布鲁氏菌IV型分泌系统效应蛋白BPE043的功能和致病机制研究

目的 研究布鲁氏菌IV型分泌系统效应蛋白BPE043在布鲁氏菌感染和胞内生存过程中的作用。方法 以羊种布鲁氏菌104株为起始菌株,通过同源重组的方法构建布鲁氏菌BPE043基因缺失株。将布鲁氏菌野生株104和突变株104ΔBPE043在相同的起始浓度下培养,观察它们生长变化的趋势;用野生株和突变株分别感染小鼠,构建小鼠体内感染模型,测定小鼠脾重并计算脾脏细菌载量;利用免疫共沉淀技术筛选BPE043蛋白的宿主互作蛋白。结果 成功构建了布鲁氏菌BPE043基因缺失株。与野生株相比,突变株104ΔBPE043在体外培养的生长趋势和野生株基本相同;小鼠感染实验显示,在感染1周后,两组小鼠脾均肿大,但突变株104ΔBPE043感染组小鼠脾重低于野生株104感染组小鼠脾重;在感染后两周,两组小鼠脾肿大现象开始减轻。在细菌载量方面,在感染1周后,突变株104ΔBPE043感染小鼠的脾脏细菌载量低于野生株104感染小鼠;在感染后2周,突变株感染组的脾细菌载量下降趋势更明显。免疫共沉淀实验显示BPE043蛋白与鼠源小分子GTP结合蛋白Rab4和Rab11存在相互作用。结论 布鲁氏菌IV型分泌系统效应蛋白BPE043是布鲁氏菌重要的毒力因子。本研究为进一步探索BPE043基因在布鲁氏菌感染和胞内生存中的作用奠定了基础。

Functional and pathogenic mechanism of Brucella type IV secretion system effector BPE043

To study the role of Brucella type IV secretion system effector BPE043 in Brucella infection and intracellular replication, a ΔBPE043 mutant strain of Brucella was constructed by homologous recombination based on Brucella melitensis wild strain 104. Brucella wild strains 104 and mutant strains 104 ΔBPE043 were cultured under the same initial concentration, and their growth rate were observed. Then the mice were infected with wild strain and mutant strain respectively, and the spleen weight of mice was measured and the bacteria load in the spleen was calculated. Co-Immunoprecipitation was used to identify the host target of BPE043. Results showed that the BPE043 deletion mutant of Brucella 104 was successfully constructed. Compared with wildtype strain, the 104 ΔBPE043 exhibited similar growth rate, with slightly different in concentration. In infected mice model, splenomegaly was observed in both 104 and 104 ΔBPE043 infected groups on day 7 post-infection (pi), while the spleen weight of mice infected with the 104 ΔBPE043 was lighter than that of mice infected with 104. In addition, the splenomegaly of both infected groups reduced after day 14 pi. The bacteria load in the spleen of mice infected with 104 ΔBPE043 was lower than that of mice infected with 104 at day 7 pi, and the bacteria load in the spleen of mice infected with the 104 ΔBPE043 reduced sharply after day 14 pi. The results of Co-Immunoprecipitation showed that BPE043 interacts with Rab4 and Rab11(small GTPases from mouse). Our results showed that Brucella type IV secretion system (T4SS) effector BPE043 is a virulence-associated factor of Brucella. This study laid a foundation for further research on the role of BPE043 in brucella infection and intracellular replication.