酸胁迫对鼠伤寒沙门氏菌抗酸性、细胞膜及膜蛋白的影响

Effect of Acid Stress on Acid Resistance,Cell Membrane and Membrane Protein of Salmonella typhimurium

目的:以鼠伤寒沙门氏菌CGMCC 1.1190为对象,研究柠檬酸反复胁迫处理对其抗酸性、细胞膜及其膜蛋白的影响。方法:将CGMCC 1.1190分别在经柠檬酸调节到pH值为3.0、2.7、2.5的胰蛋白胨大豆肉汤培养基中胁迫处理并转接12次培养后,获得3株CGMCC 1.1190的抗酸性菌株,测定了这3株抗酸性菌株的D值、菌落形态、个体形态、膜通透性、膜流动性和膜蛋白的变化。结果:这3株抗酸性菌株的D值随着酸处理次数的增加不断增大,酸胁迫的pH值越低,D值越大,菌落形态与个体形态变化越明显;与原始对照菌株相比,当这3株抗酸性菌株置于pH 2.0的强酸环境下时,其碘化丙啶染色区域的死菌比例显著降低(P<0.05),表明酸胁迫pH值越低,这3株抗酸性菌株的细胞膜对H^(+)通透性越低;随着酸胁迫pH值的降低,CGMCC 1.1190抗酸性菌株细胞膜磷脂的熔点升高,表明细胞膜磷脂的相变温度升高,细胞膜流动性降低;十二烷基硫酸钠-聚丙烯酰胺凝胶电泳结果表明,这3株抗酸性菌株分子质量35~180 kDa范围内的细胞膜蛋白表达量增加,在135 kDa和180 kDa处均增加了特异条带。结论:随着酸胁迫pH值的降低,CGMCC 1.1190抗酸性增加,菌落变小,个体形态变长,细胞膜通透性和流动性均降低,部分膜蛋白表达量和表达种类增加,这些变化有利于其适应在酸胁迫环境下生长,提高生存能力。

Objective:To investigate the effect of repeated citric acid treatment on the acid resistance,cell membrane and membrane protein of Salmonella typhimurium CGMCC 1.1190.Methods:Three acid-resistant strains were obtained after CGMCC 1.1190 was subjected to acid stress in trypticase soy broth(TSB)adjusted to pH 3.0,2.7 or 2.5 with citric acid,then transferred and incubated repeatedly for 12 times.The D-values,colony morphology,cellular morphology,membrane permeability,membrane fluidity and membrane protein of these acid-resistant strains were determined and compared with each other.Results:The D-values of the acid-resistant strains were increased with increasing number of acid stress treatments.The lower the pH,the larger the D-values and the more significant the differences in the morphology of colonies and individual cells among the three strains.Compared with the original strain,the acid-resistant strains showed a significant decrease in the percentage of dead cells stained by propidium iodide(PI)at pH 2.0(P<0.05).It was indicated that acid stress at lower pH led to lower cell membrane permeability to H^(+)together with an increase in the phase transition temperature(Tm)of cell membrane phospholipids in the acid-resistant strains,suggesting decreased cell membrane fluidity.The results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE)indicated increased expression of cell membrane proteins with molecular mass of 35 to 180 kDa in the acid-resistant strains accompanied by the appearance of new bands with molecular mass of 135 and 180 kDa.Conclusion:Acid stress at lower pH increased the acid resistance of CGMCC 1.1190,made the colonies smaller and the individual cells longer,reduced the cell membrane permeability and fluidity,and increased the expression of some cell membrane proteins and the types of expressed proteins.These changes could be beneficial for the growth of this strain under acid stress,improving its survival.