Phosphofructokinase activities within the order Spirochaetales and the characterisation of the pyrophosphate-dependent phosphofructokinase from Spirochaeta thermophila

The subtype of phosphofructokinase activity, either ATP-, ADP- or pyrophosphate-dependent, present in members of three genera from the Spirochaetales was investigated. The individual species/strains examined included Spirochaeta alkalica, S. asiatica, S. halophila, S. isovalerica, S. litoralis, S. zuelzerae, S. thermophila, two thermophilic spirochetes, Treponema bryantii, T. denticola, paragraph signT. pectinovorum, Leptospira biflexa and L. interrogans. All of the Spirochaeta strains, regardless of their phenotype, possessed primarily a pyrophosphate-dependent phosphofructokinase. In contrast, T. bryantii, T. denticola and L. biflexa had predominantly an ATP-dependent activity, whereas no activity was detected in T. pectinovorum or paragraph signL. interrogans. The results suggest that pyrophosphate-dependent phosphofructokinase activity may be a reliable phenotypic marker for the genus Spirochaeta and that there are potentially interesting differences in how the catabolism of saccharides is controlled among members of genera within the Spirochaetales. The pyrophosphate-dependent phosphofructokinase from S. thermophila strain RI 19.B1 was purified (303-fold) to homogeneity and biochemically characterised. The S. thermophila enzyme displayed hyperbolic kinetics with respect to both the forward and reverse cosubstrates and was not significantly affected by traditional activators or inhibitors of phosphofructokinase. The biochemical characterisation represents the first spirochete phosphofructokinase to be described.     

Identification and characterization of a Treponema pallidum subsp. pallidum gene encoding a DNA adenine methyltransferase

The nucleotide sequence of a DNA adenine methyltransferase gene (dam) from Treponema pallidum has been determined. Southern blot analysis of T. pallidum chromosomal DNA indicated that this gene is present as a single copy. The dam gene encodes a 303 amino acid protein whose deduced sequence has significant homology with DNA (N⁶-adenine) methyltransferases. T. pallidum Dam can be assigned to group α DNA amino methyltransferases based on the order of nine conserved motifs that are present in the protein. Digests of T. pallidum chromosomal DNA performed with isoschizomer restriction endonucleases (Sau3AI, DpnI, and MboI) confirmed the presence of methylated adenine residues in GATC sequences (Dam⁺ phenotype).     

化学发光微粒子免疫法和梅毒螺旋体颗粒凝集试验检测梅毒抗体的比较

目的评价化学发光微粒子免疫法（chemiluminescence microparticle immunoassay, CMIA）检测临床血清标本梅毒螺旋体抗体的敏感性和特异性。方法用梅毒螺旋体颗粒凝集试验（TVeponema Pallidum particie agglutination test,TPPA）法作为对照标准,采用CMIA法检测2012年11月到12月1200例住院患者的血清标本,并用卡方检验评价两种检测方法对同一个样本的化验结果的一致性。结果1200例血清标本中用CMIA法检出阳性率为11.3%,TPPA法检出阳性率为10. 9% ,以TPPA为标准,CMIA法敏感性为96. 9%,特异性为99. 2%,其中CMIA法检测血清S/CO值〉 4. 00的110例,用TPPA确认107例阳性,阳性预测值（PPV）为97.3%;CMIA法S/C0值在1.0-9.0,TPPA可出现阴性结果。结论CMIA法可替代TPPA法进行梅毒螺旋体抗体检测,对于CMIA法检测S/C0值1. 0-4.0的需进一步复检。     

免疫-PCR法检测梅毒螺旋体特异性抗体

以梅毒螺旋体重组蛋白为抗原,应用免疫-PCR方法检测梅毒螺旋体抗体,并同常规ELISA法进行比较,探讨免疫-PCR方法检测梅毒螺旋体特异性抗体的可行性。结果免疫-PCR法敏感性是常规ELISA法的104倍,阳性检出率高于ELISA法;对照血清标本梅毒螺旋体抗体检测为阴性。表明免疫-PCR方法具有较高敏感性和特异性,有一定的临床推广价值,对梅毒患者的早期诊断及时治疗等具有重要意义。     

Kinetic analysis of PPi-dependent phosphofructokinase from Porphyromonas gingivalis

We have previously cloned the gene encoding a pyrophosphate-dependent phosphofructokinase (PFK), designated PgPFK, from Porphyromonas gingivalis, an oral anaerobic bacterium implicated in advanced periodontal disease. In this study, recombinant PgPFK was purified to homogeneity, and biochemically characterized. The apparent K(m) value for fructose 6-phosphate was 2.2 mM, which was approximately 20 times higher than that for fructose 1,6-bisphosphate. The value was significantly greater than any other described PFKs, except for Amycolatopsis methanolica PFK which is proposed to function as a fructose 1,6 bisphosphatase (FBPase). The PgPFK appears to serves as FBPase in this organism. We postulate that this may lead to the gluconeogenic pathways to synthesize the lipopolysaccharides and/or glycoconjugates essential for cell viability.     

Purification and characterization of an ADP-dependent phosphofructokinase from Thermococcus zilligii

The ADP-dependent phosphofructokinase (PFK) from Thermococcus zilligii has been purified 950 fold; it had a specific activity of 190 U mg⁻¹. The enzyme required Mg²⁺ ions for optimal activity and was specific for ADP. The forward reaction kinetics were hyperbolic for both cosubstrates (pH optimum of 6.4), and the apparent K _m values for ADP and fructose-6-phosphate were 0.6 mM (apparent V _max of 243 U mg⁻¹) and 1.47 mM (apparent V _max of 197 U mg⁻¹), respectively. Significantly, the enzyme is indicated to be nonallosteric but was slightly activated by some monovalent cations including Na⁺ and K⁺. The protein had a subunit size of 42.2 kDa and an estimated native molecular weight of 66 kDa (gel filtration). Maximal reaction rates for the reverse reaction were attained at pH 7.5–8.0, and the apparent K _m values for fructose-1,6-bisphosphate and AMP were 0.56 mM (apparent V _max of 2.9 U mg⁻¹) and 12.5 mM, respectively. The biochemical characteristics of this unique ADP-dependent enzymatic activity are compared to ATP and pyrophosphate-dependent phosphofructokinases. Received: August 14, 1998 / Accepted: December 2, 1998     

基因重组抗原检测梅毒螺旋体抗体研究

梅毒是一种传染性很强的性传播疾病,早期诊断是防止其传播及治疗的关键。采用基因重组的梅毒螺旋体P47和P15抗原对289份临床标本进行梅毒螺旋体抗体的检测,并与常规方法进行了比较,结果表明：重组抗原ELISA法具有较高的敏感性和特异性,并能对梅毒进行早期诊断,可以代替常规方法检测梅毒螺旋体抗体。186份现患和已治愈梅毒患者标本,重组抗原ELISA法、TPHA法和RPR法均为阳性;60份健康献血员标本,重组抗原ELISA法和TPHA法、RPR法均为阴性;17份与梅毒患者有性接触者的标本,重组抗原ELISA法有2份阳性,而TPHA法、RPR法均为阴性,1个月后复查这2份血清TPHA和RPR均为阳性;6份RPR和类风湿因子均为阳性的血清,重组抗原ELISA法和TPHA法均为阴性,;20份肝硬化患者血清,3种方法检测均为阴性。     

三种方法检测梅毒螺旋体抗体的比较

应用ELISA法、TRUST法和TPPA法分别检测梅毒患者血清标本中梅毒螺旋体IgG抗体,比较3种方法的敏感性和特异性,选择一种适合于梅毒螺旋体抗体检测的高敏感性和高特异性的血清学检测方法。     

Expression and sequence analysis of a Treponema pallidum gene, tpn38(b), encoding an exported protein with homology to T. pallidum and Borrelia burgdorferi proteins

Abstract An Escherichia coli clone containing recombinant plasmid C19 was identified from a Treponema pallidum genomic DNA library by in situ immunoassay. E. coli maxicells containing pC19 synthesized a treponemal protein doublet of 39.2 and 38.2 kDa, designated TpN38(b). Pulse-chase and protein processing studies showed that TpN38(b) is synthesized with a cleavable amino-terminal signal peptide. A 2.0-kb fragment of pC19 containing the tpn38(b) gene was subcloned and sequenced. The tpn38(b) gene is 1029 nucleotides long and encodes a protein of 343 amino acids with a calculated molecular mass of 37.9 kDa. The deduced amino acid sequence of TpN38(b) has homology with the T. pallidum TpN35 lipoprotein and the Borrelia burgdorferi BmpA, BmpB, BmpC, and BmpD proteins.     

Two 16S-23S ribosomal DNA intergenic regions in different Treponema pallidum subspecies contain tRNA genes

Abstract The 16S-23S intergenic spacers of Treponema pallidum subspecies pallidum , Nichols strain, and Treponema pallidum subspecies pertenue , Gauthier strain, have been cloned, characterized and sequenced. Isoleucine and alanine tRNA genes have been identified within the 16S-23S intergenic regions on separate alleles of 293 and 303 bases, respectively. The two alleles are present in both T.p. pallidum and T.p. pertenue , and show no sequence differences between the bacterial subspecies. The ile-tRNA and ala-tRNA genes show 65% and 84% sequence identity, respectively, with the homologous genes of the related spirochete, Borrelia burgdorferi .     

电化学发光法检测梅毒螺旋体特异性抗体的应用研究

目的 对罗氏电化学发光免疫法（ECLIA）检测梅毒螺旋体特异性抗体的临床价值进行评估。方法 收集梅毒疑似病例血清标本132份,分别用ECLIA、梅毒螺旋体明胶凝集试验（TPPA）和免疫印迹法（WB）进行检测,以WB为金标准,计算并比较ECLIA和TPPA的灵敏度和特异性,进而比较化学发光免疫法检测低S/CO值和高S/CO值的灵敏度和特异性差异。结果 针对132份血清标本,ECLIA敏感性为100.00%,特异性为83.33%,阳性预测值为96.43%,阴性预测值为100.00%,总符合率为96.97%。TPPA敏感性为93.52%,特异性为87.50%,阳性预测值为97.12%,阴性预测值为75.00%,总符合率为92.42%。ECLIA检测1≤S/CO＜3组与S/CO≥3组的敏感性均为100.00%,特异性分别为86.96%和95.24%,结论 ECLIA检测具有较高的敏感性,适合临床大样本筛查,对S/CO值低的标本应结合TPPA、WB及临床资料确诊。     

梅毒螺旋体膜抗原基因在毕赤酵母中的表达和蛋白纯化

以梅毒螺旋体(Treponema pallidumsubsp.pallidum)Nichols菌株基因组DNA为模板,通过PCR扩增梅毒螺旋体47kDa、17kDa和15kDa 3个膜抗原基因,克隆进毕赤酵母表达载体pPICZ B,构建重组表达载体pTP47、pTP17、pTP15,转化酵母菌株GS115,甲醇诱导表达。表达菌体裂解后通过镍离子亲和层析获得3个抗原与6xHis tag的融合蛋白,重组蛋白的获得量分别为His-TP15:4.8mg/L;His-TP 17:6.6mg/L;His-TP47:25mg/L,经SDS-PAGE鉴定纯度都在96%以上,ELISA鉴定均具有很好的抗原性。从而首次在毕赤酵母中表达出梅毒螺旋体膜抗原,为梅毒血清学检测方法开辟了新的抗原制备途径。     

巢式PCR法在早期梅毒诊断中的应用评价

目的：评价巢式PCR（nPCR）法在早期梅毒诊断中的临床应用价值,以提高早期梅毒诊断的灵敏度和特异性。方法：选择2010年10月至2011年11月来我院就诊,经临床综合分析为一期梅毒的患者195例和同期就诊的120例非梅毒患者为研究对象,采用nPCR法对棉拭子标本和血液标本中梅毒螺旋体特异性基因tpp47进行扩增检测,所有标本同时做暗视野镜检和Tp—ELISA血清学检测。结果：nPCR法共检测出阳性标本176例,其灵敏度和特异性分别为90．3％和100％,明显高于暗视野镜检和Tp-ELISA法,差异有统计学意义。结论：nPCR法在早期梅毒诊断中具有较高灵敏度和特异性,可以作为暗视野镜检和血清学检测的补充试验。     

检测梅毒螺旋体抗体的纤维膜条方法的建立及其在诊断中的应用

目的：建立以纤维膜为载体的检测梅毒螺旋体抗体的方法,检查病人血清中对梅毒螺旋体多种抗原的抗体,用于梅毒感染的诊断。方法：将基因工程表达及纯化的梅毒螺旋体蛋白tp15、tp17、tp42和tp47分别结合在纤维膜上,用载抗原的纤维膜条检查血清中的抗体,抗体阳性者在相应抗原位置显示出特异条带。结果：梅毒螺旋体感染者血清中存在特异性抗体,在检查的460份临床诊断的患者血清中,对tp15、tp17、tp42和tp47抗原的抗体检出率分别为41.3%、100%、98.7%和51.7%;134份献血员血清抗体阴性。结论：建立的检测梅毒螺旋体感染的方法可同时检查对多种抗原的抗体,以纤维膜条作为诊断条检测血清抗体方法简便,用于临床诊断更特异、更敏感。     

梅毒螺旋体Tp92蛋白B细胞表位的预测与鉴定

【目的】预测和鉴定梅毒螺旋体(Tp)Tp92蛋白的B细胞表位,为深入探讨这些表位在梅毒表位疫苗中的作用奠定基础。【方法】采用Mobyle、ABCpred和IEDB在线软件综合分析预测Tp92蛋白的B细胞表位,人工合成6条表位多肽,以梅毒患者/感染兔血清(同时设健康人/兔血清对照)为标本,用间接ELISA法鉴定预测Tp92蛋白B细胞表位的免疫反应性。【结果】软件预测显示,Tp92蛋白的P1(24-39AA)、P2(332-347AA)、P3(520-536AA)、P4(575-588AA)、P5(103-118AA)、P6(694-712AA)氨基酸序列可能为其B细胞表位。间接ELISA分析表明,预测的P1、P3、P5和P6均与梅毒患者/感染兔血清呈阳性反应,而与健康人/兔血清不反应。【结论】本研究初步得出以下结论:P1、P3、P5和P6均为Tp92蛋白潜在的特异性B细胞表位,尤其是P3和P6免疫反应性最强。     

Characterization of the serum requirement for macrophage-mediated killing of Treponema pallidum ssp. pallidum: Relationship to the development of opsonizing antibodies

Abstract Serum pools were collected from rabbits bled at various times after intra-testicular infection with Treponema pallidum ssp. pallidum . These were tested for their ability to opsonize T. pallidum and promote killing of the organisms by macrophages. Compared to normal sera, significant opsonization was first seen on day 10 of infection as measured by both ingestion ( P < 0.001) and macrophage-mediated killing ( P = 0.006); significant levels of functional antibodies persisted through 300 days of infection. Although opsonic activity peaked early in infection, antibodies that promoted optimal macrophage-mediated killing developed much later, suggesting that these two functions may represent activities of antibodies with differing specificities or affinities. The initial development of antibodies that augment both phagocytosis and killing corresponds with the in vivo clearance of treponemes from the primary site of infection. These observations support the hypothesis that macrophages are the major effector mechanism for elimination of T. pallidum during early syphilis infection.     

梅毒螺旋体外膜蛋白Gpd基因的克隆、表达及其免疫活性研究

利用PCR技术从Tp Nichols株基因组模板中扩增梅毒螺旋体(Treponema pallidum,Tp)外膜蛋白Gpd基因,定向克隆构建真核表达重组体pcDNA3.1( )-Gpd,免疫印迹和免疫组化技术检测pcDNA3.1( )-Gpd在HeLa细胞中的表达;同时将真核表达重组体pcDNA3.1( )-Gpd免疫新西兰兔,检测其在兔体内的免疫应答效果。免疫印迹和免疫组化鉴定均显示重组体在HeLa细胞中能有效表达一个41kD的Gpd融合蛋白。新西兰兔接种核酸疫苗后,能产生特异性抗体,第3次免疫后2周抗体最高滴度可达1∶1024,免疫后兔脾细胞受Gpd蛋白刺激有明显增殖反应。所诱导的抗体水平和脾淋巴细胞增殖情况均显著高于空质粒对照组和空白对照组(p<0.05)。Tp真核表达重组体pcDNA3.1( )-Gpd的成功构建及能刺激新西兰兔产生较强特异的免疫应答,为Gpd蛋白生物学功能及梅毒DNA疫苗的深入研究奠定了一定的实验基础。