鸭源鸡杆菌tolC无痕敲除株的构建及生物学特性分析  

Construction and Analysis of Biological Characteristics of Gallibacterium anatis tolC Traceless Knockout Strain

作　　者：马立静 史姿聪 王新卫[1] 陈陆[1,2] 彭泽宇 李栋梁 杨霞 MA Li-Jing;SHI Zi-Cong;WANG Xin-Wei;CHEN Lu;PENG Ze-Yu;LI Dong-Liang;YANG Xia(College of Veterinary Medicine,Henan Agricultural University,Zhengzhou 450046,China;Ministry of Education Key Laboratory for Animal Pathogens and Biosafety,Zhengzhou 450046,China;Nanyang Vocational College of Agriculture of Veterinary Medicine and Engineering,Nangyang 473007,China)

机构地区：[1]河南农业大学动物医学院,郑州450046 [2]动物病原与生物安全教育部重点实验室,郑州450046 [3]南阳农业职业学院牧医工程学院,南阳473007

出　　处：《农业生物技术学报》2024年第4期974-982,共9页Journal of Agricultural Biotechnology

基　　金：河南省高等学校重点项目(22A230004)。

摘　　要：TolC家族蛋白是一种广泛存在于革兰氏阴性菌中的外膜通道蛋白,介导多种底物胞外转运过程,在细菌的生物学功能中发挥着重要作用。解析鸭源鸡杆菌(Gallibacterium anatis) TolC蛋白在该菌致病过程中发挥的功能,对于该病防控具有重要意义。本研究以临床分离株Yu-PDS-RZ-1-SLG(野生型)为研究对象,采用自然转化法构建tolC无痕敲除株ΔtolC,初步研究其生物学特性。结果显示,与野生型比较,ΔtolC的菌落及溶血环变小,对数生长期滞后2 h,整体生长速度减慢;生物被膜形成能力显著减弱(P<0.001);对高温、氧化应激及渗透压力的拮抗能力均降低(P<0.01);在浮游态及被膜态对部分抗菌药物的敏感性升高:浮游状态下ΔtolC对SDS、磷霉素和多西环素的敏感性分别增强至32、8和4倍,被膜态下ΔtolC对SDS、磷霉素和多西环素的敏感性分别增强至16、8和8倍,对硫酸新霉素和卡那霉素的敏感性没有变化;对人(Homo sapiens)宫颈癌细胞系(Hela)的黏附能力减弱。tolC无痕敲除株的构建及生物学特性分析为TolC蛋白在鸭源鸡杆菌中的生物学功能、致病机理及耐药机制等研究提供了基础资料。TolC family protein(TolC),an outer membrane channel protein,is widespread in Gram-negative bacteria,which mediates extracellular transport of various substances and plays important roles in the bacterial biological functions.The identification of the functions of TolC in the virulence of Gallibacterium anatis would play crucial roles for prevention and control of this disease.In this study,traceless deletion mutant of tolC(ΔtolC) was constructed by natural transformation using a clinical isolate Yu-PDS-RZ-1-SLG(WT) and the biological characteristics of the mutant strain was investigated.Compared with WT strain,the results of ΔtolC showed that bacteria colonies and zone of hemolysis were smaller,the logarithmic growth phase was delayed 2h with slower growth rate and weakened biofilm formation ability(P<0.001).In addition,the survival rates under high temperature,oxidative stress and osmotic pressure circumstance were significantly reduced(P<0.01).The sensitivity to some antibiotics in the planktonic and membrane states increased:in the planktonic state,the sensitivity of ΔtolC to SDS,fosfomycin,and doxycycline increased by 32,8,and 4 times,respectively;in the membrane state,the sensitivity of ΔtolC to SDS,fosfomycin,and doxycycline increased by 16,8,and 8times,respectively;while the sensitivity to neomycin sulfate and kanamycin remained unchanged.The adhesion ability of ΔtolC to human(Homo sapiens) cervical cancer cell line(Hela) reduced.The construction and analysis of biological characteristics of tolC traceless knockout strain ΔtolC provides basic data for the study of the biological function,pathogenic mechanism,and drug resistance mechanism of TolC protein in G.anatis.

关 键 词：鸭源鸡杆菌 外膜通道蛋白TolC 缺失 

分 类 号：S855.1[农业科学—临床兽医学]