Sun Lina, Chen Huarong, Lin Wenxiong, Lin Xiangmin
Fujian Provincial Key Laboratory of Agroecological Processing and Safety Monitoring, College of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou 35002, PR China; Key Laboratory of Crop Ecology and Molecular Physiology of Fujian Universities, Fujian Agriculture and Forestry University, Fuzhou 35002, PR China.
Fujian Provincial Key Laboratory of Agroecological Processing and Safety Monitoring, College of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou 35002, PR China; Key Laboratory of Crop Ecology and Molecular Physiology of Fujian Universities, Fujian Agriculture and Forestry University, Fuzhou 35002, PR China.
J Proteomics. 2017 Jan 6;150:141-148. doi: 10.1016/j.jprot.2016.09.006. Epub 2016 Sep 13.
Edwardsiella tarda is a virulent fish pathogen that causes extensive economic losses in the aquaculture industry worldwide. The antibiotic resistance status of E. tarda is high, especially in the biofilm status; however, the mechanisms underlying its resistance remain largely unknown. In this study, isobaric tag for relative and absolute quantitation (iTRAQ)-based quantitative proteomics methods were used to compare the differential expression of E. tarda in response to oxytetracycline (OXY) stress in biofilm. Additional bioinformatics analysis demonstrated an increasing abundance of translation-related proteins, especially ribosomal subunits, and a decreasing abundance of key metabolic pathways underlying the adaptation of E. tarda to OXY in biofilm. We performed Western blotting and quantitative PCR (qPCR) analyses to validate selected proteomics results, and measured enzyme activity to verify the antibiotic resistance functions of central metabolic pathways. In addition, we examined the antibiotic susceptibility of a mutant of an NADP-dependent malic enzyme (MaeB), which is involved in the bacterial tricarboxylic acid cycle, and found significantly increased resistance to OXY in biofilm. Our findings demonstrate the importance of central metabolic pathways in the antibiotic resistance of E. tarda to bacterial biofilms and provide insight into the prevention of this resistance, which would aid in disease control.
The antibiotics resistance mechanisms in E. tarda have been well documented recently; however, its response to antibiotics in biofilms remains elusive. Our current study is the first exploratory report investigating this aspect via an iTARQ-based quantitative proteomics method. Several important proteins, related processes, and metabolic pathways were found to be involved in OXY fitness in biofilm status. Most importantly, the depletion of the maeB gene decreased the susceptibility of E. tarda to OXY indicating the important role of central metabolic pathways in antibiotics resistance in biofilm.
迟缓爱德华氏菌是一种致病性很强的鱼类病原体，在全球水产养殖业中造成了巨大的经济损失。迟缓爱德华氏菌的抗生素耐药性很高，尤其是在生物膜状态下；然而，其耐药机制在很大程度上仍不清楚。在本研究中，采用基于相对和绝对定量的等压标签（iTRAQ）定量蛋白质组学方法，比较迟缓爱德华氏菌在生物膜中对土霉素（OXY）应激的差异表达。进一步的生物信息学分析表明，与翻译相关的蛋白质，特别是核糖体亚基的丰度增加，而迟缓爱德华氏菌在生物膜中适应OXY的关键代谢途径的丰度降低。我们进行了蛋白质免疫印迹和定量PCR（qPCR）分析以验证所选的蛋白质组学结果，并测量酶活性以验证中心代谢途径的抗生素耐药功能。此外，我们检测了参与细菌三羧酸循环的烟酰胺腺嘌呤二核苷酸磷酸（NADP）依赖性苹果酸酶（MaeB）突变体的抗生素敏感性，发现其在生物膜中对OXY的耐药性显著增加。我们的研究结果证明了中心代谢途径在迟缓爱德华氏菌对细菌生物膜的抗生素耐药性中的重要性，并为预防这种耐药性提供了见解，这将有助于疾病控制。
迟缓爱德华氏菌的抗生素耐药机制最近已有充分记载；然而，其在生物膜中对抗生素的反应仍不清楚。我们目前的研究是第一份通过基于iTARQ的定量蛋白质组学方法对此方面进行探索的报告。发现几种重要的蛋白质以及相关过程和代谢途径参与了生物膜状态下的OXY适应性。最重要的是，maeB基因的缺失降低了迟缓爱德华氏菌对OXY的敏感性，表明中心代谢途径在生物膜抗生素耐药性中的重要作用。
