肺炎克雷伯菌RcsAB蛋白对荚膜多糖相关基因wcaJ、wcaG和magA的调控关系

荚膜多糖(CPS)是肺炎克雷伯菌(Klebssiella pneumoniae)重要的毒力因子.Rcs系统是肠杆菌科(En-terobacteriacese)中重要的双组分信号转导系统,RcsAB是其中主要的转录调控蛋白.本研究选取了肺炎克雷伯菌NTUH-K2044 cps基因簇上的3个基因(wcaJ,wcaG和magA),研究RcsAB对上述3个基因可能存在的调控关系.通过实时荧光定量PCR(RT-qPCR)技术和LacZ报告基因融合试验来确认RcsAB是否能够调控上述3个基因的转录表达.进而用凝胶阻滞试验(EMSA)验证RcsAB能否直接结合在靶基因的启动子区,对靶基因进行直接调控.结果表明RcsAB能够正向调控上述3个基因的转录表达,EMSA的结果提示RcsAB对这3个基因可能是通过间接的方式进行调控.RcsAB蛋白可能通过间接调控荚膜多糖基因wcaJ、wcaG和magA的转录,从而影响菌株的荚膜多糖表型.     

肺炎克雷伯菌调控蛋白RcsAB的构建表达及活性鉴定

为了探讨转录调控子Rcs AB对靶基因的转录调控作用,构建肺炎克雷伯菌RcsA和RcsB的重组质粒,之后诱导蛋白表达,提取纯化后测定其活性。PCR得到rcsA、rcsB片段,分别将两DNA片段克隆至表达载体pMAL-C5X、pET28a,构建重组质粒。再将重组质粒导入E. coli BL21(DE3)菌株中,经IPTG诱导后收集菌体,超声破碎。破碎后的上清过柱、透析纯化,得到高纯度的蛋白,通过EMSA进行蛋白活性鉴定。RcsA,RcsB蛋白成功表达纯化,并能够与靶基因结合,初步证明蛋白具有生物学活性。成功制备有生物学活性的RcsA、RcsB蛋白,为进一步研究RcsAB蛋白复合物特异的生物学功能提供物质基础。     

肺炎克雷伯氏菌荚膜多糖表达量评价指标的建立及发酵条件优化

建立了特异性强的肺炎克雷伯氏菌荚膜多糖全菌ELISA检测方法,检测结果与多糖表达量相关性好;以全菌ELISA值结合菌数为评价指标,对影响荚膜多糖表达的培养基组成及发酵条件进行了优化,优化后的摇瓶培养条件下发酵液活性和生物量分别比优化前提高72.7和33倍,并经7L罐放大实验,绘制发酵动力学曲线,为肺炎克雷伯氏菌荚膜多糖进一步开发打下基础。     

肺炎克雷伯氏菌荚膜多糖的提取纯化及其对细胞免疫活性的影响

采用液体深层培养得肺炎克雷伯氏菌Kp9株发酵液,研究确立了菌体快速裂解条件:NP40 1%和胰蛋白酶25 0IUg菌体,50℃作用1h ,然后加入溶菌酶80μg/mL ,56℃作用1h ;裂解液经超滤浓缩和有机溶剂沉淀获得多糖粗品;多糖粗品先后经CTAB吸附分离,DEAE-SepharoseFastFlow离子交换和SephacrylS 30 0HR凝胶过滤纯化,得分子量分布相对均一的多糖纯品,产品得率为0.25 1g/L。采用淋巴细胞转化实验分别探讨了多糖粗品和纯品的免疫活性,研究显示荚膜多糖具有高效的体外细胞免疫活性,并具有典型的双向免疫调节作用。研究结论为肺炎克雷伯氏菌荚膜多糖的开发奠定了前期基础。     

肺炎克雷伯菌五种外膜蛋白研究进展

细菌的外膜蛋白是一种常见的毒力因子,同时也是控制抗菌药物进入细菌体内的重要通道.近年来,随着肺炎克雷伯菌(Klebsiella pneumoniae,KP)毒性和耐药性的增强,受感染人群和感染率逐年增长.为了减少感染,多采取诸如制备相关抗耐药菌药物、蛋白疫苗等预防治疗措施.该文主要针对肺炎克雷伯菌五种重要的外膜蛋白Om...     

肺炎克雷伯菌gyrA基因与parC基因的突变研究

目的探讨肺炎克雷伯菌对环丙沙星和左氧氟沙星的药物敏感性,及对喹诺酮敏感和耐药菌株中gyrA与parC基因的突变情况。方法收集肺炎克雷伯菌临床分离株231株,采用K-B纸片法测定肺炎克雷伯菌对环丙沙星和左氧氟沙星的敏感性,随机选取对环丙沙星和左氧氟沙星均耐药菌株4株和均敏感的菌株3株,分别PCR扩增gyrA基因和parC基因的耐药决定区,扩增片段长度分别为625、319bp,PCR扩增产物经纯化后测序并做序列分析。结果肺炎克雷伯菌对环丙沙星和左氧氟沙星的耐药率分别为51.1%（118/231）和45.9%（106/231）;gyrA和parC基因经序列分析显示,耐药株均有gyrA基因的突变,其中1株出现第83、87和27位氨基酸的改变,2株出现第83位氨基酸的改变,1株出现第47位点的改变;环丙沙星敏感株中未出现gyrA基因的突变。4株耐药株均有parC基因的突变,引起相应氨基酸Ser80→Arg的改变,2株环丙沙星敏感株也发生了同样的改变。结论哈尔滨地区肺炎克雷伯菌对环丙沙星和左氧氟沙星的耐药性显著,在喹诺酮耐药株中有gyrA和parC基因的同时突变,在敏感株中也发现了parC基因的突变。     

肺炎链球菌糖代谢蛋白CcpA对荚膜多糖的调控作用

摘要:【目的】研究肺炎链球菌糖代谢蛋白CcpA对肺炎链球菌荚膜多糖(CPS)的调控作用。【方法】利用大肠杆菌(Escherichia coli)BL21(DE3)工程菌原核表达CcpA蛋白,使用Ni2+亲和层析的方法纯化蛋白。利用纯化后的CcpA蛋白免疫昆明小鼠并制备多克隆抗体;采用ELISA法测定抗CcpA抗体效价。随后,利用Westertn blot方法分析CcpA蛋白在肺炎链球菌中的保守性。另外,利用EMSA方法分析CcpA与cps基因座启动子区域片段的结合。最后,构建ccpA基因缺失株和ccpA基因回复株;利用ELISA法测定野生D39菌 株、ccpA基因缺失株和ccpA基因回复株的荚膜多糖含量。【结果】Western blot结果显示CcpA蛋白在多种血清型的肺炎链球菌均有表达,CcpA蛋白可与cps基因座启动子区域结合,且呈剂量依赖性;ccpA基因缺失时,细菌CPS含量升高,回复表达CcpA蛋白后,CPS含量显著降低。【结论】CcpA是肺炎链球菌中一种保守表达的蛋白,可通过调节cps基因座启动子负性调控肺炎链球菌荚膜多糖的表达。     

肺炎克雷伯菌Hfq蛋白特性及对耐药的影响

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。     

肺炎克雷伯氏菌毒力因子的研究进展

摘要:肺炎克雷伯氏菌(Klebsiella pneumoniae)可天然合成1,3-丙二醇、2,3-丁二醇和3-羟基丙酸等大宗化学品,是近年来的研究热点,但其致病性限制了工业应用。本文综述了该菌的毒力因子,包括菌毛、受体、荚膜多糖、内毒素、铁载体,以及近年报道的其他因子。具体内容涉及毒力因子的编码基因、表达蛋白、参与的代谢活动,以及侵染宿主和抗宿主免疫反应的机制。此外,根据近年来代谢工程和合成生物学领域的新进展,提出了消除或弱化该菌毒性的策略,并对这些策略的可行性进行了讨论。     

Capsular Types of Klebsiella pneumoniae Revisited by wzc Sequencing

Capsule is an important virulence factor in bacteria. A total of 78 capsular types have been identified in Klebsiella pneumoniae. However, there are limitations in current typing methods. We report here the development of a new genotyping method based on amplification of the variable regions of the wzc gene. Fragments corresponding to the variable region of wzc were amplified and sequenced from 76 documented capsular types of reference or clinical strains. The remaining two capsular types (reference strains K15 and K50) lacked amplifiable wzc genes and were proven to be acapsular. Strains with the same capsular type exhibited ≧94% DNA sequence identity across the variable region (CD1-VR2-CD2) of wzc. Strains with distinct K types exhibited <80% DNA sequence identity across this region, with the exception of three pairs of strains: K22/K37, K9/K45, and K52/K79. Strains K22 and K37 shared identical capsular polysaccharide synthesis (cps) genes except for one gene with a difference at a single base which resulted in frameshift mutation. The wzc sequences of K9 and K45 exhibited high DNA sequence similarity but possessed different genes in their cps clusters. K52 and K79 exhibited 89% wzc DNA sequence identity but were readily distinguished from each other at the DNA level; in contrast, strains with the same capsular type as K52 exhibited 100% wzc sequence identity. A total of 29 strains from patients with bacteremia were typed by the wzc system. wzc DNA sequences confirmed the documented capsular type for twenty-eight of these clinical isolates; the remaining strain likely represents a new capsular type. Thus, the wzc genotyping system is a simple and useful method for capsular typing of K. pneumoniae.     

Adjuvant Action of Capsular Polysaccharide of Klebsiella pneumoniae on Antibody Response

Study was made to clarify the experimental conditions for the capsular polysaccharide of Klebsiella pneumoniae (CPS-K) to exhibit maximum adjuvant effect on antibody production to bovine serum albumin (BSA) in mice. To obtain the maximum primary antibody response and also the strongest priming for a secondary response to BSA, 1000 μg of CPS-K had to be injected intramuscularly into the same or adjacent site of BSA injection within the period of 1 hr before to 6 hr after the BSA injection. The optimum amount of BSA giving the maximum antibody response and also the strongest priming under these experimental conditions was 15 mg. In mice thus primed, an extremely high secondary response was induced by injecting 0.5 mg of BSA 30 days after the initial injection. The minimum amount of CPS-K, to exhibit a strong adjuvant action, was 100 μg, which was equal to the minimum amount to induce immunologic paralysis to a homologous antigen. Extremely large amounts, such as 100 to 300 mg per mouse of BSA, were also strongly immunogenic when injected together with paralyzing doses of CPS-K. In vitro admixture of BSA and CPS-K before injection did not strengthen adjuvant action of CPS-K. Alum-precipitated BSA mixed with CPS-K was not more immunogenic than native BSA mixed with CPS-K. Addition of Freund's complete adjuvant to an injection of BSA and CPS-K mixture did not enhance the adjuvant effect of CPS-K.     

Adjuvant Action of Capsular Polysaccharide of Klebsiella pneumoniae on Antibody Response

A study was performed to clarify the roles of primary and secondary injections of antigen and adjuvant (capsular polysaccharide of Klebsiella pneumoniae, CPS-K) in induction of antibody responses and in development of immunological memory in mice to bovine serum albumin (BSA). A primary injecion of BSA alone neither induced significant primary antibody response nor increased immunological memory for a secondary antibody response but, if primary injections of BSA and CPS-K were performed simultaneously, high antibody responses were induced. Moreover, a prior injection of BSA alone or CPS-K alone decreased the level of primary antibody response and the degree of increase in memory following the subsequent injection of BSA mixed with CPS-K. In contrast, a secondary injection of BSA alone into mice once primed with a mixture of BSA and CPS-K elicited very high secondary type antibody response and increased secondarily the memory for a tertiary antibody response. Injection of CPS-K simultaneously with or shortly before or after the secondary injection of BSA did not increase the level of the secondary antibody response and the degree of the secondary increase in memory. Augmentation of the secondary antibody response was elicited by simultaneous injection of CPS-K only when the secondary response was induced inadequately by a suboptimum or supraoptimum dose of antigen.     

Adjuvant Action of Capsular Polysaccharide of Klebsiella pneumoniae on Antibody Response

The neutral fraction (neutral CPS-K) of Klebsiella pneumoniae capsular polysaccharide (CPS-K) from type 1, Kasuya strain, has already been reported as the active substance responsible for the strong adjuvant effect of CPS-K. The present results demonstrate that neutral CPS-K exhibits further common biological activities with lipopolysaccharide (LPS) isolated from Salmonella enteritidis. The intensity of the lethality in mice of neutral CPS-K by the intraperitoneal route is very similar to that of LPS. Its lethality for mice by the intravenous (i.v.) route is significantly stronger than that of LPS, because the degree of increase in the sensitivity to their lethality by i.v. challenge is smaller for LPS than for neutral CPS-K. The intensity of the pyrogenicity of neutral CPS-K in rabbits is approximately one-tenth of that of LPS as judged by the minimal pyrogenic doses and fever indices. The skin-preparatory potency of neutral CPS-K for the dermal Shwartzman phenomenon in rabbits is also approximately one-tenth of that of LPS compared on the basis of the minimal skin-preparatory doses. When injected i.v., neutral CPS-K exhibits a provocative effect on hemorrhagic reactions in skin sites prepared with neutral CPS-K or LPS.