一氧化氮消除大肠杆菌对氨苄青霉素耐药性的分子机制研究

One of the most surprising and exciting developments in recent biological research is the discovery of the physiological and pathophysiological roles of nitric oxide (NO), which is a free radical gas and a multifunctional and widespread biological messenger molecule. Nowadays, with the universal utilization of various antibiotics or antimicrobials, drug resistance becomes a more and more complicated problem. At present, the relationship of NO with antibiotic resistance of Gram negative bacteria strains remains unclear. In our previous study, β-galactosyl-pyrrolidinyl diazeniumdiolates (β-Gal-NONOate) was reported as a β-galactosidase-activated and site-specific NO donor, which can efficiently accumulate intracellular NO concentration. NO was also found to possess the capability to overcome ampicillin (Amp) resistance in Escherichia coli, which may be a promising approach to combat widespread resistance in the future. In this proposal, β-Gal-NONOate will be used as an efficient NO donor to investigate the NO functions in reducing or eliminating ampicillin resistance of E. coli K-12. After successful inducement of ampicillin-resistant strains, β-Gal-NONOate will be applied to deliver different concentrations of exogenous NO into bacteria cells, restoring antibiotic susceptibility of bacteria. Relationship between NO concentrations and Amp tolerance will be studied. In order to investigate the differences in protein expression levels before and after NO treatment of bacteria, E. coli K-12 expressional proteome will be characterized with the use of two-dimensional differences gel electrophoresis (2D-DIGE). The distinct proteins will be selected and identified by MALDI-TOF/TOF-MS, followed by whole genome expression microarray analysis, carried out to determine differences among wild type, resistant and re-susceptible E.coli at the gene transcription level. Combining with measurements of carbon source metabolism by BIOLOG analysis and microstructure observation by TEM and SEM, a comprehensive explanation of NO molecule functions on genes, proteins, and metabolism in E. coli Amp resistance strains will be obtained and the mechanism for NO-reversal to Amp resistance in E. coli will be clarified. By using novel donor β-Gal-NONOate, this method can not only accurately reveal the relationship between the intracellular NO concentrations and the elimination of resistance, but also help to enrich knowledge about NO influence on microbial resistance. These results will provide support for NO as a potential tool in therapy and research to remove increasingly serious antibiotic resistances of bacteria.

一氧化氮(NO)具有复杂的生物功能，目前NO与革兰氏阴性菌的耐药性有何关系仍不清楚。针对前期发现的NO消除大肠杆菌(Escherichia coli)对氨苄青霉素(ampicillin, Amp)耐药性的现象，本研究拟利用新型NO供体-β-半乳糖基化二醇二氮烯翁(β-Gal-NONOate)向细菌胞内提供不同浓度的NO，确定外源NO浓度对大肠杆菌Amp 耐药程度的影响。利用双向差异电泳技术找到NO处理前后细菌的显著差异蛋白质并给予质谱鉴定，通过全基因组表达谱芯片技术分析在基因转录水平上的差异，结合BIOLOG法碳源代谢分析及微观形态观察，综合揭示NO对Amp 耐受大肠杆菌的基因、蛋白质、代谢层面有何影响，阐明NO逆转大肠杆菌耐药性的分子机制。这不但可以弥补前人研究中由于供体带来的误差，准确体现胞内NO浓度与耐药性的关系，还有助于增加人们对NO与微生物关系的新认识及克服细菌耐药性策略的研究。

气态小分子一氧化氮(Nitric Oxide, NO)具有广泛的生理功能。从1992年NO被Science杂志评选为明星分子，直至今天，越来越多的文章报道了NO功能的新发现，很多证据表明它与多种重要生命过程有关。本研究从前期发现的NO可降低细菌对抗生素的耐药性现象出发，利用新型供体β-半乳糖基化二醇二氮烯嗡(β-Gal-NONOate)为原料，定向胞内输送不同浓度的NO，分析了不同浓度NO对大肠杆菌(Escherichiacoli)K12氨苄青霉素(Ampicillin, Amp) 耐药性的影响。. 利用合成的β-Gal-NONOate处理E.coli K12耐药菌，通过形态学观察、BIOLOG碳源代谢分析、代谢组学分析、基因芯片表达谱分析四个水平，比对了原始菌、Amp耐药菌、NO处理菌三者之间的差异。SEM形态观察发现，耐药菌形态变化明显，长度增长，NO处理菌长度位于原始菌与耐药菌之间。BIOLOG法分析碳源代谢差异，结果表明原始菌经Amp处理后，对有机酸酯类碳源代谢明显增强，而对羧酸类碳源的利用能力明显降低。耐药菌在利用β-Gal-NONOate处理后，对糖类的代谢能力增强。采用气相色谱-质谱联用(GC-MS)技术对代谢组学数据进行检测分析，结果表明三种菌之间的主要代谢差异集中在乙胺类、葡萄糖苷、谷氨酸、亮氨酸、异亮氨酸、酪氨酸和D-2-氨基己二酸等化合物上。利用基因芯片技术对三种菌的基因表达谱进行分析，发现耐药菌经NO处理后，有16个基因显著上调，24个基因显著下调。进一步验证了NO虽然使耐药菌的耐药性显著降低，但此过程不是产生耐药性的简单逆过程，而是涉及到众多基因差异化表达和代谢物变化的非常复杂的过程。. 截止目前，很少有外源性NO与革兰氏阴性菌耐药性之间关系的准确报道，我们利用β-Gal-NONOate为工具，避免了前人研究中使用的NO供体不稳定、不能向细菌胞内提供有效、可控浓度的NO而造成的误差，提高了结果的可靠性。NO逆转大肠杆菌耐药性过程的发现，对于丰富NO分子的功能具有重要意义，本研究所得结果，为彻底阐明NO与革兰氏阴性菌抗生素耐药性的关系奠定了基础，值得在未来开展进一步深入研究。.