肺炎克雷伯菌转录调控子crp对菌株黏附能力及细胞活性的影响谭斌.docVIP

肺炎克雷伯菌转录调控子CRP对菌株黏附能力及细胞活性的影响 谭斌，白群华，罗美，杨世亚，薛健, 周锡鹏，李迎丽*，邱景富(400016重庆， [摘要] 目的 分析肺炎克雷伯菌临床分离株WT(wild type)、回补株(c-Δcrp)和突变株(Δcrp)对人肺癌上皮细胞A549细胞的黏附能力及细胞活性的影响，为进一步深入研究转录调控子CRP对菌株黏附能力和细菌对细胞毒性的调控机制奠定基础。方法 用肺炎克雷伯菌WT株、c-Δcrp株和Δcrp株感染人肺癌上皮细胞A549，经裂解液裂解后平板计数计算黏附的菌量。用LDH释放法检测细菌对细胞的毒性，并优化了感染时间和感染指数。结果 WT株及c-Δcrp株黏附的菌量分别为logCFU=5.145和logCFU=4.915，均高于Δcrp株(logCFU=4.122)，且有统计学差异(F=8.366，P=0.004)。以MOI=1000(细菌：细胞=1000)的菌量感染靶细胞，37℃孵育8h，加底物液避光显色10min，离心所得上清稀释10倍进行测定为最佳反应条件。WT株对细胞的毒性百分比(70.69%)明显高于Δcrp株(19.54%)，差异有统计学意义(t=8.843，P=0.001)。结论 crp基因敲除后，突变株的细胞毒性和黏附力明显下降，肺炎克雷伯菌转录调控子CRP对菌株黏附力及细胞活性具有正向调控作用。 [关键词]肺炎克雷伯菌；CRP；A549；黏附力；细胞活性 [中图法分类号] R378.99+6 Influence of Klebsiella pneumoniae CRP protein on adhesion and virulence Tan Bin, Bai Qunhua, Luo Mei, Yang Shiya, Xue Jian, Zhou Xipeng，Li Yingli，QJingfu(School of Public Health and Management, Chongqing Medical University, Chongqing 400016, China) [Abstract] Objective To analysis the adhesion ability and cell viability of Klebsiella?pneumonia WT strain，complemented strain c-Δcrp and crp mutant, in order to lay the foundation of the further study of crp gene on adhesion ability and cell viability. Methods After Klebsiella?pneumonia WT strain ，complemented strain c-Δcrp and crp mutant infected the A549 cells, the cells were lysed and quantified by plating appropriate dilutions on Luria-Bertani agar plates. The LDH release was detected to estimate cell activity and infection time and MOI were improved. Results The adhesion ability of Klebsiella?pneumonia WT and c-Δcrp strain higher than crp mutant. The optimal conditions to determinate the LDH release were infected cells incubated for 8 hours at condition of 37℃, the developing time for 10 min away from light, and 1:10 dilution of the supernatant used to test. Conclusions In this study, the adhesion ability and cell viability of A549 cells of crp mutant was significantly decreased comparing with the WT strain and c-Δcrp strain. Klebsiella?pneumonia CRP protein may play a positive regulatory r