O-抗原糖基转移酶对嗜水气单胞菌环境适应性及致病性的影响

Effects of O-antigen glycosyltransferase on environmental adaptability and pathogenicity of Aeromonas hydrophila

[目的]本文旨在探究4种O-抗原糖基转移酶(glycosyltransferase,GT)对嗜水气单胞菌NJ-35环境适应性和毒力的影响。[方法]采用同源重组技术构建GT基因缺失株ΔGT-1、ΔGT-2、ΔGT-3和ΔGT-4,比较缺失株与野生株在体外生长、抗应激、抗吞噬、细胞黏附能力、对斑马鱼毒力以及脂多糖(LPS)表达量等方面的差异。[结果]在含有20 g·L^(-1)NaCl的高渗LB(Luria-Bertani)培养基中,相比野生(WT)菌株,4个缺失株生长均未发生明显变化;在2 mmol·L^(-1)H _(2)O_(2)的强氧化应激条件下,4个缺失株抗氧化能力显著降低,特别是缺失株ΔGT-3存活率下降90%,ΔGT-4存活率下降86.5%,另外2株缺失株存活率下降近40%;在LB培养基(pH5.0)中作用30 min,4个缺失株存活率相比野生株均明显降低,其中ΔGT-3存活率下降近30%;4个缺失株对上皮细胞的黏附能力均显著降低,抗吞噬能力均显著增强;相比野生株,ΔGT-4对斑马鱼半数致死量(LD 50)上升近20倍,其他缺失株的毒力无明显变化;4个缺失株的LPS表达量均降低,且ΔGT-1和ΔGT-4的LPS最高分子质量条带出现缺失。[结论]O-抗原糖基转移酶有助于嗜水气单胞菌抵抗酸应激和氧化应激环境,并在该菌的黏附和毒力上发挥重要作用。

[Objectives]This study was performed to explore the effects of four O-antigen glycosyltransferases(GT)on the environmental adaptability and virulence of Aeromonas hydrophila NJ-35.[Methods]GT gene deleted strainsΔGT-1,ΔGT-2,ΔGT-3 andΔGT-4 were constructed by homologous recombination technology,and the differences of growth in vitro,anti-stress,anti-phagocytosis,cell adhesion,toxicity to zebrafish and lipopolysaccharide(LPS)expression between the wild and deleted mutant strains were compared.[Results]In LB(Luria-Bertani)medium containing 20 g·L^(-1)NaCl,the growth of four deleted strains was similar to that of the wild type(WT)strain,and no significant difference was observed in hypertonic tolerance.Under conditions of oxidative stress induced by 2 mmol·L^(-1)H _(2)O_(2),the survival rates ofΔGT-3,ΔGT-4 and the other two deleted strains decreased by 90%,86.5%and nearly 40%,respectively.Under acidic stress,the survival rate of four deleted strains was significantly lower than that of WT strains,and the survival rate ofΔGT-3 decreased by nearly 30%.The adhesion ability of the four deleted strains to epithelial cells was significantly reduced,and the anti-phagocytosis ability was significantly enhanced.Compared with the WT strain,the lethal dose(LD 50)ofΔGT-4 to zebrafish increased by nearly 20 times,and the virulence of other deleted strains did not change significantly.The expression of lipopolysaccharide(LPS)in the four deleted strains decreased,and the highest molecular weight band of LPS inΔGT-1 andΔGT-4 was deleted.[Conclusions]O-antigen glycosyltransferase was helpful for Aeromonas hydrophila to resist acid stress and oxidative stress,and played an important role in the bacterial adhesion and virulence.