Lin Xiangmin, Lin Ling, Yao Zujie, Li Wanxin, Sun Lina, Zhang Danfeng, Luo Ji, Lin Wenxiong
Fujian Provincial Key Laboratory of Agroecological Processing and Safety Monitoring, School of Life Sciences, Fujian Agriculture and Forestry University , Fuzhou 350002, PR China.
J Proteome Res. 2015 Mar 6;14(3):1515-25. doi: 10.1021/pr501188g. Epub 2015 Feb 4.
To date, above ten thousand tons of antibiotics are used in aquaculture each year that lead to the deterioration of natural resources. However, knowledge is limited on the molecular biological behavior of common aquatic pathogens against antibiotics stress. In this study, proteomics profiles of Aeromonas hydrophila, which were exposed to different levels of oxytetracycline (OXY) stress, were displayed and compared using iTRAQ labeling and SWATH-MS based LC-MS/MS methods. A total 1383 proteins were identified by SWATH-MS method, and 2779 proteins were identified from iTRAQ labeling samples. There are 152 up-regulated and 52 down-regulated proteins overlapped in 5 μg/mL OXY stress and both 83 up- and down-regulated proteins overlapped in 10 μg/mL OXY stress in both methods, respectively. Results show that many protein synthesis and translation related proteins increased, while energy generation related proteins decreased in OXY stress. The varieties of selected proteins involved in both pathways were further validated by sMRM(HR), q-PCR, and enzyme activity assay. Furthermore, the concentrations of NAD+ and NADH were measured to verify the characteristic of energy generation process in OXY stress and OXY resistance strain. We demonstrate that the down-regulation of energy generation related metabolic pathways and up-regulation of translation may play an important role in antibiotics fitness or resistance of aquatic pathogens.
迄今为止，每年有超过万吨的抗生素被用于水产养殖，这导致了自然资源的恶化。然而，关于常见水生病原体对抗生素应激的分子生物学行为的了解有限。在本研究中，使用iTRAQ标记和基于SWATH-MS的LC-MS/MS方法展示并比较了嗜水气单胞菌在不同水平的土霉素（OXY）应激下的蛋白质组学图谱。通过SWATH-MS方法共鉴定出1383种蛋白质，从iTRAQ标记样本中鉴定出2779种蛋白质。在两种方法中，分别有152种上调和52种下调的蛋白质在5μg/mL OXY应激中重叠，以及83种上调和下调的蛋白质在10μg/mL OXY应激中重叠。结果表明，在OXY应激下，许多与蛋白质合成和翻译相关的蛋白质增加，而与能量产生相关的蛋白质减少。通过sMRM(HR)、q-PCR和酶活性测定进一步验证了参与这两条途径的所选蛋白质的种类。此外，测量了NAD+和NADH的浓度，以验证OXY应激和OXY抗性菌株中能量产生过程的特征。我们证明，能量产生相关代谢途径的下调和翻译的上调可能在水生病原体对抗生素的适应性或抗性中起重要作用。
