布鲁氏菌16M <i>bvfA</i>突变株的构建及其对环境应激耐受与巨噬细胞内存活研究

王浩; 张凤姣; 塔娜; 塞击拉呼; 贾芳

doi:10.3784/jbjc.202507220519

布鲁氏菌16M bvfA突变株的构建及其对环境应激耐受与巨噬细胞内存活研究

Construction of Brucella 16M bvfA mutant strain and its stress response and survival in macrophage

摘要

摘要:
目的探讨bvfA基因对布鲁氏菌16M的生长、应激耐受及胞内存活的影响。
方法利用基因重组技术构建bvfA基因缺失和回补菌株，利用静置比浊法测定菌株的生长曲线；利用平板菌落计数法测定菌株对环境的应激耐受、对巨噬细胞的侵入及其在胞内生存能力的影响。
结果与野生株相比，缺失株16M△bvfA对数生长期明显延长，生长速率显著下降；与缺失株相比，回补株16M△bvfA:bvfA生长速度显著提高。缺失株16M△bvfA在高渗、高盐及氧化压力下存活率低于野生株16M（高盐P=0.025；高渗P=0.002；氧化压力P=0.046），其中氧化压力下存活率仅为野生型的11.01%，回补株16M△bvfA:bvfA在以上条件下存活率均高于缺失株16M△bvfA（高盐P=0.045；高渗P=0.006；氧化压力P = 0.004）。在细菌感染巨噬细胞30 min、45 min和60 min后，缺失株16M△bvfA对巨噬细胞的侵袭率均低于野生株16M（30 min P<0.001；45 min P=0.004；60 min P<0.001），而回补株16M△bvfA:bvfA在上述时间条件下对巨噬细胞的侵袭率高于缺失株16M△bvfA（30 min P<0.001；45 min P=0.011；60 min P<0.001）。在细菌侵入巨噬细胞12 h和24 h后，缺失株16M△bvfA在胞内的存活率均低于野生株16M（12 h P=0.016 ；24 h P=0.001），且缺失株在胞内存活率逐渐下降；在上述时间条件下，回补株16M△bvfA:bvfA的胞内存活率均高于缺失株16M△bvfA（12 h P=0.049 ；24 h P=0.001），且回补株16M△bvfA:bvfA和野生株16M在胞内存活率逐渐增加。
结论 bvfA参与调控布鲁氏菌16M的生长动力学和应激反应，并参与调节16M对巨噬细胞的侵入和胞内存活能力，其在布鲁氏菌对环境适应中发挥关键作用。

Abstract:
Objective To explore the effects of the bvfA gene on the growth, stress tolerance and intracellular survival of Brucella 16M.
Methods bvfA deletion and complement strains were constructed by using genetic recombination technology. The growth curves of 16M, 16M bvfA mutant and complement strains were determined by using static turbidimetric method. The stress tolerance, macrophage invasion, and intracellular survival of three strains were evaluated by using plate colony counting method.
Results Compared with the wild strains, the logarithmic growth period of the deletion strain 16M△bvfA was significantly prolonged, and the growth velocity significantly decreased. Compared with the deletion strains, the complement strain 16M△bvfA:bvfA had a significantly increased growth velocity. The survival rate of the deletion strains16M△bvfA was significantly lower than that of the wild strain 16M under conditions of high osmolarity (P=0.002), high salinity (P = 0.025) and oxidative stress (P = 0.046), but the survival rate was only approximately 11.01% of that of the wild strain under oxidative stress. However, the survival rate of the complement strain 16M△bvfA:bvfA was always higher compared with the deletion strain 16M△bvfA under the conditions mentioned above (P=0.045, P=0.006, P=0.004). The invasion rates of the deletion strain 16M△bvfA in macrophages was significantly lower than those of the wild strain 16M at 30 min (P < 0.001), 45 min (P=0.004) and 60 min (P<0.001). And the invasion rates of the complement strain 16M△bvfA:bvfA in macrophages were higher than those of the deletion strain 16M△bvfA at 30 min (P<0.001), 45 min (P=0.011) and 60 min (P<0.001) after the infection. The survival rates of the deletion strain 16M△bvfA in macrophages was lower than the wild strain 16M at 12 h (P=0.016) and 24 h (P=0.001) after the infection, and the survival rate of the deletion strain 16M△bvfA gradually decreased within the cells. Whereas the survival rates of the complement strain 16M△bvfA:bvfA were higher than deletion strain 16M△bvfA at 12 h (P=0.049) and 24 h (P=0.001) after the infection, and the complement strain 16M△bvfA:bvfA and the wild-type strain 16M showed a gradual increase in cell survival rate.
Conclusion bvfA is involved in regulating the growth kinetics and stress response of Brucella 16M, and also participates in modulating, invasion and intracellular survival of Brucella 16M in macrophages, indicating that bvfA plays a crucial role in the adaptation of Brucella to the environment.

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