家蚕BmPGRP-S5蛋白的抑菌活性及其在细胞免疫中的作用

Antibacterial activity of BmPGRP-S5 of Bombyx mori and its role in cellular immunity

【目的】肽聚糖识别蛋白(peptidoglycan-recognition proteins,PGRP)可以特异性识别细菌细胞壁中的肽聚糖(peptidoglycan,PGN),并通过免疫缺陷(immune deficiency,IMD)途径和Toll途径诱导昆虫抗菌肽的产生。本研究旨在探讨家蚕Bombyx mori肽聚糖识别蛋白BmPGRP-S5的抑菌活性及在引发家蚕细胞免疫中的作用。【方法】用果蝇胚胎S2细胞表达BmPGRP-S5蛋白;通过细菌生长曲线法检测BmPGRP-S5蛋白对大肠杆菌Escherichia coli K 12 D 31、金黄色葡萄球菌Staphylococcus aureus和巨大芽孢杆菌Bacillus megaterium的抑菌活性;ELISA检测BmPGRP-S5蛋白与大肠杆菌、金黄色葡萄球菌、枯草芽孢杆菌Bacillus subtilis胞壁组分的结合力;通过测定吸光值和观察家蚕血淋巴黑化反应分析BmPGRP-S5对细菌胞壁组分激活酚氧化酶原(prophenoloxidase,PPO)的影响,并通过异硫氰酸荧光素(FITC)标记法检测BmPGRP-S5对家蚕血细胞吞噬细菌的影响。【结果】获得表达纯化的BmPGRP-S5蛋白。BmPGRP-S5蛋白对金黄色葡萄球菌、大肠杆菌K 12 D 31和巨大芽孢杆菌无抑菌效果,加入40μmol/L的Zn 2+后,其对巨大芽孢杆菌的抑菌效果明显增强。ELISA结果表明,BmPGRP-S5蛋白与来自金黄色葡萄球菌的PGN和枯草芽孢杆菌脂磷壁酸(LTA)的结合能力较强,同时也促进了这两种细菌胞壁组分激活家蚕血淋巴酚氧化酶原,加快胞璧组分介导的家蚕血淋巴黑化反应。加入BmPGRP-S5蛋白后,家蚕血细胞对金黄色葡萄球菌的吞噬率提高到53.33%左右,对大肠杆菌K 12 D 31的吞噬率在25.83%左右,对巨大芽孢杆菌的吞噬率达30.83%左右,与对照组相比显著增强。【结论】BmPGRP-S5抑菌作用依赖于Zn 2+,可能与其酰胺酶活性有关。BmPGRP-S5可以通过识别细菌胞壁组分PGN或LTA在家蚕的黑化反应和细胞吞噬中发挥作用。本研究使用的BmPGRP-S5蛋白是通过在果蝇S2细胞进行重组表达获得,更能真实地反映其在昆虫体内生理状态下的功能活性。因此,本研究的结果对进一步开发利用BmPGRP-S5有指导意义。

【Aim】Peptidoglycan-recognition proteins(PGRP)can specifically recognize peptidoglycan(PGN)in bacterial cell walls,and trigger the production of antimicrobial peptides through the immune deficiency(IMD)pathway and Toll pathway in insects.This study aims to explore the antibacterial activity of the Bombyx mori PGRP-S5(BmPGRP-S5)and its function in initiating the cellular immunity in B.mori.【Methods】BmPGRP-S5 protein was expressed in Drosophila embryonic S2 cell line.The antibacterial activities of BmPGRP-S5 protein to Escherichia coli K 12 D 31,Staphylococcus aureus and Bacillus megaterium were assayed based on the bacterial growth curve.The binding ability of BmPGRP-S5 protein to bacterial cell wall components of E.coli,S.aureus and Bacillus subtilis was detected by ELISA.The effect of BmPGRP-S5 on the activation of prophenoloxidase(PPO)by bacterial cell wall components was analyzed by measuring the absorbance value and observing the hemolymph melanization reaction of B.mori.The effect of BmPGRP-S5 on the phagocytosis to bacteria by B.mori hemocytes was detected by fluorescein isothiocyanate isomer(FITC)labeling method.【Results】The expressed and purified BmPGRP-S5 protein was obtained.BmPGRP-S5 had no antibacterial effect on S.aureus,E.coli K 12 D 31 and B.megaterium.However,after adding 40μmol/L Zn 2+,the antibacterial effect of BmPGRP-S5 protein on B.megaterium was significantly enhanced.The ELISA results showed that the binding ability of BmPGRP-S5 with PGN from S.aureus and lipoteichoic acid(LTA)from B.subtilis was strong,which also promoted the activation of phenoloxidase in B.mori hemolymph by these two bacterial cell wall components.The melanization reaction of B.mori hemolymph mediated by cell wall components was accelerated by BmPGRP-S5.After adding BmPGRP-S5 protein,the phagocytic rates of B.mori hemocytes to S.aureus,E.coli K 12 D 31 and B.megaterium increased to about 53.33%,25.83%,and 30.83%,respectively,which were significantly higher than those of the control groups.【Conclusion】The antibacterial activity of BmPGRP-S5 is dependent on Zn 2+and may be related to its amidase activity.BmPGRP-S5 can play a role in melanization and phagocytosis in B.mori by recognizing bacterial cell wall components PGN or LTA.The BmPGRP-S5 protein used in this study was obtained by the recombinant expression in Drosophila S2 cells and can more truly display its functional activity in the physiological state of insects.Therefore,the results are instructive for the further development and utilization of BmPGRP-S5.