肺炎克雷伯菌cAMP受体蛋白基因缺失株的构建及其在生物膜形成中的作用  

Construction the cAMP receptor protein gene deletion mutant and its role in the biofilm formation of Klebsiella pneumoniae

作　　者：雷旭[1,2] 谭华炳[1] 

机构地区：[1]湖北医药学院附属人民医院感染性疾病科,肝病研究室,十堰市442000 [2]湖北中医药大学临床学院

出　　处：《中华实验和临床感染病杂志（电子版）》2014年第2期1-4,共4页Chinese Journal of Experimental and Clinical Infectious Diseases(Electronic Edition)

基　　金：湖北省教育厅青年人才项目(No.Q20132101);湖北医药学院优秀中青年科技创新团队(No.2011 CXX01)

摘　　要：目的构建肺炎克雷伯菌cAMP受体蛋白基因(crp基因)缺失突变株与回补株,了解crp基因在肺炎克雷伯菌生物膜形成中的作用。方法设计位于crp基因(566 bp)上游698 bp片段及下游576 bp片段的两对引物,分别扩增出相应片段并通过融合PCR构建一个1 274 bp片段的突变盒,利用双酶切克隆的方法将突变盒克隆至温度敏感性自杀载体pKO3-Km的NotⅠ位点间,电转化至肺炎克雷伯菌中构建肺炎克雷伯菌crp基因无痕缺失突变株(Δcrp)。扩增包含crp基因编码、启动子结合区及转录终止区的2 063 bp基因片段并克隆至pGEM-T-easy质粒上,将此质粒电转至crp基因缺失突变株中构建crp基因回补株(C-crp)。采用RT-PCR方法检测crp基因在基因缺失株与回补株的表达情况,确定crp基因缺失突变株与回补株构建成功。利用结晶紫染色法检测crp基因对肺炎克雷伯菌生物膜形成的影响,并通过酵母凝集试验检测crp基因对肺炎克雷伯菌的菌毛形成影响。结果成功构建了肺炎克雷伯菌crp基因缺失突变株与回补株,RT-PCR结果显示,crp基因在缺失突变株中无表达,在回补株重新表达。体外试管静止培养48 h后,crp基因缺失株无明显生物膜形成,而在野生株及回补株的液体培养基表面形成宽厚生物膜。结晶紫染色定量发现,crp缺失突变株的生物膜相对形成量显著低于野生株(0.063±0.011 vs 1.020±0.056,P<0.001)。酵母凝集试验发现在crp基因缺失突变株中细菌菌毛生成能力下降,提示crp基因影响肺炎克雷伯菌菌毛的生成。结论肺炎克雷伯菌crp基因可能通过调控肺炎克雷伯菌菌毛生成而正调控细菌生物膜的形成。Objective To construct the cAMP receptor protein （crp） gene deletion mutant strain and the crp gene complemented strain and study the role of crp gene in the bioiflm formation of K. pneumoniae. Methods The two DNA fragments （698 bp and 576 bp in length, respectively） lfanking the 566 bp deletion region （crp gene） were ampliifed by PCR, puriifed, and used as the templates to create a 1 274 bp deletion construct that was subsequently inserted between the Not Ⅰsites of pKO3-Km. The resulting plasmid was then electroporated into WT strain to construct the deletion mutant of K. pneumoniae. Then a PCR-generated 2 063 bp DNA fragment containing the crp coding region together with its upstream region and downstream region were cloned into the Km-pGEM-T-easy vector to construct the complement strain. The RT-PCR experiments were performed to detected the crp mRNA in WT,Δcrp, and C-crp strains. The effect of crp gene in the bioiflm formation was evaluated by crystal violet stain and the presence and activity of ifmbriae at the bacterial cell surface was assessed using yeast cell agglutination test. Results The crp mutant as well nbsp;as the complemented mutant was constructed from the K. pneumoniae causing the primary pyogenic liver abscess. The crp transcript was lacking inΔcrp strain but detectable in both WT and C-crp strains by RT-PCR. A mass of bioiflms of WT and C-crp attached to the liquid-solid interface and cold be steadily stained by crystal violet;in contrast, theΔcrp strain stained only a little crystal violet. The WT and C-crp strains induced a considerable agglutination of yeast cells, and in contrast almost no agglutination was observed for theΔcrp strain. Conclusion CRP is important to control the bioiflm formation and ifmbria production in K. pneumoniae .

关 键 词：肺炎克雷伯菌 cAMP受体蛋白基因(crp基因) 生物膜形成 菌毛 

分 类 号：R563.1[医药卫生—呼吸系统]