类鼻疽伯克霍尔德菌基因BPSL1972缺失株的构建及其生物学特性分析  

Construction of BPSL1972 null mutation strain of Burkholderia pseudomallei and its biological characterization

作　　者：朱侨 田申 王彦双[1,2] 李雪苗 章越 李欣泽 夏乾峰[1] ZHU Qiao;TIAN Shen;WANG Yanshuang;LI Xuemiao;ZHANG Yue;LI Xinze;XIA Qianfeng(School of Tropical Medicine,Hainan Medical University,Haikou,Hainan 571199,China;The Second Affiliated Hospital of Hainan Medical University,Haikou,Hainan 570216,China)

机构地区：[1]海南医学院热带医学院,海南海口571199 [2]海南医学院第二附属医院,海南海口570216

出　　处：《中国热带医学》2024年第6期644-651,679,共9页China Tropical Medicine

基　　金：国家自然科学基金项目(No.82260003)。

摘　　要：目的 研究可能的外排泵基因BPSL1972在类鼻疽伯克霍尔德菌(Burkholderia pseudomallei, Bp)中的生物学功能,以完善Bp的耐药形成机制。方法 以Bp海南标准菌株HNBP001为对象,通过同源重组和电击转化分别构建BPSL1972基因敲除株及其回补株,通过酶切酶联的方法构建BPSL1972编码蛋白表达载体,通过His标签蛋白表达试剂盒对BPSL1972编码的蛋白表达,通过Western Blot技术对表达的蛋白进行验证,通过微量肉汤法、K-B法、比浊法、试管法、结晶紫染色和CCK-8试剂盒,依次比较了其在药敏、生长、生物膜形成和细胞毒力上的变化,通过Protscale在线网站、Protparam在线网站预测理化性质,利用SignalP 5.0在线网站预测BPSL1972编码蛋白的信号肽以及Smart在线网站预测了BPSL1972编码蛋白的跨膜结构域及基因功能,通过Phyre 2在线网站对BPSL1972编码蛋白3D结构进行构建并进行同源性分析。结果 预测结果显示,BPSL1972编码蛋白是一类中性稳定的疏水性蛋白,其具有一个脂蛋白信号肽及一个跨膜结构区,属于RND外排泵系统,具有2个脂蛋白外膜外排蛋白,可能介导大环内酯类抗生素的耐药,其3D建模结果显示其与VceC和镰刀酸外排蛋白高度同源,可能介导离子活化剂的外排。测序结果显示缺失株和回补株构建成功。但缺失株在各生物学特性上与野生株无差异,并且对头孢类、喹诺酮类、碳青霉烯类、氨基糖苷类、四环素类等抗生素及脱氧胆酸钠等表面活性剂也无明显差异。结论 在Bp中可能存在能够代替BPSL1972的基因,或者BPSL1972可能为LysR家族的辅助基因,而这种辅助基因是次要的,因此导致了其缺失后没有发生功能变化,其对其他抗生素如镰刀酸是否存在耐药性及具体的代替或辅助功能还有待后续研究。Objective To study the biological function of the possible efflux pump gene BPSL1972 in Burkholderia pseudomallei(Bp),so as to improve the drug resistance mechanism of Bp.Methods BPSL1972 gene knockout strain and its complement strain were constructed by homologous recombination and electric shock transformation,respectively.BPSL1972 protein expression vector was constructed by enzyme digestion and enzyme-linked method.The BPSL1972-encoded protein was expressed using a His-tag protein expression kit and the expressed proteins were verified by Western Blot.The changes in drug sensitivity,growth,biofilm formation,and cytotoxicity were compared by microbroth dilution assay,Kirby-Bauer assay,turbidimetric assay,tube assay,crystal violet staining,and CCK-8 kit.The physicochemical properties of BPSL1972 were predicted by Protscale and Protparam online websites,the signal peptide of BPSL1972 was predicted by SignalP 5.0 online website,and the transmembrane domain and gene function of BPSL1972 were predicted by Smart online website.The 3D structure of BPSL1972-encoded protein and its homology were analyzed through Phyre 2 online website.Results The predictions suggested that the BPSL1972-encoded protein is a class of neutral-stable hydrophobic protein with a lipoprotein signal peptide and a transmembrane region,classified under the RND efflux pump system with two lipoprotein outer membrane efflux proteins,potentially mediating macrolide resistance.Its 3D modeling results show high homology to VceC and fusaric acid efflux protein,possibly mediating the efflux of ion activators.Sequencing results confirmed the successful construction of both the deletion mutant strain and complement strain.However,there were no significant differences in biological characteristics between the deletion mutant strain and the wild-type strain,and there was also no significant difference in resistance to antibiotics such as cephalosporins,quinolones,carbapenems,aminoglycosides,tetracyclines,and surfactants such as sodium deoxycholate.Conclusio

关 键 词：类鼻疽伯克霍尔德菌 外排泵 耐药 

分 类 号：R378[医药卫生—病原生物学]