金葡菌和大肠埃希菌细菌外囊泡的生物学特性比较研究

Biological properties of bacterial extracellular vesicles of Staphylococcus aureus versus Escherichia coli

背景近年来研究发现细菌可分泌细菌外囊泡(bacteria extracellular vesicles,BEVs),并发挥重要的生物学功能。工程化修饰细菌外囊泡从而进行药物递送载体构建、新型疫苗研发已成为研究新热点。然而目前缺乏对不同来源细菌外囊泡相关特性的比较研究。目的探索以金葡菌为代表的革兰阳性菌和以大肠埃希菌为代表的革兰阴性菌来源细菌外囊泡的理化性质和生物学特性。方法本研究通过切向流过滤系统结合超速离心法提取金葡菌和大肠埃希菌的细菌外囊泡,并通过电镜形貌、粒径、电位、蛋白产量、免疫细胞激活情况对金葡菌和大肠埃希菌细菌外囊泡进行比较。结果透射电镜下细菌外囊泡呈现杯状、茶托状的双层膜结构,大小为20~300 nm;金葡菌外囊泡37.84~396.10 nm,大肠埃希菌外囊泡13.54~78.82 nm;金葡菌和大肠埃希菌的细菌外囊泡带负电荷,分别为(-19.7±0.17)mV和(-24.1±2.55)mV;金葡菌和大肠埃希菌外囊泡的蛋白产量分别为(0.94±0.04)mg和(1.71±0.19)mg;颗粒数分别为4.00×10^(11)(±1×10^(10))/mL和9.47×10^(11)(±2.52×10^(10))/mL;颗粒数/蛋白量比值分别为5.59×10^(11)(±1.6×10^(10))和4.24×10^(11)(±6.43×10^(10));加入金葡菌囊泡后,巨噬细胞系RAW264.7中CD86阳性细胞占比为20.23%,比对照组(PBS组)升高45.9倍(P<0.001);CD206阳性细胞占比为4.5%,相比对照组升高34.6倍(P<0.001)。加入大肠埃希菌外囊泡后RAW264.7的CD86阳性细胞占比为11.16%,相比对照组升高25.4倍(P<0.001);CD206阳性细胞占比为2.09%,相比对照组升高16.1倍(P<0.05)。结论本研究通过切向流过滤系统结合超速离心法成功提取出金葡菌和大肠埃希菌的细菌外囊泡,且金葡菌外囊泡直径略大于大肠埃希菌外囊泡,Zeta电位相比大肠埃希菌略高。同等培养条件下,金葡菌外囊泡产量比大肠埃希菌外囊泡低,但对免疫细胞的激活能力更强。

Background In recent years,it has been found that bacteria can secrete bacterial extracellular vesicles(BEVs)to play important biological functions.Engineering modified BEVs for drug delivery carrier construction and new vaccine development have become new research hotspots.However,there is currently a lack of comparative studies on the relevant properties of BEVs from different sources.Objective To investigate the physicochemical properties and biological characteristics of BEVs derived from G+bacteria represented by Staphylococcus aureus and G-bacteria represented by Escherichia coli.Methods In this study,a tangential flow filtration system(TFF)and ultracentrifugation were used to construct a method for the extraction of BEVs.The BEVs of Staphylococcus aureus and Escherichia coli were compared by electron microscope morphology,particle size,potential,protein production,and immune cell activation.Results BEVs with double-membrane structure in cup-shaped and saucer-packed structures could be seen under transmission electron microscope,and the size was about 20-300 nm.Most of the BEVs of Staphylococcus aureus were 50.75±0.00 nm in diameter,and the particle size range was 37.84-396.1 nm;Most of the E.coli outer vesicles were 22.15±1.92 nm in diameter,and the particle size range was 13.54-78.82 nm;The BEVs of Staphylococcus aureus and Escherichia coli were negatively charged at(-19.7±0.17)mV and(24.1±2.55)mV,respectively;The protein yields of BEVs of Staphylococcus aureus and E.coli were(0.94±0.04)mg and(1.71±0.19)mg,respectively;The number of particles was 4.00×10^(11)(±1×1010)/mL,9.47×10^(11)(±2.52×10^(10))/mL;The ratio of particle number/protein amount was 5.59×10^(11)(±1.6×10^(10)),4.24×10^(11)(±6.43×10^(10)),respectively;After adding S.aureus vesicles,the proportion of CD86-positive cells in the macrophage cell line RAW264.7 was 20.23%,which was 45.9 times higher than that of the control group(PBS group)(P<0.001);The proportion of CD206-positive cells was 4.5%,which was 34.6 times higher than that in the control group(P<0.001).After adding E.coli-derived BEVs,the proportion of CD86-positive cells in RAW264.7 was 11.16%,which was 25.4 times higher than that in the control group(P<0.001);The proportion of CD206-positive cells was 2.09%,which was 16.1 times higher than that in the control group(P<0.05).Conclusion In this study,BEVs from Staphylococcus aureus and Escherichia coli are successfully extracted by the tangential flow filtration system and ultracentrifugation.And the diameter of BEVs from S.aureus is slightly larger than that of BEVs from E.coli,and the zeta potential is slightly higher than that of E.coli.Under the same condition,the output of BEVs from Staphylococcus aureus is lower than that of Escherichia coli,but it has a stronger ability to activate immune cells.