嗜水气单胞菌Sec分泌系统亚基SecD的生理功能研究

Physiological Functions of SecD in Aeromonas hydrophila

【目的】系统研究嗜水气单胞菌Sec分泌系统亚基SecD(Protein-export membrane protein,SecD)的生理功能。【方法】利用pRE112自杀载体,通过同源重组的方法,构建secD基因缺失株(标记为∆secD)。以嗜水气单胞菌野生型菌株为对照组,分别采用绵羊血、牛奶固体培养基测定∆secD的溶血性和胞外蛋白酶活性;结晶紫染色法结合多功能酶标仪测定生物被膜形成能力;利用全自动生长曲线仪进行细菌酸碱及高渗透压耐受性分析;采用二倍稀释法系统评估细菌抗生素耐药性。【结果】与嗜水气单胞菌野生型菌株相比,发现∆secD生物被膜形成能力降低,溶血性、胞外蛋白酶活性显著增强,碱性和高渗透压环境胁迫的耐受性增强,对盐酸土霉素、四环素、依诺沙星和美罗培南的MIC提高4倍,对环丙沙星和诺氟沙星的MIC提高2倍,而对红霉素和头孢噻肟钠的MIC分别降低了2倍和4倍。【结论】发现Sec分泌系统的亚基SecD参与嗜水气单胞菌毒力因子、抗生素耐药蛋白等的跨膜转运,对以Sec分泌系统的亚单位为靶标开发新一代抗菌药物提供理论依据,对预防和控制嗜水气单胞菌引起疾病的发生和传播可能具有重要的科学意义。

【Objective】Physiological functions of the Sec pathway subunit secD of Aeromonas hydrophila were studied.【Method】An secD-knockout A.hydrophila was constructed by homologous recombination with pRE112 suicide vector.Effects of the genetic deletion on the physiological phenotypes including hemolytic and extracellular protease activities were determined on sheep’s blood and skim milk agar plates;on the biofilm formation,by crystal violet staining combined with SpectraMax®i3;on the bacterial acid-base and hyperosmolarity tolerance,according to the growth curve;and on the minimal inhibitory concentration(MIC)of antibiotics,using the broth microdilution method.【Result】Compared to wild-type,the constructed∆secD A.hydrophila showed no significant differences in growth and tolerance to pH 5,6,7,and 8.The activities of hemolytic and extracellular protease,as well as the resistance to high osmotic pressure or pH 9 were enhanced.The biofilm forming ability significantly declined.And the resistance to antibiotics varied,as the MIC increased 4 times on oxytetracycline,tetracycline,enoxacin,or meropenem and two times on ciprofloxacin and norfloxacin,but decreased 2 times on erythromycin and 4 times on cefotaxime.【Conclusion】The Sec pathway subunit secD was confirmed to be involved in the transport of virulence factors and resistance to antibiotics in A.hydrophila.It could be a key element in studies for developing antimicrobial applications and prevention of disease infected by the pathogen.