdadei cells contain an acid phosphatase (ACP2) which blocks superoxide anion production by human neutrophils stimulated with formyl-Met-Leu-Phe (fMLP) [A. K. Saha, et al. (1985) Arch. Biochem. Biophys. 243, 150-160]. In the present study, we have purified the Legionella phosphatase to homogeneity as indicated by the finding of a single 68,000-Da band following sodium dodecyl sulfate-polyacrylamide gel electrophoresis. We explored the possibility that ACP2 acts by interfering with polyphosphoinositide hydrolysis and the production of the intracellular second messengers, inositol trisphosphate (IP3) and diacylglycerol, following neutrophil stimulation. Phosphatidylinositol 4,5-bisphosphate (PIP2) was hydrolyzed rapidly by ACP2 in vitro. The rate of hydrolysis of PIP2 was higher at pH 7.0 (Km 2.0 microM; 4 X 10(3) units/mg protein; 1 unit equals 1 nmol of Pi released/h) than at lower pH. IP3 was also a good substrate for ACP2 in vitro. When human neutrophil phosphoinositides were prelabeled with 32Pi, subsequent incubation with ACP2 resulted in an 85% loss of the labeled PIP2 over 2 h. Following fMLP stimulation of [3H]inositol-labeled neutrophils, the quantity of IP3 produced by ACP2-treated cells was reduced by 44%. Prior treatment of neutrophils with ACP2 also reduced by 45% the amount of diacylglycerol they produced when stimulated by fMLP. These results indicate that the Legionella phosphatase may compromise the neutrophils' microbicidal response to the organism by hydrolyzing PIP2, the progenitor of IP3 and diacylglycerol, and by hydrolyzing IP3 itself.
嗜肺军团菌是兼性细胞内细菌病原体，可在宿主吞噬细胞中繁殖。米德戴军团菌细胞含有一种酸性磷酸酶（ACP2），它可阻断由甲酰甲硫氨酸亮氨酸苯丙氨酸（fMLP）刺激的人中性粒细胞产生超氧阴离子[A.K.萨哈等人（1985年），《生物化学与生物物理学档案》243卷，第150 - 160页]。在本研究中，我们已将军团菌磷酸酶纯化至同质，这可通过十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳后出现单一的68,000道尔顿条带得以证明。我们探究了ACP2通过干扰多磷酸肌醇水解以及中性粒细胞受刺激后细胞内第二信使肌醇三磷酸（IP3）和二酰甘油产生的可能性。磷脂酰肌醇4,5 - 二磷酸（PIP2）在体外被ACP2快速水解。PIP2在pH 7.0时的水解速率（Km为2.0微摩尔；4×10³单位/毫克蛋白质；1单位等于每小时释放1纳摩尔无机磷酸）高于较低pH值时。IP3在体外也是ACP2的良好底物。当人中性粒细胞磷酸肌醇用³²Pi预标记后，随后与ACP2一起孵育导致标记的PIP2在2小时内损失85%。在用fMLP刺激[³H]肌醇标记的中性粒细胞后，经ACP2处理的细胞产生的IP3量减少了44%。用ACP2预先处理中性粒细胞也使其在受fMLP刺激时产生的二酰甘油量减少了45%。这些结果表明，军团菌磷酸酶可能通过水解PIP2（IP3和二酰甘油的前体）以及IP3本身来损害中性粒细胞对该生物体的杀菌反应。
