摘要
志贺氏菌(Shigella spp.)是一种最常见的引起人类腹泻的病原菌,严重威胁人类健康。抗生素作为一种见效快、成本低和易推广的治疗志贺氏菌的方法具有其现实意义。但是随之产生的细菌耐药性问题也困扰着抗生素治疗的发展。因此研究抗生素对志贺氏菌作用的分子机理和志贺氏菌致病机理具有较大的理论意义和应用价值。论文以福氏2a志贺菌301为材料,探究黄连素对福氏2a志贺菌301的作用机理,得到了以下主要结果:
首先,在对数早期和平台早期的福氏2a志贺菌301培养物中分别添加160μg/mL浓度的黄连素,发现黄连素添加时间不同,作用效果不同。志贺氏菌在黄连素孵育情况下的存活率:对数期实验组为62.45 %,对数期对照组为152.6 %;平台期实验组为91.22 %,平台期对照组为93.01 %。可见黄连素在对数早期对志贺氏菌生长表型的影响大于在平台早期时的影响。
应用双向电泳技术分析了添加160μg/mL浓度的黄连素后对福氏2a志贺菌301蛋白质表达谱的影响。结果表明,不同时期添加黄连素,对福氏2a志贺菌301蛋白质表达的影响也不尽相同,对数生长早期可引起9个蛋白质表达差异:谷氨酸脱羧酶B(GadB),谷氨酸脱羧酶A(GadA),高渗诱导周质蛋白(OsmY),超氧化物歧化酶前体(SodC)、细菌铁蛋白(Bfr),假想蛋白(YgiW),yhbH的sigma调节因子(YfiA),可能为Mxi-Spa途径分泌蛋白(OspC2)和假想蛋白(YqhE),它们均被下调;其中OsmY与细胞渗透压调控有关;GadB和GadA参与调节细胞抗酸性;OspC2与志贺氏菌毒力有关, SodC与细胞抗氧化胁迫有关。平台早期处理的引起3个蛋白质表达差异,其中上调的为:球面调节蛋白(Dps)和3-磷酸甘油醛脱氢酶A(GapA),下调的为:3-氧酰基还原酶(FabG)。有趣的是,谷氨酸脱羧酶(GadB)的表达量在黄连素处理后发生较大的变化,它可能在福氏2a志贺菌301致病中具有重要作用。
继而,利用λ-Red重组系统构建了gadB缺失突变株,研究了GadB在豚鼠角膜侵袭中的可能作用。结果表明,gadB基因缺失株不能利用甘油作为碳源代谢,豚鼠角膜炎症反应稍轻于野生株。基因缺失导致其全菌蛋白表达谱出现10个差异蛋白,主要包括6个与能量和生物大分子代谢有关的蛋白质:葡糖磷酸异构酶(Pgi)、磷酸甘油酸激酶(Pgk)、葡萄糖酸激酶(Gntk)、琥珀酸脱氢酶(SdhB)、硫辛酰胺脱氢酶(LpdA)和L-乳酸脱氢酶(LldD)。2个应激性激活的蛋白质烷基氢过氧化物还原酶(AphF)和FK结合蛋白型肽酰-脯氨酰-顺反异构酶(FkpA)。上述发现的差异蛋白为GadB蛋白潜在功能的进一步研究提供了线索和可能性。
Shigella spp. is one of the most common causes of diarrhea of human pathogen. It is still a serious threat to human health. Since antibiotics have some advantages such as a quick effect and low cost, antibiotics treatment of Shigella spp. has its practical significance. But the antibiotic resistance plagued the development of antibiotic treatment. So to study the molecular mechanism of antibiotic resistance in Shigella spp. and pathogenesis posseses a certain theoretical significance and has potential applicable value. In the present thesis, Shigella flexneri 2a stain 301 was utilized as biomaterials, the effect of berberine on the growth and its mechanisms were investigated, and the data were showed as follows:
First, effect of berberine on the growth of Shigella flexneri 2a stain 301 was dependent on the time of berberine treatment. The survival ratio of strains treated by berberine in the early logarithmic phase was much lower than that treated in early stationary phase, which was 62.45% (control, 152.6%) and 91.22% (control, 93.01%) respectively.
The two-dimensional gel electrophoresis (2-DE) was applied to detect the effect of berberine on the expressing of proteins of Shigella flexneri 2a stain 301. The results revealed that effect of berberine on the expressing of proteins was dependent on the addition time. Nine proteins were identified as berberine influenced proteins when the strain was treated by 160μg/mL in early logarithmic phase, e.g. GadB, GadA, OsmY, SodC, Bfr, YgiW, YfiA, OspC2 and YqhE, which were down regulated. In these proteins, OsmY is involved in osmotic regulation; GadB and GadA are related to acid resistance, OspC2 is correlated to virology, and SodC is involved in antioxidant. Three proteins were identified as berberine influenced proteins when the strain was treated by 160μg/mL in early stationary phase, e.g. Dps and GapA were up-regulated and FabG was down regulated. It was interested that the expression of GadB, the glutamate decarboxylase was significantly down regulated by berberine. The enzyme might have important role in the pathogenesis of Shigella flexneri 2a stain 301.
The null-mutant strain of gadB was successfully constructed. The mutant could not use glycerol as the only carbon source. Sereny test revealed that the virulence of gadB mutant were slightly weaker than wild-type strain. Deletion of gadB would influence the expressions of some proteins.A total of 10 proteins were identified as differentially expressed proteins according to the comparative proteomic results. The six proteins, Pgi, Pgk, Gntk, SdhB, LpdA and LldD were involved in energy and macromolecule metabolisms and two proteins, AphF and FkpA were related to shock responses. These data provided some trails for the function research of GadB.
引文
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