肺炎克雷伯菌CRP调控子对细菌毒力影响与机制研究

The Role of CRP Regulator in the Virulence of Klebsiella Pneumoniae and the Pathogenesis

目的:了解肺炎克雷伯菌调控因子CRP对细菌毒力的影响及其对毒力相关因子磷酸转移酶系统的作用及其机制。方法:构建肺炎克雷伯菌CRP调控因子的基因缺失突变株与回补株,小鼠毒力试验检测CRP对细菌LD50的影响及小鼠生存率的影响,qRT-PCR与lac Z报告基因检测CRP对果糖磷酸转移酶系统EIIC蛋白frwC基因的影响并通过凝胶阻滞试验(EMSA试验)检测CRP调控frwC基因的机制。结果:CRP基因敲除菌株的小鼠毒力明显下降,野生株、CRP基因缺失株及回补株的LD50分别为<100,3.2×10~5和4.5×10~4CFUs。qRT-PCR与lac Z报告基因发现CRP基因敲除后,果糖磷酸转移酶系统EIIC蛋白frwC基因表达量明显下降,EMSA试验发现CRP蛋白能够结合在frwC启动子区。结论:肺炎克雷伯菌CRP调控子与细菌毒力密切相关,并且能够直接正调控frwC基因的表达。除影响细菌荚膜、菌毛、生物膜的形成而影响细菌毒力外,CRP调控子可能通过影响磷酸转移酶系统而影响细菌毒力。

Objective Understanding the effect of CRP regulator in the virulence of Klebsiella pneumoniae and the role of it in the virulence factor phosphotransferase system（PTS）. Methods Construction the c AMP receptor protein（CRP） gene（crp gene） deletion mutant strain and the crp gene complemented strain and study the effect of crp gene in the virulence in mouse model. The regulation roles of CRP in frwC gene,the EIIC encoded gene of PTS,were assessed using qRT-PCR and lac Z reporter. Electrophoretic mobility shift assay（EMSA） was used to study the mechanism of CRP in frwC. Results The LD50 values was 100,3.2×10^5,and 4.5×10^4CFUs for WT,Δcrp,and C-crp,respectively. The expression of frwC,the fructose-specific enzyme IIC of fructose phosphotransferase system（PTS）,was repressed in the mutant. EMSA show CRP can directly bind to the promoter of frwC. Conclusion pneumoniae CRP regulator was required for lethality in mouse and CRP could control the virulence through acting on fructose phosphotransferase system directly.