糖基转移酶WekM参与禽致病性大肠杆菌脂多糖合成和环境适应  

The glycosyltransferase WekM is involved in the lipopolysaccharide biosynthesis and environmental adaptation of avian pathogenic Escherichia coli

作　　者：曹启予 高宇杰 张晓荟 陈歆丹 罗平 翟瑞东 韩先干[2] 宋厚辉 程昌勇 于纪棉[3] 韩月 CAO Qiyu;GAO Yujie;ZHANG Xiaohui;CHEN Xindan;LUO Ping;ZHAI Ruidong;HAN Xiangan;SONG Houhui;CHENG Changyong;YU Jimian;HAN Yue(Key Laboratory of Applied Technology on Green-Eco-Healthy Animal Husbandry of Zhejiang Province,Zhejiang Provincial Engineering Research Center for Animal Health Diagnostics&Advanced Technology,Zhejiang International Science and Technology Cooperation Base for Veterinary Medicine and Health Management,China-Australia Joint Laboratory for Animal Health Big Data Analytics,College of Animal Science and Technology&College of Veterinary Medicine,Zhejiang A&F University,Hangzhou 311300,Zhejiang,China;Shanghai Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Shanghai 201199,China;Ningbo College of Health Sciences,Ningbo 315100,Zhejiang,China)

机构地区：[1]浙江农林大学动物科技学院·动物医学院、浙江省畜禽绿色生态健康养殖应用技术研究重点实验室、动物健康互联网检测技术浙江省工程研究中心、浙江省动物医学与健康管理国际科技合作基地、中澳动物健康大数据分析联合实验室,浙江杭州311300 [2]中国农业科学院上海兽医研究所,上海201199 [3]宁波卫生职业技术学院,浙江宁波315100

出　　处：《微生物学报》2024年第8期2702-2712,共11页Acta Microbiologica Sinica

基　　金：国家重点研发计划(2023YFD1801000);国家自然科学基金(31902280,32102671);浙江省自然科学基金(LQ24C010005)。

摘　　要：【目的】研究O_(1)血清型禽致病性大肠杆菌(avian pathogenic Escherichia coli,APEC)的O抗原糖基转移酶WekM在脂多糖合成和环境适应中的作用。【方法】采用Red同源重组方法,构建APEC O_(1)菌株的wekM基因缺失株,并构建wekM回补株。随后分析wekM基因对APEC O_(1)菌株生长和运动能力的影响,通过银染和Western blotting鉴定细菌脂多糖(lipopolysaccharide,LPS)图谱以及与兔抗O_(1)血清的反应能力,通过实时荧光定量PCR测定细菌鞭毛相关基因转录水平,使用溴化乙锭测定细菌细胞膜通透性。最后,通过药敏试验检测细菌对环丙沙星等抗生素敏感性。【结果】PCR验证及DNA测序结果表明ΔwekM缺失株和回补株构建成功。银染鉴定ΔwekM缺失株较野生株LPS图谱不完整,部分O-抗原条带缺失;同时,Western blotting检测未见到ΔwekM与O因子血清的反应条带,这说明O-抗原糖基转移酶wekM基因缺失后影响LPS合成。生长运动能力分析显示,ΔwekM缺失株的运动能力较野生株显著减弱,生长速率与野生株一致。实时荧光定量PCR检测发现,ΔwekM缺失株的flgC等鞭毛相关基因转录水平降低,表明wekM基因影响细菌鞭毛的合成。此外,ΔwekM的细胞膜通透性较野生株显著增加(P<0.01),药敏结果也显示,ΔwekM缺失株较野生株对多黏菌素等7种抗生素敏感性增加,这说明wekM缺失后细菌细胞膜理化性质改变,适应环境的能力降低。【结论】本研究揭示了禽致病性大肠杆菌糖基转移酶wekM基因缺失导致细菌LPS完整性受损,运动能力降低,鞭毛合成受阻,鞭毛形成基因转录水平下降,细胞膜通透性增强,对抗生素敏感性增加。这些结果为解析wekM基因的功能奠定了研究基础,有助于深入了解禽致病性大肠杆菌O_(1)的环境适应机制。[Objective]To investigate the role of WekM,the O-antigen glycosyltransferase of avian pathogenic Escherichia coli(APEC)O_(1),in lipopolysaccharide biosynthesis and environmental adaptation.[Methods]The wekM-deleted strainΔwekM of APEC O_(1)was constructed by Red homologous recombination,and then the complementary strain CΔwekM was constructed.The impacts of wekM on bacterial growth and motility were examined.The lipopolysaccharide(LPS)profile and reactivity with rabbit anti-O_(1)serum of each strain were identified by silver staining and Western blotting.Real-time fluorescence quantitative PCR was conducted to determine the transcriptional levels of flagellum-related genes,and ethidium bromide was used to measure the bacterial cell membrane permeability.Finally,the drug sensitivity test was carried out to identify the bacterial susceptibility to antibiotics such as ciprofloxacin.[Results]The constructedΔwekM and CΔwekM were verified by PCR amplification and DNA sequencing.Compared with the wild type,ΔwekM showed incomplete LPS profile and absence of some O-antigen bands.Western blotting results showed thatΔwekM did not react with the anti-O_(1)serum,suggesting that the loss of WekM impaired the LPS production.The deletion of wekM reduced the swimming motility and did not impact the bacterial growth rate compared with the wild type.The transcription levels of flagellum-related genes such as flgC were down-regulated inΔwekM.The results implied that the reduced motility ofΔwekM was caused by the decrease in flagellar production.In addition,ΔwekM demonstrated increased cell membrane permeability compared with the wild type(P<0.01),andΔwekM improved bacterial sensitivity to 7 antibiotics including polymyxin.This result suggested that the adaptability ofΔwekM to the environment was inhibited due to the increased cell membrane permeability.[Conclusion]The deletion of wekM in APEC results in diminished swimming motility,increased antibiotic resistance,improved cell membrane permeability,and damaged LPS integri

关 键 词：禽致病性大肠杆菌O_(1) 脂多糖 O-抗原 糖基转移酶 环境适应 

分 类 号：S852.61[农业科学—基础兽医学]