Biochemical and functional characterization of membrane blebs purified from Neisseria meningitidis serogroup B
Biochemical and functional characterization of membrane blebs purified from Neisseria meningitidis serogroup B
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DOI：
10.1074/jbc.m508063200
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发表时间：
2005-11-18
影响因子：
4.8
通讯作者：
Weiss, JP
中科院分区：
文献类型：
--
作者：
Post, DMB;Zhang, DS;Weiss, JP
对内毒素纯化聚集体的研究揭示了脂多糖结合蛋白（LBP）依赖的提取和个体内毒素分子转移到CD14在toll样受体4 （TLR4）依赖的细胞活化中的重要性。内毒素通常嵌入完整革兰氏阴性菌的外膜并脱落膜泡（“水泡”）。然而，LBP和CD14通过膜相关内毒素有效促进tlr4依赖性细胞活化的能力尚未得到广泛研究。在这项研究中，我们使用脑膜炎奈瑟菌血清B组的醋酸盐营养不良剂来促进细菌内毒素的代谢标记，并比较纯化的内毒素聚集体和膜相关内毒素与LBP、CD14和内毒素反应细胞的相互作用。内毒素、磷脂和蛋白质组成的恢复水泡表明，水泡来自细菌外膜。蛋白质组学分析显示高阳离子（pI >9）蛋白异常富集。纯化的内毒素聚集体和水泡都以LBP-、CD14-和TLR4/ md -2依赖的方式激活单核细胞和内皮细胞，但当内毒素添加量正常化时，水泡的效力降低了3 - 10倍。效价的差异与lbp依赖性递送和CD14提取内毒素的效率差异相关。通过LBP/可溶性CD14 （sCD14）处理提取膜磷脂和内毒素，但只有内毒素中心点sCD14与MD- 2反应并激活细胞。这些发现表明，内毒素的促炎效力可能不仅受内毒素的内在结构特性的调节，还受其与外膜邻近分子的结合的调节。
Studies with purified aggregates of endotoxin have revealed the importance of lipopolysaccharide-binding protein (LBP)-dependent extraction and transfer of individual endotoxin molecules to CD14 in Toll-like receptor 4 (TLR4)-dependent cell activation. Endotoxin is normally embedded in the outer membrane of intact Gram-negative bacteria and shed membrane vesicles ("blebs"). However, the ability of LBP and CD14 to efficiently promote TLR4-dependent cell activation by membrane-associated endotoxin has not been studied extensively. In this study, we used an acetate auxotroph of Neisseria meningitidis serogroup B to facilitate metabolic labeling of bacterial endotoxin and compared interactions of purified endotoxin aggregates and of membrane-associated endotoxin with LBP, CD14, and endotoxin-responsive cells. The endotoxin, phospholipid, and protein composition of the recovered blebs indicate that the blebs derive from the bacterial outer membrane. Proteomic analysis revealed an unusual enrichment in highly cationic (pI > 9) proteins. Both purified endotoxin aggregates and blebs activate monocytes and endothelial cells in a LBP-, CD14-, and TLR4/MD-2-dependent fashion, but the blebs were 3 - 10-fold less potent when normalized for the amount of endotoxin added. Differences in potency correlated with differences in efficiency of LBP-dependent delivery to and extraction of endotoxin by CD14. Both membrane phospholipids and endotoxin are extracted by LBP/soluble CD14 (sCD14) treatment, but only endotoxin center dot sCD14 reacts with MD- 2 and activates cells. These findings indicate that the proinflammatory potency of endotoxin may be regulated not only by the intrinsic structural properties of endotoxin but also by its association with neighboring molecules in the outer membrane.