β-毒素促进金黄色葡萄球菌破坏血脑屏障的实验研究

Experimental Research on β-toxin of Staphylococcus aureus promotes breaking the blood-brain barrier in mice

目的 探讨β-毒素(Hlb)在金黄色葡萄球菌通过血脑屏障(blood-brain barrier, BBB)中的作用。方法 通过血琼脂平板划线筛选金葡菌MW2的Hlb高产菌株,验证其对hCMEC/D3模拟BBB细胞模型通透性的影响。采用生物信息学分析筛选hlb基因完整的金葡菌菌株,利用同源重组的方法构建hlb敲除株,血琼脂平板法检测溶血活性改变。利用BBB细胞模型比较不同菌株对其通透性的影响,再将野生株与hlb敲除株经尾静脉注射构建小鼠血源性脑膜炎/脑微脓肿模型并比较感染72 h脑组织中菌载量。结果 金葡菌MW2的Hlb高产菌株对BBB细胞模型破坏导致BBB细胞模型通透性增加的能力较野生株明显增强。NCTC8325-4、COL和RN4220菌株hlb基因无噬菌体插入。成功构建NCTC8325-4Δhlb株,血平板证实敲除株的β-溶血缺失。NCTC8325-4Δhlb株对BBB模型细胞破坏导致BBB细胞模型通透性增加的作用较野生株下降,在小鼠血源性脑膜炎/脑微脓肿模型脑组织中菌载量也较野生株明显降低。结论 β-毒素具有促进金葡菌破坏BBB的作用。

Objective To investigate the role ofβ-toxin(Hlb)in the penetration of Staphylococcus aureus(S.aureus)through blood-brain barrier(BBB).Methods The high Hlb-producing strain of S.aureus MW2 was screened by streaking on blood agar plate,and its effect on the permeability of hCMEC/D3 simulated BBB cell model was verified.Bioinformatics analysis was used to screen the strains with complete hlb gene,and then the hlb knockout strain was constructed with homologous recombination.The hemolytic activities of strains of interest were verified by streaking on blood agar plate.The permeability of different strains of S.aureus was compared in BBB cell model.A hematogenous meningitis/brain microabscess model was generated by injection of the wild-type or the hlb-deletion strain individually into CD1 mice through tail vain,and the bacterial load in the brain tissue was determined in 72 h after infection.Results The Hlb high-producing strain of MW2(Hyper-β)led to increased permeability of the BBB model cells than the wild-type strain.The NCTC8325-4,COL,and RN4220 strains had intact hlb gene without phage insertion.The NCTC8325-4Δhlb strain was successfully constructed,and the lack ofβ-hemolysis of the mutant was verified by blood agar plate.A reduced permeability of NCTC8325-4Δhlb on the BBB cell model was observed in comparison to that of the wild-type strain.The bacterial load of NCTC8325-4Δhlb strain in the brain tissue of the mouse hematogenous meningitis/brain microabscess model was obviously lower than that of the wild-type strain.Conclusionβ-toxin from S.aureus promotes the bacteria destroying BBB.