组织蛋白酶B基因敲除抑制布鲁菌胞内感染

Deficiency of cathepsin B suppresses Brucella intracellular infection

目的研究组织蛋白酶B(cathepsin B,CTSB)在布鲁菌感染中的作用。方法利用CRISPR/Cas9技术构建ctsb基因敲除的人宫颈癌HeLa细胞株,并检测ctsb敲除对细胞增殖的影响。通过CCK-8法和胞内菌计数检测布鲁菌感染中ctsb敲除对细胞增殖活性和细菌胞内存活的影响,并通过Western印迹检测揭示ctsb调控布鲁菌感染的分子机制。结果成功构建了ctsb基因敲除细胞株;CCK-8结果表明敲除ctsb不影响布鲁菌感染前后的细胞增殖率;胞内菌计数结果显示KO细胞感染4 d后胞内布鲁菌数量显著减少;Western印迹检测结果提示KO细胞在布鲁菌长期感染中自噬相关蛋白表达水平显著下调。结论ctsb基因敲除能够抑制长期感染中布鲁菌的胞内存活,为慢性布鲁菌病的治疗提供了潜在的靶点。

Objective To investigate the role of cathepsin B(CTSB)inhuman cervical cancer HeLa cellswith Brucella infections.Methods The ctsb knockout(KO)HeLa cell line was constructed using CRISPR/Cas9.The effects of ctsbKO on cell proliferation and survival of intracellular bacteria in case of Brucella infection were detected by CCK-8 assay and intracellular bacteria count while the molecular mechanism of ctsb regulation on Brucella infection was revealed by Western blotting.Results The ctsbKO cell line was constructed.CCK-8 results showed that KO of ctsb had no impact on cell proliferationwith or without Brucella infection.The results of intracellular bacteria count showed a significant decrease in intracellular Brucella four days after infection in the KO cell line.Western blotting assay suggested that the expression levels of autophagy-related proteins were significantly down-regulated in the KO cell line with long-term Brucella infection.Conclusion KO of ctsb inhibits the intracellular survival of Brucella in long-term infections,providing a potential target for the treatment of chronic brucellosis.