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

摘要:

【目的】 研究O₁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 O₁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 O₁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O₁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O₁的环境适应机制。

关键词:禽致病性大肠杆菌O₁;脂多糖;O-抗原;糖基转移酶;环境适应

Abstract:

[Objective] To investigate the role of WekM, the O-antigen glycosyltransferase of avian pathogenic Escherichia coli (APEC) O₁, in lipopolysaccharide biosynthesis and environmental adaptation. [Methods] The wekM-deleted strain ΔwekM of APEC O₁ 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-O₁ 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 constructed ΔwekM 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 that ΔwekM did not react with the anti-O₁ 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 in ΔwekM. The results implied that the reduced motility of ΔwekM was caused by the decrease in flagellar production. In addition, ΔwekM demonstrated increased cell membrane permeability compared with the wild type (P<0.01), and ΔwekM improved bacterial sensitivity to 7 antibiotics including polymyxin. This result suggested that the adaptability of ΔwekM 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 integrity. The findings lay a foundation for mining the role of wekM and enrich our understanding of the stress resistance mechanism of APEC.

Key words:avian pathogenic Escherichia coli O₁;lipopolysaccharide;O-antigen;glycosyltransferase;environmental adaptation