基于转录组学与蛋白质组学对猪丹毒丝菌耐药性的研究

Study on the resistance of Erysipelothrix rhusiopathiae based on transcriptomics and proteomics

摘要:

[背景] β-内酰胺类（β-lactams）抗生素是防治猪丹毒的常用药物，其中青霉素（Penicillin，PG）更是首选药物。[目的] 运用转录组学与蛋白质组学方法初步探究猪丹毒丝菌（Erysipelothrix rhusiopathiae，E.rhusiopathiae）产生PG抗性的机制，为进一步开展E.rhusiopathiae耐药机制的研究奠定基础。[方法] 采用K-B纸片法和微量稀释法分别测定受试菌株AEr51、AEr31和AErS对26种抗生素的感受性及对PG、阿莫西林（AMX）和氨苄西林（AMP）的最小抑菌浓度（Minimun Inhibitory Concentration，MIC）；然后通过转录组学测序（RNA-Seq）技术和串联质谱标签法（Tandem Mass Tag，TMT）定量蛋白质组学技术进一步探究猪丹毒丝菌产生PG抗性的分子机制。[结果] AEr51、AEr31和AErS对26种抗生素耐药率分别为34.62%、26.92%和34.62%，其中AErS和AEr31对所有β-lactams抗生素敏感，而AEr51除对PG耐药外，对其他β-lactams抗生素均敏感；AEr51、AEr31和AErS对PG、AMX、AMP的MIC分别为32、4、2 μg/mL，0.25、0.50、0.50 μg/mL和0.125、0.500、0.250μg/mL；RNA-Seq分析显示AEr51/AErS比较组中共筛到668个差异基因，其中上调434个、下调234个，AEr51/AEr31比较组中共筛到403个差异基因，其中上调275个、下调128个，差异表达基因主要富集于代谢途径、ABC转运系统、β-内酰胺抗性、双组分信号传导系统等通路，而且与RT-qPCR验证结果基本一致；TMT分析显示AEr51/AErS比较组中共筛到167个差异蛋白，其中上调86个、下调81个，AEr51/AEr31比较组中共筛到159个差异蛋白，其中上调80个、下调79个，差异表达蛋白显著富集于微生物、氨基酸、碳、硫、嘧啶代谢等相关的代谢通路，而且与平行反应监视（Parallel Reaction Monitoring，PRM）靶向验证结果基本一致。[结论] ABC转运系统、双组分信号传导系统、β-内酰胺抗性等通路在猪丹毒丝菌对青霉素耐药性产生过程中发挥重要作用，同时伴随微生物、氨基酸、碳、硫、嘧啶代谢等生命过程。

Abstract:

[Background] β-lactam antibiotics are commonly used drugs for the prevention and treatment of swine erysipelas, and penicillin (PG) is the preferred drug. [Objective] The use of transcriptomics and proteomics methods to preliminarily explore the mechanism of Erysipelothrix rhusiopathiae (E. rhusiopathiae) producing penicillin resistance, laying a foundation for further research on the resistance mechanism of E. rhusiopathiae. [Methods] The susceptibility to 26 antibiotics and the minimum inhibitory concentration (MIC) to PG, amoxicillin (AMX) and ampicillin (AMP) of the tested strains AErS, AEr51 and AEr31 were determined by the Kirby-Bauer disk diffusion method and the microdilution method; Then through transcriptomics sequencing (RNA-Seq) technology and tandem mass tag (TMT) quantitative proteomics technology to further explore the molecular mechanism of E. rhusiopathiae producing penicillin resistance. [Results] The resistance rates of AErS, AEr51 and AEr31 to 26 antibiotics were 34.62%, 34.62%, and 26.92%, respectively. AErS and AEr31 were sensitive to all β-lactams antibiotics, while AEr51 was sensitive to other β-lactams antibiotics except PG; the MICs of AErS, AEr51 and AEr31 to PG, AMX and AMP were 0.125, 0.500, 0.250 μg/mL, 32, 4, 2 μg/mL and 0.25, 0.50, 0.50 μg/mL, respectively; RNA-Seq analysis showed a total of 668 differential genes were screened in the AEr51/AErS comparison group, of which 434 were up-regulated and 234 were down-regulated. In the AEr51/AEr31 comparison group, a total of 403 differential genes were screened, of which 275 were up-regulated and 128 were down-regulated. The differentially expressed genes were mainly enriched in the metabolic pathway, ABC transport system, two-component signal transduction system, β-lactam resistance and other pathways, and the results of RT-qPCR verification were basically the same; TMT analysis showed that a total of 167 differential proteins were screened in the AEr51/AErS comparison group, among which 86 were up-regulated and 81 were down-regulated. A total of 159 differential proteins were screened in the AEr51/AEr31 comparison group, of which 80 were up-regulated and 79 were down-regulated. Differentially expressed proteins were significantly enriched in metabolic pathways related to microorganisms, amino acids, carbon, sulfur, and pyrimidine metabolism, and the results of PRM targeting verification were basically the same [Conclusion] ABC transport system, two-component signal transduction system, β-lactam resistance and other pathways played an important role in the development of E. rhusiopathiae resistance to PG, and were accompanied by life processes, such as microorganisms, amino acid, carbon, sulfur, and pyrimidine metabolism, etc.