糖基转移酶WekO参与禽致病性大肠杆菌感染和致病

The glycosyltransferase WekO is involved in the infection and pathogenicity of avian pathogenic Escherichia coli

摘要:

目的 探讨糖基转移酶WekO基因对禽致病性大肠杆菌(avian pathogenic Escherichia coli, APEC)生物学特性的影响。方法 以O₁血清型APEC为亲本株，利用Red同源重组技术构建wekO基因缺失株(ΔwekO)和回补株(CΔwekO)。通过硝酸银染色和蛋白质免疫印迹(Western blotting)分析细菌脂多糖(lipopolysaccharide, LPS)图谱及血清反应性。同时，检测ΔwekO的生长速率、运动能力，并采用结晶紫染色法评估其生物被膜形成能力。此外，利用鸡成纤维细胞(DF-1)测定ΔwekO的体外黏附和侵袭能力，并以萧山蛋鸡为动物模型评估其致病力。结果 成功构建了缺失株ΔwekO和回补株CΔwekO；与野生株相比，ΔwekO的LPS图谱不完整，O-抗原梯状条带缺失，且与O₁血清无反应。在生长速率方面，ΔwekO与野生株无显著差异(P>0.05)，但其运动能力和生物被膜形成能力显著降低(P<0.001)，对DF-1细胞的黏附率显著降低(P<0.001)、侵袭率显著降低(P<0.01)，对7日龄雏鸡的致病力也显著降低(P<0.05)。结论 本研究表明，wekO基因缺失导致APEC的O-抗原合成受阻，进而影响LPS的完整性。此外，wekO基因缺失还导致细菌的鞭毛形成和生物被膜构建能力丧失，致病力显著下降。这些结果对于深入理解参与APEC O-抗原合成的糖基转移酶的生物学功能具有重要意义。

Abstract:

Objective To study the effects of the glycosyltransferase WekO involved in O-antigen synthesis on the biological characteristics of avian pathogenic Escherichia coli (APEC).Methods The mutant strain ΔwekO of APEC O₁ was constructed by Red homologous recombination, and the complementary strain CΔwekO was then constructed. The lipopolysaccharide (LPS) profile of each strain was identified by silver staining. Simultaneously, the growth rate and swimming motility were measured. The reactivity of each strain with rabbit anti-O₁ serum was determined by Western blotting. The ability of ΔwekO to form biofilms was measured by the crystal violet staining method. DF-1 cells were used to evaluate the adhesion and invasion of ΔwekO in vitro. Subsequently, chicks were selected as an animal model to evaluate the pathogenicity of ΔwekO.Results The mutant strain ΔwekO and the complementary strain CΔwekO were constructed. The LPS profile of ΔwekO was incomplete compared with that of the wild-type strain. The mutant lacked O-antigen bands and showed no reactivity to anti-O₁ serum. There was no significant difference in growth rate between different strains (P>0.05). However, the motility and biofilm formation capabilities of ΔwekO decreased (P<0.001). Additionally, ΔwekO demonstrated weakened adhesion to DF-1 cells (P<0.001) and demonstrated weakened invasion to DF-1 cells (P<0.01). Also, ΔwekO reduced pathogenicity to 7-day-old chicks (P<0.05).Conclusion The deletion of wekO results in impaired O-antigen synthesis, incomplete LPS profile, loss of flagellar and biofilm formation capabilities, and reduced pathogenicity of APEC. These findings are highly significant for improving the understanding of the role of glycosyltransferases involved in APEC O-antigen synthesis.