Bacterial cell membrane hydrolysis by secreted phospholipases A2:: a major physiological role of human group IIa sPLA2 involving both bacterial cell wall penetration and interfacial catalysis
Bacterial cell membrane hydrolysis by secreted phospholipases A2:: a major physiological role of human group IIa sPLA2 involving both bacterial cell wall penetration and interfacial catalysis
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DOI：
10.1016/s1388-1981(00)00018-4
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发表时间：
2000-04-12
影响因子：
4.8
通讯作者：
Wilton, DC
中科院分区：
文献类型：
--
作者：
Buckland, AG;Heeley, EL;Wilton, DC
使用连续荧光置换测定法在真实的时间内证明了人IIa族分泌型磷脂酶A（2）（人sPLA（2））水解革兰氏阳性细菌全细胞悬浮液的磷脂膜的能力。将藤黄微球菌用作模型系统，并与猪胰和眼镜蛇毒sPLA（2）s相比，证明了该人类酶几乎绝对的特异性。这种特异性是由于高度阳离子的人sPLA（2）通过高度阴离子的细菌细胞壁的选择性渗透。通过用溶菌酶处理来破坏肽聚糖细胞壁允许所有三种酶对阴离子细菌细胞膜表达类似的水解活性。观察到广泛（>50%）的磷脂水解，并通过电喷雾质谱法证实，该质谱法允许鉴定几种磷脂酰甘油分子种类作为水解的靶点。然而，在这些测定条件下人酶的杀菌活性较低，突出了生物体在主要磷脂损伤中存活的能力。除纯酶外，还使用M.以黄体为底物。与M相比。金黄色葡萄球菌的细胞悬液对人sPLA的水解具有高度抗性（2），对胰腺酶和毒液酶也具有高度抗性。用特定的细胞壁蛋白酶溶葡萄球菌酶处理这种微生物，导致所有三种sPLA（2）的细胞膜磷脂水解显著增强。总体而言，结果突出了人sPLA（2）作为体内革兰氏阳性菌的选择性抗菌剂的潜力，因为这种酶对哺乳动物质膜基本上是无活性的。然而，该酶与其他抗微生物剂组合将是最有效的，所述其他抗微生物剂增强细菌细胞壁的渗透性，并且预期会增强其他抗生素的有效性。(C)2000 Elsevier Science B.V.保留所有权利。
The ability of human group IIa secreted phospholipase A(2) (human sPLA(2)) to hydrolyse the phospholipid membrane of whole cell suspensions of Gram-positive bacteria is demonstrated in real time using a continuous fluorescence displacement assay. Micrococcus luteus is used as a model system and demonstrates an almost absolute specificity for this human enzyme compared with porcine pancreatic and Naja naja venom sPLA(2)s. This specificity is due to selective penetration of the highly cationic human sPLA(2) through the highly anionic bacterial cell wall. Disruption of the peptidoglycan cell wall by treatment with lysozyme allows all three enzymes to express similar hydrolytic activity against the anionic bacterial cell membrane. Extensive (>50%) phospholipid hydrolysis was observed and this was confirmed by electrospray mass spectrometry that allowed the identification of several molecular species of phosphatidylglycerol as the targets for hydrolysis. However, the bactericidal activity of the human enzyme under these assay conditions was low, highlighting the capacity of the organism to survive a major phospholipid insult. In addition to pure enzyme, the human sPLA(2) activity in tears was demonstrated using M. luteus as substrate. In comparison to M. luteus, cell suspensions of Staphylococcus aureus were highly resistant to hydrolysis by human sPLA(2) as well as to the pancreatic and venom enzymes. Treatment of this organism with the specific cell wall protease lysostaphin resulted in a dramatic enhancement in cell membrane phospholipid hydrolysis by all three sPLA(2)s. Overall, the results highlight the potential of the human sPLA(2) as a selective antimicrobial agent against Gram-positive bacteria in vivo because this enzyme is essentially inactive against mammalian plasma membranes. However, the enzyme will be most effective in combination with other antimicrobial agents that enhance the permeability of the bacterial cell wall and where potentiation of the effectiveness of other antibiotics would be expected. (C) 2000 Elsevier Science B.V. All rights reserved.