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目的 E.tarda能够在体内外许多环境包括在巨噬细胞内生存和繁殖,eha基因是该菌一个重要的转录调控基因,本研究探讨该基因在细菌抵抗巨噬细胞内外压力中的作用及机制。方法采用体外实验模拟巨噬细胞内氧化和酸杀菌条件、体内血清和胆汁杀菌条件、以及细菌对SDS和多粘菌素B敏感性试验,比较ET-13毒力株及其Δeha缺失株和ehaComp互补株在这些压力下存活率,利用RT-PCR和SDS-PAGE电泳,比较上述3种细菌相关基因的转录和表达的差异。结果eha基因的缺失使ET-13对酸、H2O2、SDS和多粘菌素B敏感性提高(P<0.05);经小鼠血清或鱼胆汁处理后,3种菌株的存活率没有明显区别(P>0.05);RT-PCR结果显示,eha基因的缺失使得E.tarda内的超氧化物歧化酶基因sodC、过氧化氢酶基因katB和鞭毛蛋白基因fliC、Ⅲ型分泌系统分泌蛋白基因eseC的转录水平下降。SDS-PAGE电泳结果显示,缺失株的主要外膜蛋白比野生株表达降低。结论 eha基因通过调控E.tarda相关基因的表达,参与了该菌抵抗巨噬细胞内氧化和酸等的压力,有助于细菌在巨噬细胞内生存和繁殖。
Purpose E. tarda can survive and multiply in many environments both in vitro and in vivo, including in macrophages. The eha gene is an important transcriptional regulatory gene of this bacterium. This study explored the role and mechanism of this gene in the intracellular and extracellular pressure of bacteria against macrophages . Methods In vitro experiments were performed to simulate the conditions of oxidative and acid bactericidal activity of macrophages, serum and bile bactericidal conditions in vivo and bacterial sensitivity to SDS and polymyxin B, and to compare ET-13 virulence strains with Δeha deletion and ehaComp The survival rates of the complementary strains under these pressures were compared using RT-PCR and SDS-PAGE electrophoresis to compare the differences in transcription and expression of the three bacterial-associated genes. Results The deletion of eha gene increased the sensitivity of ET-13 to acid, H2O2, SDS and polymyxin B (P <0.05). After treatment with mouse serum or fish bile, the survival rates of the three strains were not significantly different P> 0.05). The results of RT-PCR showed that deletion of eha gene led to the change of SOD gene sodC, catalase gene katB and flagellin gene fliC in E. tarda, secretion of type III secretion gene eseC Transcriptional decline. The results of SDS-PAGE showed that the major outer membrane protein of the deletion strain was lower than that of the wild-type strain. Conclusion The eha gene is involved in the resistance of macrophages to oxidative stress and acid stress by regulating the expression of E. tarda related genes, which contributes to the survival and multiplication of bacteria in macrophages.