大肠杆菌O157:H7氟苯尼考耐药菌株与敏感菌株的蛋白组学差异

Differential proteomics analysis in Escherichia coli O157∶H7 between florfenicol-resistant strain and florfenicol-sensitive strain

【目的】分析大肠杆菌O157:H7氟苯尼考耐药菌株与敏感菌株的差异表达蛋白,从蛋白组学层面揭示大肠杆菌O157:H7对氟苯尼考耐药性的产生机制。【方法】提取大肠杆菌O157:H7氟苯尼考耐药菌株(RS2-256)和敏感菌株(RS2)的全菌蛋白,经同位素标记相对和绝对定量(iTRAQ)标记后进行液相色谱串联质谱(LC-MS/MS)分析,质谱数据通过Mascot v2.2鉴定,并根据COGs数据库对差异表达蛋白进行GO功能注释和分类。【结果】RS2菌株获得3385个肽段,RS2-256菌株获得3955个肽段,合计7340个肽段,其中6975个肽段为特有肽段,分属于1039个蛋白。与RS2菌株相比,RS2-256菌株有145个蛋白表达差异显著(P<0.05),占检测蛋白总数的13.96%,其中,上调表达蛋白有87个、下调表达蛋白有58个。表达差异蛋白按照功能进行分类,可分为外排泵、转运蛋白、细胞膜形成、应激调控、核糖体结构、DNA复制、转录、翻译、翻译后修饰、能量产生和转化、氨基酸转运和代谢、辅酶转运和代谢、糖转运和代谢、脂肪代谢、离子转运和代谢、细菌运动性、转座及信号通路共18类;多药物外排泵亚基AcrA和AcrB、外膜蛋白TolC、外膜蛋白X等52个蛋白的表达变化在3.00倍以上,占表达差异蛋白总数的35.86%。【结论】外排泵AcrAB-TolC家族、ABC家族、外膜蛋白、毒性调节器B、饥饿蛋白等在大肠杆菌O157:H7对氟苯尼考耐药性产生过程中发挥重要作用,同时涉及细菌糖代谢、氨基酸代谢、离子转运、DNA损伤与修复、生物膜及毒素与抗毒素系统等生命活动。

【Objective】This study was to identify differentially expressed proteins in Escherichia coli O157:H7 between florfenicol-resistant strain and florfenicol-sensitive strain,and reveal the resistance mechanism of E. coli O157:H7 against flunicol from protein level.【Method】Complete bacterial proteins were extracted from florfenicol-resistant strain (RS2-256)and florfenicol-sensitive strain(RS2)of E. coli O157:H7,and labelled with isobaric tags for relative and absolute quantification(iTRAQ)reagent. Different expression proteins were identified and quantified with liquid chromatography tandem mass spectrometry(LC-MS/MS). The mass spectrometry data was identified and analyzed with Mascot v2.2. GO functional annotation and classification analysis of differentially expressed proteins were performed according to COGs database.【Result】The RS2 strain obtained 3385 peptides,and the RS2-256 strain obtained 3955 peptides,a total of 7340 peptides,of which 6975 peptides were specific peptides belonging to 1039 proteins. Compared with RS2 strain, expressions of 145 proteins of RS2-256 strain were significantly different(P<0.05),accounting for 13.96% of the total protein detected,among which 87 proteins were up-regulated and 58 proteins were down-regulated. The differentially expressed proteins could be divided into 18 categories according to their functions:efflux pump,transporters,cell membrane biogenesis,stress control,the structure of the ribosome,DNA replication,transcription,translation,posttranslational modification,energy production and conversion,amino acid transport and metabolism,coenzyme transport and metabolism,sugar transport and metabolism,fat metabolism,ion transport and metabolism,bacterial motility,translocation,signaling pathways and metabolism. The expression of 52 proteins,including multidrug efflux pump subunits acrA and acrB,outer membrane protein TolC and outer membrane protein X,varied more than 3.00 times,accounting for 35.86% of the total number of differentially expressed proteins.【Conclusion】The results show that various mechanisms such as the efflux pump AcrAB-TolC family,ABC family,outer membrane protein,toxicity regulator B,starvation protein play important roles in the development of resistance of E. coli O157:H7 to fluorbenicol. At the same time,it involves bacterial glucose metabolism,amino acid metabolism,ion transport,DNA damage and repair,biofilm,toxin and antitoxin systems and other life activities.