对硝基苯酚对细菌产生持留菌的影响及其相关机制

Influence of p-nitrophenol on bacteria persisters

摘要:

[目的]研究对硝基苯酚（PNP）对大肠杆菌和铜绿假单胞菌产生持留菌的影响，并对转录组进行分析，阐明对硝基苯酚影响持留菌形成的相关机制。[方法]采用氧氟沙星抗生素探究对硝基苯酚对细菌产生持留菌的影响，并通过检测细菌自溶情况和呼吸抑制剂羰酰氰氯苯腙（CCCP）对持留菌比例的影响，然后通过转录组分析其相关基因的表达，最后通过实时荧光定量PCR和反义核酸进行相关功能基因的验证。[结果]PNP可以通过抑制大肠杆菌和铜绿假单胞菌的呼吸作用使其产生持留菌的比例增加，PNP不同浓度、作用不同时间和作用不同生长时期的菌体都会影响细菌产生持留菌的比例。PNP和呼吸抑制剂CCCP均能够抑制2个菌体的自溶情况，包括溶解氧含量的变化、蛋白质降解情况、细胞尺寸的变化和RNA完整性。转录组分析和实时荧光定量PCR实验结果表明加入PNP后，cyoA、appC两个基因在大肠杆菌和铜绿假单胞菌中的表达量均显著下降，再通过反义核酸抑制cyoA、appC的表达发现持留菌的比例和原始菌株相比均有所增加。[结论]PNP可以通过抑制细胞呼吸作用来增加细菌产生持留菌的比例。

关键词:持留菌;大肠杆菌;铜绿假单胞菌;对硝基苯酚;细胞呼吸

Abstract:

[Objective] To study the effect of p-nitrophenol (PNP) on persisters of Escherichia coli and Pseudomonas aeruginosa and to analyze the transcriptome to illustrate the impact of PNP on persister formation.[Methods] Ofloxacin was used to determine the bacteria persisters number. Cell self-digestion assay was done and the effect of respiratory inhibitors carbonyl cyanide chlorobenzene hydrazone (CCCP) on bacteria persisters was studied as well. Based on analysis of bacterial transcriptome, two genes (cyoA and appC) associated with the formation of persistence were screened. Their expressions were confirmed by Real-time Fluorescence Quantitative Polymerase Chain Reaction (PCR). The association between the two genes and the formation of persistence was also checked by antisense oligodeoxynucleotide assay.[Results] PNP inhibited the respiration of E. coli and P. aeruginosa, thus increased the proportion of bacterial persisters. PNP concentration, PNP function time and the bacterial growth period affected the proportion of bacteria persisters. PNP and CCCP inhibited the self-digestion of E. coli and P. aeruginosa, including the changes in dissolved oxygen, protein degradation and cell size, and the integrity of RNA. The results from transcriptome analysis and Real-time fluorescent quantitative PCR showed that PNP addition decreased the expression of cyoA and appC in E. coli and P. aeruginosa. Through the antisense oligodeoxynucleotide inhibitory expression of cyoA and appC was found that bacteria persister proportion increased compared with the original strain.[Conclusion] PNP can increase the proportion of bacteria persisters by inhibiting the cellular respiration.

Key words:persisters;Escherichia coli;Pseudomonas aeruginosa;nitrophenol;cell respiration