铜绿假单胞菌在常规培养与美罗培南压力下培养产生的外膜囊泡表征差异分析

甘奕箫 李梦雪 王志成

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微生物与感染 ›› 2024, Vol. 19 ›› Issue (3) : 129-139.

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铜绿假单胞菌在常规培养与美罗培南压力下培养产生的外膜囊泡表征差异分析

甘奕箫,李梦雪,王志成

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Comparison in the characterization of outer membrane vesicles from Pseudomonas aeruginosa in normal culture and under meropenem pressure

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本研究旨在通过透射电子显微镜、液相色谱-质谱联用多种技术手段分析铜绿假单胞菌（Pseudomonas aeruginosa , P.aeruginosa）在正常培养条件以及美罗培南刺激下产生的外膜囊泡（OMV，Outer membrane Vesicle）的物理表征和蛋白质成分的区别。同时体外培养小鼠巨噬细胞系Raw264.7细胞，采用酶联免疫吸附法（ELISA）检测细胞与OMV共孵育48小时后的多种炎性细胞因子的分泌情况。结果显示，一定浓度的美罗培南（Meropenem，MEM）压力培养下P.aeruginosa产生的OMV（MEM-OMV）在形态和尺寸上与正常培养产生的OMV（Normal-OMV）相比无明显变化，但是同样的培养时间下，MEM-OMV的离子浓度显著高于Normal-OMV（20±0.53×108/mL vs 1.50±0.76×108/mL）。蛋白质组学分析显示，两种OMV的蛋白质表达也有差异，MEM-OMV中与外排泵相关的MexA和MexB更多。细胞实验结果表明，MEM-OMV能够比Normal-OMV刺激Raw264.7细胞产生更高浓度的IL-6、TNF-α、IL-8和IFN-γ。综上，美罗培南的加入能够改变P.aeruginosa分泌OMV的产率、内含蛋白质以及对巨噬细胞的免疫刺激作用。

Abstract

This aim of this study was to distinct the physical characterization and protein components of outer membrane vesicle (OMV) secreted from Pseudomonas aeruginosa under normal culture condition and meropenem (MEM) stimulated culture by a variety of technological means including transmission electron microscopy (TEM), Liquid chromatography tandem mass spectrometry (LC-MS/MS). At the same time, mouse macrophage cell line Raw264.7 was cultured and incubated with OMV for 48 hours in vitro, the secretion level of various inflammatory cytokines in Raw264.7 cells was detected by enzyme-linked immunosorbent assay (ELISA). The results showed that, compared with normal culture samples (normal-OMV), there was no significant difference in the morphology and size of OMV derived from P. aeruginosa under pressure culture with a certain concentration of MEM (MEM-OMV). The particle concentration of MEM-OMV was significantly higher than the particle concentration of Normal-OMV (20 ± 0.53×108/mL vs 1.50 ± 0.76×108/mL). The results of proteomics analysis showed that the protein expression of the two kind of OMVs was also heterogeneous, MEM-OMV containing more MexA and MexB proteins related to efflux pumps. The results of cell experiments showed that MEM-OMV could stimulate Raw264.7 cells to produce significantly higher concentrations of IL-6, TNF-α, IL-8 and IFN-γ than Normal-OMV. In conclusion, the addition of meropenem can change the production rate of OMV secreted by P. aeruginosa, the inclusion of protein and the immune stimulation effect on macrophages.

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甘奕箫李梦雪王志成. 铜绿假单胞菌在常规培养与美罗培南压力下培养产生的外膜囊泡表征差异分析[J]. 微生物与感染. 2024, 19(3): 129-139

Comparison in the characterization of outer membrane vesicles from Pseudomonas aeruginosa in normal culture and under meropenem pressure[J]. Journal of Microbes and Infections. 2024, 19(3): 129-139

中图分类号： R378.99+1

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