基于蛋白质组学的肺炎支原体耐药机制调控网络的探索研究

发布时间：2018-02-25 00:00

本文关键词： 比较蛋白质组学肺炎支原体大环内酯类耐药　出处：《中国人民解放军医学院》2015年博士论文　论文类型：学位论文

【摘要】：目的肺炎支原体(Mp)是社区获得性肺炎(CAP)最常见致病原之一,目前世界范围内Mp对大环内酯类药物的耐药形势日趋严峻,但对其耐药机制的深入研究还很少,多数研究只是针对基因水平上药物结合位点的突变检测以及流行病学方面的调查。本研究拟从蛋白质水平探讨Mp对大环内酯类的耐药机制,以期能鉴定注释新的耐药相关蛋白,并探讨其相关的基因调控网络。方法对Mp临床分离株进行大环内酯类的药物敏感性测定。应用基于液相色谱-串联质谱(LC-MS/MS)的iTRAQ定量蛋白质组组学技术,对高MIC的临床耐药株和敏感株进行高通量蛋白质测序及鉴定。运用比较蛋白质组学原理,以敏感株为对照,找出2株临床耐药株共有的差异蛋白。再对这些差异蛋白进行包括GO、COG、KEGG、富集分析等的生物信息学分析,然后在大样本的Mp菌株中,对目标差异蛋白进行RT-PCR验证,最终确定可能与耐药相关的潜在蛋白。结果本研究共鉴定出蛋白496种。敏感株FH和耐药株S20比较差异表达蛋白为10种；敏感株FH和耐药株S56比较差异表达蛋白有18种。这两个比较组共有的差异蛋白为7种,且这7种蛋白无一出现在S20和S56比较组的差异蛋白中。通过生物信息学分析,确定了这些蛋白的分子功能和参与的代谢调控网络,并进行富集分析,但未发现这7种差异蛋白与其他的大环内酯类耐药机制相关。对上述菌体差异蛋白,在53株Mp样本中进行RT-PCR验证,发现差异蛋白MPN480、MPN277的编码基因在红霉素耐药菌株中的表达量较敏感株组同样显著上调。结论MPN480、MPN277可以作为蛋白质水平上潜在的Mp耐药分子标识,但尚需在更大的样本中进一步验证。本研究首次利用蛋白质组学方法研究Mp耐药机制,为耐大环内酯类Mp的机制研究开辟新的领域。
[Abstract]:Objective Mycoplasma pneumoniae (MP) is one of the most common pathogens of community-acquired pneumonia (CPAP). At present, the drug resistance of MP to macrolides is becoming more and more serious, but the mechanism of drug resistance is seldom studied. Most studies have focused on mutation detection and epidemiological investigation of drug binding sites at the gene level. This study is intended to explore the mechanism of MP resistance to macrolides at the protein level in order to identify and annotate new drug-resistance related proteins. Methods the drug sensitivity of macrolides in clinical isolates of MP was determined. ITRAQ quantitative proteomics technique based on liquid chromatography-tandem mass spectrometry (LC-MS / MS) was used. The high throughput protein sequencing and identification of clinical resistant and sensitive strains with high MIC were carried out. Using the principle of comparative proteomics, the sensitive strains were used as control. The differential proteins shared by two clinical resistant strains were identified. The differential proteins were analyzed by bioinformatics, including gocog, KEGG, enrichment analysis, and then the target differential proteins were tested by RT-PCR in a large sample of MP strains. Results in this study, 496 proteins were identified, and 10 differentially expressed proteins were identified between FH and S20. There were 18 differentially expressed proteins in FH and S56. There were 7 differentially expressed proteins in these two groups, and none of them appeared in the differential proteins of S20 and S56. The molecular function and metabolic regulatory network of these proteins were determined, and the enrichment analysis was carried out. However, the seven differential proteins were not found to be related to other macrolide resistance mechanisms. The expression of MPN480- MPN277 was also significantly up-regulated in erythromycin resistant strains by RT-PCR validation in 53 strains of MP. Conclusion MPN480- MPN277 can be used as a potential molecular marker of MP resistance at protein level. In this study, proteomics was used to study the mechanism of MP resistance for the first time, which opened up a new field for the study of the mechanism of macrolide resistance.
【学位授予单位】：中国人民解放军医学院
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R446.5

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