活体内磷脂酰胆碱替代磷脂酰乙醇胺影响E. coli细胞的功能

In vivo replacement of phosphatidylethanolamine by phosphatidylcholine affects biological function of E. coli

摘要:

摘要:[目的] 通过磷脂酰胆碱(PC)阳性大肠杆菌菌株与磷脂酰乙醇胺(PE)阳性野生型和PE阴性突变型菌株比较，并用PC+PE+双阳性菌株佐证，从细菌形态、生理生化以及巨噬细胞对细菌的吞噬作用等方面探讨活体内磷脂酰胆碱能否替代磷脂酰乙醇胺。[方法] 光学和电子显微镜观察细菌形态和结构；不同条件培养细菌并使用分光光度计测定OD600值，评估细菌的生长情况；使用SDS-PAGE和2-D电泳法测定细菌间质蛋白组分；用小鼠RAW264.7 巨噬细胞系检测细菌的粘附和吞噬作用。[结果]与短棒状的PE+野生型细菌AD93/pDD72相比，PC+细菌AD93/ptac67中仍有25%丝状体；与PE-突变体AD93一样，PC+细菌AD93/ptac67仍需要添加二价离子Mg2+或Ca2+才能生长；与野生型AD93/pDD72相比，PC+细菌AD93/ptac67的周质蛋白组分、粘附率与相对吞噬效率呈现明显的差异；与野生型细菌Top10/ptac85相比，PE+PC+双阳性细菌Top10/ptac66的细胞壁外层、抗逆性和周质蛋白组分也显示差别。[结论]在活体内，膜磷脂中PC替代PE不能使PE-突变体细胞的功能完全恢复至野生型状态，PE和PC在功能上存在明显的差别，两者在功能上不能相互替代。

Abstract:

Abstract: [Objective] We investigated differences in morphology, physiology, periplasmic proteins and phagocytosis by macrophage among E. coli strains AD93(PE-PC-), AD93/ptac67(PE-PC+), Top10/ptac66(PE+PC+) and the wild types in order to understand if phosphatidylcholine (PC) can substitute phosphatidylethanolamine (PE) in vivo. [Methods] Bacterial cells were observed under microscope after staining with Gram-staining kit or by electron microscope. Bacterial growth under different conditions was monitored by measuring the absorbance at the wavelength of 600 nm. Periplasmic proteins were analyzed using SDS-PAGE and 2-D electrophoresis. Bacterial adherence and phagocytosis by macrophage were also examined by using murine RAW264.7 macrophage. [Results] 100% bacterial cells in AD93/pDD72 were bar-shaped but 25% AD93/ptac67 cells came out as long filaments. Different from AD93/pDD72, AD93/ptac67 and AD93 required Mg2+or Ca2+ for growth. Moreover, AD93/ptac67 displayed a different pattern of periplasmic proteins on a 2-D gel and a low relatively phagocytic efficiency in the phagocytosis test when compared to AD93/pDD72 and AD93. Both Top10/ptac66 and the wild-type Top10/ptac85 cells were bar-shaped under microscope, but the former showed noticeably difference in the outer-layer structure of cell wall, and its stress resistance and periplasmic protein composition were also different from those of the latter. [Conclusion] Substitution of phosphatidylethanolamine with phosphatidylcholine in E. coli cells is unable to restore the phonotype of PE- mutant to the wild type. Biological functions of PE and PC are different, and phosphatidylcholine cannot substitute phosphatidylethanolamine in vivo.