肺炎克雷伯菌Hfq蛋白特性及对耐药的影响

Hfq protein characteristics and effects on drug resistance of Klebsiella pneumoniae

Hfq(host factor for RNA phage QB replicase)蛋白是一个全局性调节因子,广泛参与细菌生长、趋化、毒力、耐药及应对外界选择压力等方面的调节,但在肺炎克雷伯菌(Klebsiella pneumoniae,KP)中的功能尚不清楚。本研究从临床病例中分离到59株KP,将其hfq基因与11例常见临床感染菌株hfq基因〔从美国国立生物技术信息中心(National Center for Biotechnology Information,NCBI)数据库下载〕进行了比较。所有hfq基因经EMBOSS Transeq翻译成氨基酸序列,用MAFFT软件进行多序列比对,并通过NCBI数据库中的保守结构域预测Hfq蛋白结构域。分别采用ESPript3.0、Phyre2分析Hfq蛋白的二、三级结构。59株KP中仅3株hfq基因的5个密码子位点存在差异,而其蛋白质氨基酸序列完全一致。KP与大肠埃希菌、阴沟肠杆菌、痢疾志贺菌之间,Hfq蛋白的氨基酸序列相似度较高,主要区别在C末端上;与金黄色葡萄球菌、产单核细胞李斯特菌相比,KP Hfq蛋白在N末端和C末端上差别较大;所有菌株C末端均呈酸性。三级结构预测提示68(66.67%)个氨基酸与模板序列一致,较为保守的功能结构为54-VYKHAI-59序列。采用CRISPR/Cas9同源重组技术敲除KP的hfq基因,并对其进行药物敏感性测试,结果显示,基因敲除菌株对抗生素的耐药性较野生株有显著下降(P<0.05),差异有统计学意义,提示KP的Hfq蛋白氨基酸序列非常保守,可能参与了KP的耐药调节。

Host factor for RNA phage QB replicase(Hfq)is a global regulator that regulates sRNA-mRNA binding and stability and is involved in bacterial growth,chemotaxis,virulence,drug resistance,and response to external stress.The hfq gene of 59 clinical isolates of Klebsiella pneumoniae(KP)collected from Aug 2017 to Aug 2018 in our hospital were extracted and compared with the ones downloaded from NCBI database.Transeq,multiple sequence alignments of amino acid sequences were performed with MAFFT software,and the secondary structure of the protein were analyzed using the online website ESPript3.0.The physicochemical properties of the amino acid sequences were calculated using the online website ProtParam.Hfq domains were predicted from Conserved domains in the NCBI database.The tertiary structure of Hfq protein was predicted using Phyre2.The CRISPR/Cas9 homologous recombination technology was used to knock out the hfq gene in KP strain.The wild strain and the hfq knockout strain were diluted by 1∶100,and 0.1μg/mL,0.2μg/mL,and 0.3μg/mL Imipenem/cilastatin sodium were added to co-culture respectively.The growth curve was drawn by taking the 1 h,2 h,3 h,4 h bacteria liquid count respectively.Despite 5 hfq gene mutations detected in 3 strains,the amino acid sequences of all strains were identical.The tertiary structure prediction indicated that 68(66.67%)amino acids were consistent with the template sequence,and the more conserved functional domain was located at the 54-59 sites(VYKHAI).The hfq gene of one clinical strain was successfully knocked out by CRISPR/Cas9 homologous recombination.The resistance of the hfq knockout strain to antibiotic significantly decrease than that of the wild type(P<0.05).The results of this study suggest that KP’s Hfq amino acid sequence is conserved,and the gene may play a role in carbapenems resistance.