金属离子抑制细菌整合子捕获耐药基因盒的机制研究

Mechanism of metal ions against capturing drug resistance gene cassette by bacterial integron

摘要:

【目的】探索金属离子对整合子捕获耐药基因盒的影响及其相关机制。【方法】在大肠埃希菌中构建一个1类整合子捕获耐药基因盒的体内模型。通过实时荧光定量PCR (real-time fluorescence quantitative PCR，RT-qPCR)测定不同浓度银(0.3、0.9、1.5 μg/mL的Ag⁺)和铜离子(5、50、100、150、210 μg/mL的Cu²⁺)干预后实验组和无金属离子干预对照组整合子整合频率，并用表型筛选法验证。利用质谱法测量细菌吸收的金属离子浓度，并进一步通过转录组测序方法分析银离子抑制细菌整合子捕获耐药基因盒的分子机制。【结果】qPCR和表型筛选法的结果表明，0.9 μg/mL银离子组整合频率为9.42×10^‒5 (6.49×10^‒5，1.44×10^‒4)，1.5 μg/mL银离子组整合频率为7.29×10^‒5 (4.45×10^‒5，9.03×10^‒5)，与对照组2.59×10^‒4 (2.24×10^‒4，3.33×10^‒4)相比整合频率明显降低，差异有统计学意义(P<0.001)。而不同浓度铜离子组与对照组没有明显差异。转录组测序方法对银离子作用前后大肠杆菌基因表达水平进行对比分析，通过GO功能和KEGG通路富集发现，差异表达基因与甲基半乳糖苷转运(methylgalactoside transport)、麦芽糖转运(maltose transport)、磷酸烯醇式丙酮酸-甘油磷酸转移酶活性(phosphoenolpyruvate-glycerone phosphotransferase activity)和甘油激酶活性(glycerone kinase activity)等功能有关以及涉及氨基酸糖和核苷酸糖代谢(amino sugar and nucleotide sugar metabolism)、鞭毛组装(flagellar assembly)、阳离子抗菌肽(CAMP)耐药性[cationicantimicrobial peptide (CAMP) resistance]、果糖和甘露糖代谢和磷酸糖转移酶系统[phosphotransferase system (PTS)]等代谢通路。通过蛋白互作网络筛选出前12个枢纽基因(ptsG、malE、lamB、lacZ、malK、basR、ais、ugd、nagE、metN、malQ和malF)。【结论】一定浓度银离子可以抑制整合子整合耐药基因盒。铜离子对整合频率影响不明显，因此不是所有具有杀菌性的金属离子都能对整合频率产生影响。银离子抑制细菌整合子捕获耐药基因盒可能是通过影响麦芽糖转运和碳分解代谢来调控。然而，碳分解代谢和麦芽糖转运过程很复杂，需要进一步研究。目前尚无细菌整合子转录组学相关研究，这为解决细菌耐药性问题提供了一个新的途径。

Abstract:

[Objective] To explore the effect of metal ions against capturing drug resistance gene cassette by integron and the related mechanism.[Methods] An in vivo model of class 1 integron capturing resistance gene cassette was constructed in Escherichia coli.Different concentration of silver ions (0.3,0.9,and 1.5 μg/mL of Ag⁺) and copper ions (5,50,100,150,and 210 μg/mL of Cu²⁺) were used to intervene in the experimental group.The integration frequency of the experimental group and the control group was determined by real-time fluorescence quantitative polymerase chain reaction (RT-qPCR) and validated by phenotypic screening.The concentration of metal ions absorbed by bacteria was measured by mass spectrometry,and the molecular mechanism of silver ion against capturing drug resistance gene cassette by bacterial integron was further analyzed by transcriptome sequencing.[Results] The integration frequencies were 9.42×10^‒5(6.49×10^‒5,1.44×10^‒4) in the 0.9 μg/mL silver ion group and 7.29×10^‒5(4.45×10^‒5,9.03×10^‒5) in the 1.5 μg/mL silver ion group,which were lower the 2.59×10^‒4(2.24×10^‒4,3.33×10^‒4) in the control group (P<0.001).In contrast,there was no significant difference between the groups with different concentration of copper ions and the control group.The gene expression level of E.coli before and after silver ion treatment was compared and analyzed by transcriptome sequencing.The differentially expressed genes were related to the Gene Ontology (GO) terms of methylgalactoside transport,maltose transport,phosphoenolpyruvate-glycerone phosphotransferase activity,and glycerone kinase activity,and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways of amino sugar and nucleotide sugar metabolism,flagellum assembly,cationic antimicrobial peptide (CAMP) resistance,fructose and mannose metabolism,and phosphotransferase system (PTS).The top 12 hub genes (ptsG,malE,lamB,lacZ,malK,basR,ais,ugd,nagE,metN,malQ and malF) were screened out by protein-protein interaction (PPI) network.[Conclusion] A certain concentration of silver ions inhibited the integration of drug resistance gene cassettes by integron,while the effect of copper ions was not obvious.Therefore,not all metal ions with bactericidal properties had the effect on integration frequency.The mechanism of silver ions against capturing drug resistance gene cassette by bacterial integron was possibly through maltose transport and carbon catabolism.However,the processes of carbon catabolism and maltose transport were complex and deserved further study.At present,there is no related study on bacterial integron transcriptome.This study provides a new way to solve the problem of bacterial drug resistance.