重组金黄色葡萄球菌次黄嘌呤单核苷酸脱氢酶与脂蛋白(a)的相互作用

次黄嘌呤单核苷酸脱氢酶(IMPDH)是金黄色葡萄球菌(S.aureus)表面的纤溶酶原(Plg)受体之一,它可以通过赖氨酸结合位点(LBS)与Plg相结合。脂蛋白(a)[Lp(a)]中的载脂蛋白(a)[Apo(a)]与Plg有很高的同源性,即两者的Kringle结构域都含有LBS,其中Apo(a)的KIV10含有强的LBS。因此本实验提出了Lp(a)应该能够与S.aureus表面的Plg受体相结合,进而可能竞争性抑制S.aureus与Plg结合的假说。本研究克隆了S.aureus的IMPDH基因,酶切后将其连接到表达载体pASK-IBA37中,并在大肠杆菌BL21中表达了该重组蛋白(rIMPDH)。通过酶联免疫吸附试验(ELISA)、亲和色谱层析及Western blot对rIMPDH与Lp(a)的相互作用机制进行了研究。结果表明rIMPDH可以通过LBS与Lp(a)和rKIV10发生特异性结合,而且一定浓度的赖氨酸类似物6-氨基己酸(EACA)可以抑制这种结合,然而本研究并未发现Lp(a)和rKIV10对rIMPDH与Plg的相互作用有明显的抑制。     

猴肝细胞膜脂蛋白(a)受体的研究

猴肝细胞膜蛋白经 SDS- PAGE和蛋白转移电泳后用免疫印迹法测定 ,硝酸纤维素膜分别与抗牛肾上腺皮质 LDL受体的抗体、LDL、Lp( a)和 PLG温育后见有 3条不同的蛋白条带 ,分子量分别为 370 kd、2 90 kd和 80 kd.用酶标法测定猴肝细胞膜蛋白 ,发现经 Lp( a) + PLG温育后用 Lp( a)抗体反应的结果与 Lp( a)温育后用 Lp( a)抗体反应的结果比较无显著差异 ,而经 Lp( a) + PLG温育后用 PLG抗体反应的结果比单独用 PLG温育后用 PLG抗体反应的作用强 ;同样经 Lp( a) +LDL温育后用 Lp( a)抗体反应的结果与经 Lp( a)温育后用 Lp( a)抗体反应的结果比较无显著差异 ,而经 Lp( a) + LDL温育后用 LDL抗体反应比单独经 LDL温育后用 LDL抗体的反应强 ,排除Lp( a)与 PLG和 LDL的交叉反应 ,实验认为猴肝细胞膜上可能同时存在 LDL受体、PLG受体和Lp( a)受体     

重组不可分型流感嗜血杆菌E蛋白与血浆脂蛋白(a)的相互作用

目的:E蛋白(Protein E,PE)是不可分型流感嗜血杆菌(Nontypeable Haemophilus influenza,NTHi)表面的一种纤溶酶原(plasminogen,Plg)受体,其C末端含有两个赖氨酸残基。NTHi可通过其表面的PE与Plg结合,从而利用机体的纤溶系统深层入侵宿主。基于脂蛋白(a)[Lipoprotein(a),Lp(a)]中载脂蛋白(a)[Apolipoprotein(a),Apo(a)]与Plg高度的同源性,拟证明Lp(a)是否会与重组表达的PE(r PE)结合。方法:原核表达并纯化r PE及敲除C末端两个赖氨酸残基的r PEΔKK,密度梯度离心结合阴离子交换层析分离人血浆Lp(a),通过ELISA、Pull down、Western blot等方法研究r PE与Lp(a)的相互作用。结果:r PE与Lp(a)结合,但不与LDL结合,且r PEΔKK与Lp(a)的结合能力明显低于r PE;赖氨酸类似物6-氨基乙酸(EACA)能有效抑制r PE与Lp(a)的结合;Lp(a)对r PE与Plg的结合具有微弱的抑制作用。结论:r PE能够与Lp(a)结合,其中r PE的C末端赖氨酸残基和Apo(a)的赖氨酸结合位点(lysine binding sites,LBS)是r PE与Lp(a)结合的主要位点。     

重组铜绿假单胞菌外毒素A工程菌的高密度发酵与表达

高密度培养表达大肠杆菌生产重组铜绿假单胞菌外毒素A(rEPA)。用上海高机公司的30L自控发酵罐,采用分批补料培养技术,维持葡萄糖的浓度始终处于较低的水平,并分批补加氮源,同时溶氧控制在30%～40%,pH自动调控至7.0,培养至对数中期进行诱导。重组菌最终发酵液光密度(A600)未诱导时达到44,诱导时达到36,在上清液中表达的rEPA蛋白的含量占总蛋白的28.1%,在菌体中表达蛋白含量占总蛋白的4.7%。本实验为rE-PA的大规模生产奠定了基础。     

铜绿假单胞菌的双组分系统

双组分系统是存在于原核和少部分真核生物细胞中的信号转导系统,主要由组氨酸蛋白激酶和反应调节蛋白组成,通过感应外界环境信号、信号输入、磷酸基团传递、信号输出等环节调节基因表达,使细胞能更加适应环境变化。铜绿假单胞菌为条件致病菌,其双组分系统构成多样、功能复杂且参与介导耐药性产生,因此铜绿假单胞菌的双组分系统日益引起人们关注。本文对铜绿假单胞菌双组分系统的组成、信号转导机制、种类、研究方法及其临床意义进行了综述。     

重组大肠杆菌表达铜绿假单胞菌溶血性磷脂酶C

[目的]构建产溶血性磷脂酶C (Hemolytic Phospholipase C,PLCH)的重组大肠杆菌(Escherich coli菌株,并初步优化其发酵条件.[方法]首先利用卵黄硼砂平板分离法筛选到一株产磷脂酶C(Phospholipase C,PLC)活性较高的菌株,命名为铜绿假单胞菌(Pseudomonas aeruginosa)41;进一步以P.aeruginosa 41基因组DNA为模板设计引物,PCR扩增获得溶血性磷脂酶C(PLCH)基因,构建重组大肠杆菌表达质粒并转化大肠杆菌E.coli BL21 (DE3);筛选转化子并检测PLC活性和溶血能力,并初步优化其发酵条件.[结果]成功构建了重组大肠杆菌E.coli BL21(DE3) /pET28a-plcH;在硼砂卵黄平板上对重组菌进行PLC活性测定,显示重组菌有明显的磷脂酶C活性;在哥伦比亚血琼脂平板上对重组菌进行溶血性试验,表明PLCH具有较强的溶血活性;初步优化摇瓶发酵条件为:5％转接量,37℃、200 r/min下培养4h添加IPTG至终浓度为0.9 mmol/L,转为25℃、150 r/min诱导培养14 h;优化后重组菌的酶活可达到722.89±0.47 U/mL.[结论]本文成功构建了一株产溶血性磷脂酶C活性较高的重组大肠杆菌菌株,并通过优化发酵条件使其酶活达到了722.89±0.47 U/mL,本实验在国内首次实现了铜绿假单胞菌来源的溶血性磷脂酶C基因在大肠杆菌的胞内表达,该研究为研究磷脂酶C产业化奠定了一定的基础.     

铜绿假单胞菌异质性耐药的研究进展

异质性耐药是指细菌中的同源亚群对某种抗生素表现出不同的敏感性,被认为是细菌由敏感进化成完全耐药的中间阶段.常规的临床检验无法有效检测出异质性耐药,这对临床治疗用药造成了巨大的威胁,引起患者的反复感染和用药失败.铜绿假单胞菌作为医院内感染的主要条件致病菌之一,其耐药机制已被广泛研究,而异质性耐药研究则相对较少.本文主要就...     

铜绿假单胞菌生物降解特性的研究进展

近年来在环境污染物的生物降解研究方面有了很大进展。铜绿假单胞菌(Pseudomon asaeruginosa,PA)作为重要的降解菌株之一,具有较强的降解能力,可降解物质种类广泛,在环境污染物的生物降解中具有重要作用并占据重要地位。本文综述了PA的降解特性、代谢途径、遗传基础与酶系及促降解物质在生物降解方面的研究进展。     

铜绿假单胞菌与机体免疫的相互关系

铜绿假单胞菌是导致许多急性和慢性感染的重要细菌,在机械通气或某些免疫缺陷的患者中常常导致严重的感染。在其所造成的感染中,随着感染的持续发展,铜绿假单胞菌极易形成生物膜。生物膜是一个具有结构性、协调性和功能性的高度组织群体,生物膜细菌的生物学特性与浮游菌显著不同,其环境适应能力更强,不仅可以防止抗生素的杀灭,还能逃避宿主免疫,使得铜绿假单胞菌相关感染的临床治疗变得更加棘手。本文主要综述铜绿假单胞菌如何逃避宿主免疫和机体在铜绿似单胞菌感染时的免疫反应及在免疫方面的治疗进展。     

NAD-dependent glutamate dehydrogenase from Pseudomonas aeruginosa is a membrane-bound enzyme

Measurements of the deaminating activity of NAD-dependent glutamate dehydrogenase (NAD-GDH) in Pseudomonas aeruginosa strain 8602 (PAC 1) showed an initially constant rate that gave way to a 3.5-fold increased rate on prolonged incubation. Only the faster rate was observed when assay mixtures were preflushed with nitrogen or were treated with the detergent Triton X-100. Comparison of the intracellular distribution of NAD-GDH with marker enzymes showed it to be associated with the cytoplasmic membrane. The results suggest that NAD-GDH may be linked to oxygen through an electron-transport system.     

Identification and purification of a distinct dihydrolipoamide dehydrogenase from pea chloroplasts

Two distinct dihydrolipoamide dehydrogenases (E3s, EC 1.8.1.4) have been detected in pea (Pisum sativum L. cv. Little Marvel) leaf extracts and purified to at or near homogeneity. The major enzyme, a homodimer with an apparent subunit M_r value 56 000 (80–90% of overall activity), corresponded to the mitochondrial isoform studied previously, as confirmed by electrospray mass spectrometry and N-terminal sequence analysis. The minor activity (10–20%), which also behaved as a homodimer, copurified with chloroplasts, and displayed a lower subunit M_r value of 52 000 which was close to the M_r value of 52 614±9.89 Da determined by electrospray mass spectrometry. The plastidic enzyme was also present at low levels in root extracts where it represented only 1–2% of total E3 activity. The specific activity of the chloroplast enzyme was three-to fourfold lower than its mitochondrial counterpart. In addition, it displayed a markedly higher affinity for NAD⁺ and was more sensitive to product inhibition by NADH. It exhibited no activity with NADP⁺ as cofactor nor was it inhibited by the presence of high concentrations of NADP⁺ or NADPH. Antibodies to the mitochondrial enzyme displayed little or no cross-reactivity with its plastidic counterpart and available amino acid sequence data were also suggestive of only limited sequence similarity between the two enzymes. In view of the dual location of the pyruvate dehydrogenase multienzyme complex (PDC) in plant mitochondria and chloroplasts, it is likely that the distinct chloroplastic E3 is an integral component of plastidic PDC, thus representing the first component of this complex to be isolated and characterised to date.Abbreviations E1 pyruvate dehydrogenase - E2 dihydrolipoamide acetyltransferase - E3 dihydrolipoamide dehydrogenase - PDC pyruvate dehydrogenase complex - OGDC 2-oxoglutarate dehydrogenase complex - GDC glycine decarboxylase complex - SDS-PAGE sodium dodecyl sulphate/polyacrylamide gel electrophoresis - TDP thiamine diphosphate - M_r relative molecular mass J.G.L. is grateful to the Biotechnology and Biological Sciences Research Council (BBSRC), U.K. for continuing financial support. M.C. is the holder of a BBSRC-funded earmarked Ph.D. studentship.     

The Crystal Structure of L-Leucine Dehydrogenase from Pseudomonas aeruginosa

Leucine dehydrogenase (LDH, EC 1.4.1.9) catalyzes the reversible deamination of branched-chain L-amino acids to their corresponding keto acids using NAD⁺ as a cofactor. LDH generally adopts an octameric structure with D4 symmetry, generating a molecular mass of approximately 400 kDa. Here, the crystal structure of the LDH from Pseudomonas aeruginosa (Pa-LDH) was determined at 2.5 _Å resolution. Interestingly, the crystal structure shows that the enzyme exists as a dimer with C2 symmetry in a crystal lattice. The dimeric structure was also observed in solution using multiangle light scattering coupled with size-exclusion chromatography. The enzyme assay revealed that the specific activity was maximal at 60°C and pH 8.5. The kinetic parameters for three different amino acid and the cofactor (NAD⁺) were determined. The crystal structure represents that the subunit has more compact structure than homologs’ structure. In addition, the crystal structure along with sequence alignments indicates a set of non-conserved arginine residues which are important in stability. Subsequent mutation analysis for those residues revealed that the enzyme activity reduced to one third of the wild type. These results provide structural and biochemical insights for its future studies on its application for industrial purposes.     

Simple and rapid procedure for the purification of lipoprotein(a)

Lipoprotein(a) [Lp(a)] is a low-density lipoprotein-like particle displaying strong athero-thrombotic properties. Highly purified Lp(a) is increasingly requested for standardization of Lp(a) measurements and for biological studies. Several procedures have been described for Lp(a) separation and purification but none of them appear completely suitable. We present here a procedure for Lp(a) purification based on sequential elutions after lysine-Sepharose affinity chromatography. We were able to identify four distinct subspecies of Lp(a) showing different affinity to ε-amino groups of lysine-Sepharose, simply by modifying molarity and pH of the eluents; the fraction obtained in highly purified state represented the major form and could be eluted with 0.5 M sodium phosphate buffer (pH 4.4). Advantages of this procedure are represented by simplicity, rapidity and final yield.     

The Cysteine Dioxygenase Homologue from Pseudomonas aeruginosa Is a 3-Mercaptopropionate Dioxygenase

Thiol dioxygenation is the initial oxidation step that commits a thiol to important catabolic or biosynthetic pathways. The reaction is catalyzed by a family of specific non-heme mononuclear iron proteins each of which is reported to react efficiently with only one substrate. This family of enzymes includes cysteine dioxygenase, cysteamine dioxygenase, mercaptosuccinate dioxygenase, and 3-mercaptopropionate dioxygenase. Using sequence alignment to infer cysteine dioxygenase activity, a cysteine dioxygenase homologue from Pseudomonas aeruginosa (p3MDO) has been identified. Mass spectrometry of P. aeruginosa under standard growth conditions showed that p3MDO is expressed in low levels, suggesting that this metabolic pathway is available to the organism. Purified recombinant p3MDO is able to oxidize both cysteine and 3-mercaptopropionic acid in vitro, with a marked preference for 3-mercaptopropionic acid. We therefore describe this enzyme as a 3-mercaptopropionate dioxygenase. Mössbauer spectroscopy suggests that substrate binding to the ferrous iron is through the thiol but indicates that each substrate could adopt different coordination geometries. Crystallographic comparison with mammalian cysteine dioxygenase shows that the overall active site geometry is conserved but suggests that the different substrate specificity can be related to replacement of an arginine by a glutamine in the active site.     

Proteomic analysis of outer membrane vesicles derived from Pseudomonas aeruginosa

Pseudomonas aeruginosa, an opportunistic human bacterial pathogen, constitutively secretes outer membrane vesicles (OMVs) into the extracellular milieu. Although recent progress has revealed that OMVs are essential for pathogenesis of P. aeruginosa, their proteins have not been comprehensively analyzed so far. In this study, we identified 338 vesicular proteins with high confidence by five separate LC-MS/MS analyses. This global proteome profile provides a basis for future studies to elucidate the pathological functions of OMVs from P. aeruginosa.     

Biochemical characterization of isovaleryl-CoA dehydrogenase (LiuA) of Pseudomonas aeruginosa and the importance of liu genes fora functional catabolic pathway of methyl-branched compounds

Growth of Pseudomonas aeruginosa on acyclic terpene alcohols (citronellol) and on other methyl-branched compounds such as leucine or isovalerate requires a functional leucine/isovalerate utilization (Liu) pathway. In this study, we investigated the liuABCDE gene cluster by insertion mutant analysis, heterologous expression of liuA in Escherichia coli and by biochemical characterization of purified LiuA protein. Mutants with insertion in any of the liu genes were unable to utilize acyclic terpenes or leucine/isovalerate and confirmed the importance of the liu genes for catabolism of methyl-branched compounds. An insertion mutant in liuA was complemented by a liuA copy in trans , indicating that possible polar downstream effects of the insertion are not essential for growth. LiuA purified from recombinant E. coli revealed acyl-CoA dehydrogenase activity with isovaleryl-CoA ( K _M 2.3 μM) and butyryl-CoA as substrates. Other acyl-CoA compounds such as isobutyryl-CoA, 3-hydroxybutyryl-CoA, octanoyl-CoA, citronellyl-CoA or 5-methyl-hex-4-enoyl-CoA were not utilized. Experimental evidence for expression and essential functions of other Liu proteins in metabolism of methyl-branched compounds is provided.     

连续七年铜绿假单胞菌的临床分布及耐药性变化分析

了解铜绿假单胞菌在医院的动态分布及耐药情况变化,防止耐药菌株的传播及暴发流行。细菌鉴定采用法国生物梅里埃公司的API 20NE和VITEK2系统,药敏试验采用K-B纸片扩散法,结果判定按照2005版CLSI的标准,药敏结果分析采用WHONET5.3。7 a共分离铜绿假单胞菌2 220株,主要来源于病房的呼吸道标本,分离的细菌数逐年增加,连续7 a监测了14种抗菌药物,均有不同程度的耐药,且耐药率有逐年增加的趋势,尤其是从2003年起ICU病房成立后,ICU病房分离的PA数占总数的30%以上,引起总体耐药率的明显增加。动态监测铜绿假单胞菌的耐药情况,对临床治疗其引起的感染具有十分重要的意义。     

铜绿假单胞菌生物被膜与宿主免疫的关系

铜绿假单胞菌(Pseudomonas aeruginosa,P.aeruginosa)是一种常见的革兰阴性条件致病菌,能引起严重的院内感染,可从支气管扩张、肺囊性纤维化(CF)等患者体内分离。机体免疫系统可以通过识别不同的病原体相关分子模式(PAMPs)来抵御P.aeruginosa的感染,但P.aeruginosa生物被膜(BF)的形成可以导致这些成分被遮蔽从而引起免疫逃逸,导致疾病的迁延难愈。BF是一种与游离细菌相对立的生活方式,能帮助细菌有效适应外部环境,其可以通过藻酸盐的屏障作用,抵抗吞噬细胞的吞噬,干扰多核白细胞(PMNs)的激活,从而逃避宿主免疫。研究P.aeruginosa-BF的免疫逃逸机制,发现有效清除P.aeruginosa-BF的方法,从而为临床治疗P.aeruginosa引起的感染性疾病提供科学依据。现以P.aeruginosa为例对近年来国内外BF的免疫逃逸机制的研究进展进行综述。