Lin Ling, Sun Lina, Ali Farman, Guo Zhuang, Zhang Liang, Lin Wenxiong, Lin Xiangmin
1 Fujian Provincial Key Laboratory of Agroecological Processing and Safety Monitoring, College of Life Sciences, Fujian Agriculture and Forestry University , Fuzhou, People's Republic of China .
2 Key Laboratory of Crop Ecology and Molecular Physiology of Fujian Universities, Fujian Agriculture and Forestry University , Fuzhou, People's Republic of China .
Microb Drug Resist. 2018 Oct;24(8):1067-1074. doi: 10.1089/mdr.2017.0324. Epub 2018 Jan 22.
In Gram-negative bacteria, the outer membrane proteins (OMPs) perform a crucial role in antibiotic resistance, but it is largely unknown how they behave in response to antibiotic stress. In this study, we treated Aeromonas hydrophila with two different doses of oxytetracycline (OXY) to induce antibiotic stress. Proteins were isolated from sarcosine-insoluble fractions and quantitatively examined by using tandem mass tag labeling-based mass spectrometry to identify differentially expressed proteins. As a result, we identified 125 differential proteins in the 5 μg/ml OXY treatment group, including 20 OMPs, and 150 proteins from the 10 μg/ml OXY group, including 22 OMPs. Gene ontology analysis showed that translation-related proteins, including 30S and 50S ribosome proteins, were significantly enriched in increasing abundance under OXY stress; whereas the downregulated proteins were associated with the transport process, such as maltodextrin, maltose, and oligosaccharide transport. We then validated a subset of the identified differential proteins by using Western blot and quantitative polymerase chain reaction analyses. Finally, the quantitative real-time PCR (qPCR) results showed that at the transcription level, the expression of five OMP genes, including AHA_1280 (protein name A0KHS0), AHA_1281 (A0KHS1), AHA_1447 (A0KI84, BamE), AHA_1861 (A0KJE1), and AHA_2766 (A0KLX3), and one lipoprotein gene AHA_1740 (A0KJ25) was consistent with proteomic results under 5 and 10 μg/ml OXY treatment, respectively. In addition, the Western blotting also demonstrated that two altered OMP proteins A0KHS1 and A0KHH2 were upregulated for both OXY treatment groups. This study indicates that bacteria regulate the expression levels of OMPs in response to antibiotic stress and further contribute to our understanding of the functions of OMPs in antibiotic resistance. Moreover, our results suggest that the upregulation of translation and downregulation of the transport process may affect bacterial fitness during OXY stress. These findings may provide new clues to the antibiotic resistance mechanism in A. hydrophila.
在革兰氏阴性菌中，外膜蛋白（OMPs）在抗生素耐药性方面发挥着关键作用，但它们在抗生素应激下的行为在很大程度上尚不清楚。在本研究中，我们用两种不同剂量的土霉素（OXY）处理嗜水气单胞菌以诱导抗生素应激。从肌氨酸不溶性组分中分离蛋白质，并使用基于串联质量标签标记的质谱法进行定量检测，以鉴定差异表达的蛋白质。结果，我们在5μg/ml OXY处理组中鉴定出125种差异蛋白，包括20种OMPs，在10μg/ml OXY组中鉴定出150种蛋白，包括22种OMPs。基因本体分析表明，在OXY应激下，与翻译相关的蛋白质，包括30S和50S核糖体蛋白，丰度显著增加；而下调的蛋白质与转运过程相关，如麦芽糖糊精、麦芽糖和寡糖转运。然后，我们通过蛋白质免疫印迹和定量聚合酶链反应分析验证了一部分鉴定出的差异蛋白。最后，定量实时PCR（qPCR）结果表明，在转录水平上，包括AHA_1280（蛋白名称A0KHS0）、AHA_1281（A0KHS1）、AHA_1447（A0KI84，BamE）、AHA_1861（A0KJE1）和AHA_2766（A0KLX3）在内的5种OMP基因以及一种脂蛋白基因AHA_1740（A0KJ25）的表达分别与5μg/ml和10μg/ml OXY处理下的蛋白质组学结果一致。此外，蛋白质免疫印迹还表明，两种变化的OMP蛋白A0KHS1和A0KHH2在两个OXY处理组中均上调。本研究表明，细菌会响应抗生素应激调节OMPs的表达水平，这进一步有助于我们理解OMPs在抗生素耐药性中的功能。此外，我们的结果表明，翻译上调和转运过程下调可能会影响OXY应激期间细菌的适应性。这些发现可能为嗜水气单胞菌的抗生素耐药机制提供新线索。
