Defining the interaction of the Treponema pallidum adhesin Tp0751 with laminin

Various invasive pathogens attach to host tissues via the extracellular matrix component laminin, the major glycoprotein found within basement membranes. Previous investigations identified the laminin-binding adhesin Tp0751 within the spirochete bacterium Treponema pallidum. In the current study, Tp0751 was shown to attach to a variety of laminin isoforms that are widely distributed throughout the host, including laminins 1, 2, 4, 8, and 10. Such universal attachment is conducive for an adhesin present within a highly invasive pathogen that encounters a variety of tissue sites during the course of infection. Additional studies systematically identified the amino acid residues within Tp0751 that contribute to laminin binding using synthetic peptides designed from the mature protein sequence. The minimum laminin-binding region of the adhesin was localized to 10 amino acids; peptides containing these residues inhibited attachment of Tp0751 and T. pallidum to laminin. Further, Tp0751-specific antibodies inhibited attachment of T. pallidum to laminin. This study furthers our knowledge of the interaction of T. pallidum with laminin, an association that is proposed to facilitate bacterial traversal of basement membranes and subsequent entry into the circulation and tissue invasion. As such, these investigations will reveal new targets for possible prevention of bacterial dissemination and establishment of chronic infection.     

梅毒螺旋体黏附素Tp0751 核酸菌影的构建及免疫原性研究

目的:构建梅毒螺旋体(Tp)黏附素Tp0751 的重组真核大肠埃希菌菌影(EBG)并检测其在免疫鼠中的免疫原性,为探讨新型梅毒疫苗奠定基础。方法:构建pcDNA3.1(+) / Tp0751 真核表达载体,将其装载入已构建的空EBG 中,形成重组核酸菌影pcD/ Tp0751-BG,计算装载率;将核酸菌影转染鼠源性巨噬细胞RAW264.7,Western blot(WB)鉴定目的蛋白表达。将雌性BALB/ c 鼠随机分为A(PBS)、B(空EBG)、C(空pcDNA3.1)三个对照组和D(pcD/ Tp0751)、E(pcD/ Tp0751-BG)、F(pcD/ Tp0751-BG+rTp0751)三个实验组,各组间隔两周肌注免疫共三次,检测特异性血清IgG 及生殖道黏膜SIgA、小鼠脾细胞增殖水平和分泌IFN-γ水平。结果:重组真核质粒对菌影的装载率为76.1%;WB 显示此转染细胞能有效表达重组目的蛋白。D、E、F 实验组小鼠特异性血清IgG 与生殖道SIgA 效价均随免疫次数增加而增加,各时间点均显著高于三个对照组(P<0.01),于末次加免后第8 周达到峰值,此时F 组IgG 与SIgA 效价分别为1 :102 400 与1 :12 800;首次加免2 周后,E、F 组均显著高于D 组(P<0.01);末次加免2 周后,F 组显著高于E 组(P<0.01)。末次加免后第8 周,D、E、F 组的刺激指数(SI)值与IFN-γ水平均分别显著高于三个对照组(P<0.01);E、F 组均分别显著高于D 组(P<0.01);F 组分别均高于E 组(P<0.05)。结论:Tp0751 真核质粒菌影具有良好的免疫原性,在小鼠体内诱生了有效的系统和黏膜的体液应答以及系统细胞免疫应答;异源加免较同源加免免疫效果更好。     

梅毒螺旋体Tp0751黏附蛋白的表达、纯化及免疫活性研究

目的 表达梅毒螺旋体黏附蛋白Tp0751,纯化表达产物并进行免疫活性分析,为探索Tp0751重组蛋白在梅毒致病过程中的作用奠定基础.方法 通过生物信息学分析,去除Tp0751信号肽序列,构建原核表达体进行诱导表达;Ni亲和层析柱纯化重组蛋白,Western blot检测其免疫反应性,用重组蛋白免疫新西兰家兔,评价其免疫原性.结果 成功构建了pET-28a(+)-0751原核表达载体,经表达、纯化后获得了相对分子质量约为26×103的融合蛋白;Western blot检测其能与梅毒患者阳性血清发生特异性反应;利用纯化的Tp0751重组蛋白免疫新西兰家兔,能诱导家兔产生特异性免疫应答,ELISA法测定免疫血清中特异性抗体滴度在1∶10 240以上.结论 重组表达的Tp0751黏附蛋白具有良好的免疫活性,为进一步研究其在梅毒致病过程中的作用和生物学功能奠定了基础.     

Enhanced levels of attachment of fibronectin-primed Treponema pallidum to extracellular matrix.

Freshly extracted Treponema pallidum organisms treated with exogenous human fibronectin (Fn) (Fn-primed treponemes) showed a 6- to 15-fold increased level of attachment to Fn-coated cover slips and to extracellular matrix (ECM) when compared with unprimed treponemes. Treponemes primed with collagen or laminin showed no similar enhanced binding to immobilized Fn or ECM. Preexposure of immobilized Fn and ECM to anti-Fn serum but not to anti-collagen or anti-laminin serum prevented treponemal adherence. Also, the presence of proteoglycanlike molecules such as dextran sulfate or heparan sulfate inhibited Fn-primed treponemal attachment to Fn or ECM. In contrast Fn-primed treponemes did not exhibit elevated levels of attachment to eucaryotic cell monolayers. To understand the increased tropism of Fn-primed T. pallidum organisms for Fn and ECM-like surfaces, we radiolabeled freshly extracted treponemes with [35S]methionine and examined them for the presence of surface immunoreactive Fn. Magnetic protAspheres and glass beads coated with monospecific anti-Fn serum bound only 20 to 30% of radiolabeled treponemes. Nonadherent treponemes failed to bind to gelatin-agarose, further confirming the absence of surface Fn or Fn-like material. Fn-free organisms, however, did attach to Fn-coated cover slips and to cell monolayers like treponemes of the original population. Incubation of Fn-free treponemes with human Fn resulted in almost total binding of organisms to anti-Fn antibody on glass beads and also produced increased attachment to Fn-coated cover slips and ECM. These results suggest that enhanced interactions between T. pallidum and the host are dependent on the presence of Fn on syphilis spirochetes and the specific location and orientation of Fn in vivo.     

重组梅毒螺旋体蛋白Tp47通过诱导uPA增加血管内皮细胞通透性

目的探讨重组梅毒螺旋体蛋白Tp47(rTpp47)对单核-巨噬细胞系THP-1合成尿激酶型纤溶酶原激活物(uPA)的调控及其对人脐静脉/血管内皮细胞(HUVECs)通透性的影响。方法用rTpp47刺激THP-1细胞24 h后,分别收集细胞培养上清和THP-1细胞,用ELISA和Western blot检测THP-1细胞表达的uPA含量;用THP-1细胞培养上清刺激人单层血管内皮细胞后,使用FITC-葡聚糖评价单层内皮细胞通透性的变化,用Western blot检测uPA对HUVECs细胞紧密连接蛋白claudin-5表达的影响以及PKC信号通路是否参与rTpp47诱导的THP-1细胞表达uPA。结果重组蛋白rTpp47刺激THP-1细胞合成和分泌的uPA显著高于对照组(P<0.05,P<0.001);用rTpp47与THP-1细胞共培养24 h后收集的细胞培养上清刺激单层血管内皮细胞12和24 h,实验组血管内皮细胞相对通透性显著高于对照组(P<0.05,P<0.000 1);uPA活性抑制剂阿米洛利(amiloride)抑制了rTpp47刺激THP-1细胞分泌...     

梅毒螺旋体Tp0136活性肽段的可溶性表达、纯化及鉴定

目的 筛选梅毒螺旋体特异性抗原Tp0136的活性肽段,可溶性表达和纯化该肽段,并鉴定其免疫活性,探索Tp0136活性肽段在早期梅毒诊断中的价值.方法 通过生物信息学方法对Tp0136亲水性、B细胞表位和二级结构等进行分析,筛选出Tp0136活性肽段(Tp0136B)替代全蛋白.将Tp0136B基因插入到pET22b(+)上,在E.coli BL21中表达.镍离子亲和色谱纯化表达产物,Western blot检测其免疫反应性,免疫日本大耳白兔评价其免疫原性,免疫双扩检测其效价,以重组Tp0136B蛋白为包被抗原的间接ELISA检测早期梅毒血清抗体.结果 重组工程菌可溶性表达相对分子质量约为28×103的rTp0136B,表达率为21%,制备得到纯度大于98%的rTp0136B.纯化的rTp0136B能诱导大耳白兔产生特异性免疫应答,免疫双扩测得其效价为1:16.Western blot检测重组蛋白能与兔抗Tp0136多克隆抗体发生特异性反应.间接ELISA检测正常人血清均为阴性,而早期梅毒血清抗体的阳性率为85.5%.结论 重组表达的Tp0136活性肽段具有良好的免疫活性,预示其在早期梅毒血清学诊断中具有良好的前景.

Abstract：

Objective To express and purify recombinant Tp0136 epitope fragment, and study the immunity activity. Methods The Tp0136 selective fragment(Tp0136B) gene was devised by the surface property analysis, solvent-accessible suface calculateions, secondary structure function region analysis, and was inserted between the sites of Nde Ⅰ and Not Ⅰ in pET22b ( + ) . The recombinant plasmid was expressed in E. coli BI21. After nickel ion metal affinity chromatography, the antigenic and immune reactivity of rTp0136B was confirmed. Then indirect ELISA with the rTp0136B as coating antigen was performed to detect the anti-Tp0136 antibody in sera from 100 normal human controls and 131 primary syphilis patients. Results The rTp0136B was soluble expressed with a molecular weight of about 28 000 and was obtained with a purity of ＞98% by chromatography. Western blot proved that the rTp0136B could specifically react with anti-Tp0136 polyclonal antibody. Specific humoral response was elicited by the recombinant protein in Japan negative. The positive detection rate in sera from primary syphilis patients was 85.5%. Conclusion This result suggested that the recombinant Tp0136 epitope fragments have a satisfactory immunocompetence,which may have applications in the serodiagnosis of primary syphilis.     

Expression and purification of the recombinant outermembrane protein Tp0453 of Treponema pallidum and its characterization of immuno-competence

ELISA test has been shown to have some advan tages in relation to the tests used for the diagnosisof syphilis because of its easy and quick perfor mance and result readings. With recent develop ment of the gene engineering technology and eluci dation of the whole genome of Nichols strain ofTreponema pallidum, new protein coding openreading frames (ORFs) are available for testing,and the study on the serological tests based on therecombinant protein have been become the focus ofinter…     

Mucopolysaccharidase of Treponema pallidum

Treponema pallidum (Nichols strain) exhibited mucopolysaccharidase activity. Acidic mucopolysaccharides were broken down more rapidly by viable treponemes than by heat-inactivated treponemes or membrane filtrates of treponemal suspensions. Ouchterlony immunodiffusion demonstrated the occurrence of antibodies to the hyaluronidase-like enzyme within syphilitic sera. After intratesticular inoculation of 2 x 10(7) to 6 x 10(7) treponemes, these anti-mucopolysaccharidase antibodies were detected between 9 and 35 days postinoculation. In addition, acidic mucopolysaccharides were present in the serum of infected animals 9 and 16 days postinoculation. Immune serum that contained antibodies to the mucopolysaccharidase restricted treponemal breakdown of acidic mucopolysaccharides. It has been previously demonstrated that immune rabbit serum contains a factor that blocks attachment of T. pallidum (Nichols strain) to cultured mammalian cells. This factor was effectively absorbed by prior incubation with bovine hyaluronidase. It is postulated that T. pallidum attaches to acidic mucopolysaccharides on the surface of cultured cells through the mucopolysaccharidase enzyme at the surface of the organisms. These findings are discussed in terms of the histopathogenesis of T. pallidum with applications to the healing immune response.     

The Treponema pallidum outer membrane protein Tp92 activates endothelial cells via the chemerin/CMKLR1 pathway

Endothelium damage caused by Treponema pallidum is the key step in the systemic dissemination and pathophysiology of syphilis, particularly cardiovascular syphilis and neurosyphilis. However, the molecular mechanisms supporting endothelium damage of syphilis are undefined. The outer membrane proteins were thought to be involved. Tp92 was first identified as an outer membrane protein of T. pallidum. Homologous proteins to Tp92 play important roles in cell attachment, inflammation, and tissue destruction in other bacterial species. In this study, we investigated the effect of Tp92 on endothelial cells activation. The data showed that Tp92 induced chemerin production in activated endothelial cells. Endothelial cell-derived chemerin upregulated the expression of TNF-α and ICAM-1 in endothelial cells via CMKLR1. In addition, endothelial cell-derived chemerin promoted THP-1-derived macrophage migration towards endothelial cells. These findings suggest that Tp92 may play an important role in mediating endothelial cell activation by inducing the secretion of chemerin.     

Serodiagnosis of syphilis: antibodies to recombinant Tp0453, Tp92, and Gpd proteins are sensitive and specific indicators of infection by Treponema pallidum

Syphilis serodiagnosis relies on a combination of nonspecific screening tests (antilipoidal antibodies) and Treponema pallidum-specific tests (anti-T. pallidum antibodies). We studied a group of six recombinant T. pallidum antigens for their sensitivities and specificities with sera from individuals with syphilis (n = 43), relapsing fever (n = 8), Lyme disease (n = 8), and leptospirosis (n = 9) and from uninfected individuals (n = 15). Three recombinant proteins, Tp0155, Tp0483, and Tp0751, demonstrated sensitivity values that ranged from 28 to 42%. In contrast, three other recombinant proteins exhibited the following sensitivity and specificity values: Tp0453, 100% sensitivity and 100% specificity; Tp92 (Tp0326), 98% sensitivity and 97% specificity; and Gpd (Tp0257), 91% sensitivity and 93% specificity. Tp0453, Tp92, and Gpd also were recognized by sera from individuals with early primary syphilis that were nonreactive with the antilipoidal Venereal Disease Research Laboratory test. The reactivities of syphilis patient sera with Tp0453, Tp92, and Gpd were proportional to the titers of these sera with the treponemal test MHA-TP (microhemagglutination assay for T. pallidum). Thus, the recombinant T. pallidum antigens Tp0453, Tp92, and Gpd show promise as diagnostic antigens in the enzyme-linked immunosorbent assay-based assay.     

Surface Mucopolysaccharides of Treponema pallidum

The viscous mucoid fluid that accumulates within syphilitic lesions may be due to breakdown of host tissue during infection, or may be synthesized by Treponema pallidum. Experiments were performed to investigate the acidic mucopolysaccharides that occur at the surface of T. pallidum (Nichols strain). These mucopolysaccharides were demonstrated by reaction with acidified bovine serum albumin and by agglutination with wheat germ agglutinin and soybean agglutinin. The polycations ruthenium red and toluidine blue influenced treponemal survival. Concentrations of both compounds at 200 mug/ml inhibited survival, whereas concentrations at 0.1mug/ml enhanced survival. The mucopolysaccharide concentration within the mucoid fluid that accumulates during intratesticular infection was determined by reaction with acidified bovine serum albumin; it ranged from 10,000 mug/ml to less than 8 mug/ml. The addition of this mucoid fluid to treponemal suspensions resulted in differing effects on T. pallidum survival. Some preparations were inhibitory, and others were stimulatory. Commercial preparations of hyaluronic acid and chondroitin sulfate at 400, 200, 100, and 50 mug/ml were detrimental to treponemal survival. The organisms exhibited pronounced clumping in the presence of the higher concentrations of hyaluronic acid. These clumps of treponemes were comprised of mucopolysaccharides as shown by acidified bovine serum albumin and toluidine blue reactions and by hyaluronidase degradation. Results are discussed in terms of the derivation and potential role of acidic mucopolysaccharides at the surface of T. pallidum.     

Antibody responses elicited against the Treponema pallidum repeat proteins differ during infection with different isolates of Treponema pallidum subsp. pallidum

Variation in the expression of the different Tpr proteins in the syphilis spirochete, Treponema pallidum subsp. pallidum, may have important implications in its ability to evade host immune detection and cause persistent infection. In the present study we examined the pattern of antibody responsiveness to different Tpr members during infection with three isolates of T. pallidum. There was variability in the specificities and temporal patterns of reactivity of the antibodies elicited against the individual Tpr proteins, suggesting that isolates may express different repertoires of Tpr proteins during infection.     

Activation of the classical and alternative pathways of complement by Treponema pallidum subsp. pallidum and Treponema vincentii

Both in vivo and in vitro studies have indicated that complement plays an important role in the syphilitic immune responses. Few quantitative data are available concerning activation of the classical pathway by Treponema pallidum subsp. pallidum, and no information is available on treponemal activation of the alternative pathway. Activation of both pathways was compared by using T. pallidum subsp. pallidum and the nonpathogen T. vincentii. With rabbit and human sources of complement, both organisms rapidly activated the classical pathway, as shown by hemolysis of sensitized sheep erythrocytes and by the generation of soluble C4a. With human sources of complement, both organisms also activated the alternative pathway, as shown by hemolysis of rabbit erythrocytes and by the generation of soluble C3a in the presence of magnesium ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA). During incubation, organisms remained actively mobile and did not lyse, indicating that activation was a function of complement reactivity with the intact outer treponemal surface. In addition, freshly harvested T. pallidum subsp. pallidum immediately activated both pathways of complement; preincubation of organisms did not enhance complement reactivity. T. vincentii was a more potent activator of this pathway. T. pallidum subsp. pallidum contained almost four times as much surface sialic acid as T. vincentii did. When sialic acid was enzymatically removed from T. pallidum subsp. pallidum, enhanced activation of the alternative pathway was detected. It is proposed that T. pallidum subsp. pallidum retards complement-mediated damage by the alternative pathway through surface-associated sialic acid. This may be an important virulence determinant that enables these organisms to readily disseminate through the bloodstream to infect other tissues.     

Outer Envelope of Virulent Treponema pallidum

Ultrathin sections of virulent Treponema pallidum (Nichols strain) were examined with the electron microscope, and the presence of an outer cell envelope was documented.     

Lipids of Treponema pallidum Kazan 5

The lipid composition of Treponema pallidum Kazan 5 cultivated in a lipid-defined medium was investigated. Lipids comprised 18 to 20% of the dry weight of the treponeme. Glycolipid and phospholipids accounted for 90 to 95% of the total lipids and free fatty acids made up the remaining 5 to 10%. The major polar lipids were the glycolipid, 1-(O-alpha-d-galactopyranosyl)-2,3-diglyceride (45 to 55%), and phosphatidylcholine (30 to 40%). Phosphatidylethanolamine (5 to 10%), an unidentified compound (1 to 2%), and occasional trace amounts of diphosphatidylglycerol (cardiolipin) were also found. The monogalactosyldiglyceride was also a major component (50%) of the lipids of the Reiter, Noguchi, and Nichols strains of T. pallidum. The fatty acid composition of Kazan 5 usually consisted of saturated and unsaturated fatty acids ranging from 14 to 18 carbons depending upon the fatty acids added to the culture medium. When the cells were cultivated on elaidic acid (trans-9-octadecenoic acid), their lipids contained only elaidic acid.     

Surface Characterization of Virulent Treponema pallidum

Characterization of the surface of Treponema pallidum was accomplished by [¹²⁵I]lactoperoxidase-catalyzed iodination of intact organisms and sensitive radioimmunoprecipitation and gel electrophoresis technology. At least 11 outer membrane proteins with molecular weights ranging from 89,000 (89K) to 20K were identified, and all elicited high titers of antibody in experimentally infected rabbits. Proteins of 89.5K, 29.5K, and 25.5K previously implicated as ligands involved in attachment (J. B. Baseman and E. C. Hayes, J. Exp. Med. 151:573-586, 1980) were found to reside on the treponemal surface. Low levels of the 89.5K treponemal protein were released by high salt concentrations, whereas the remaining comigrating material was neither radioiodinated nor released with selective detergents. Other lower-molecular-weight (60K, 45K, and 30K) surface proteins were extracted with octyl glucoside detergent, suggesting their hydrophobic interaction with the external membrane. The molecular organization of surface proteins was studied by employing the cross-linker dithiobis(succinimidyl)-propionate, and data suggested the presence of a highly fluid envelope resulting in random collisions by the surface proteins. The biological function of the treponemal outer envelope proteins was evaluated using, as the indicator system, adherence of T. pallidum to monolayer cultures of eucaryotic cells. Trypsin treatment of motile, freshly harvested organisms decreased the extent of surface parasitism to normal rabbit testicular cells, reinforcing the idea of the proteinaceous nature and role of treponemal ligands for attachment. Other data supported functional and antigenic relatedness among the implicated ligands. Finally, brief periodate treatment of human epithelial (HEp-2) and normal rat testicular cells as well as casein-elicited rabbit peritoneal macrophages significantly reduced the extent of treponemal parasitism, suggesting a role of specific host membrane molecules as mediators of attachment.     

Molecular differentiation of Treponema pallidum subspecies

Treponema pallidum includes three subspecies of antigenically highly related treponemes. These organisms cause clinically distinct diseases and cannot be distinguished by any existing test. In this report, genetic signatures are identified in two tpr genes which, in combination with the previously published signature in the 5' flanking region of the tpp15 gene, can differentiate the T. pallidum subspecies, as well as a simian treponeme.     

Penicillin-binding proteins and peptidoglycan of Treponema pallidum subsp. pallidum.

Penicillin-binding proteins (PBPs) of Treponema pallidum subsp. pallidum (T. pallidum) were characterized by using [3H]penicillin G and a conjugate consisting of ampicillin and 125I-labeled Bolton-Hunter reagent. Both antibiotics specifically radiolabeled proteins with molecular masses of 94, 80, 63, and 58 kilodaltons (kDa); 125I-labeled Bolton-Hunter reagent-ampicillin also radiolabeled several polypeptides with lower molecular masses. The 94- and 58-kDa proteins demonstrated the highest binding affinities for [3H]penicillin G and were radiolabeled at concentrations of 8 and 40 nM, respectively. Radiolabeling of PBPs was detectable after 1 min of incubation in 1 microM [3H]penicillin G and was nearly maximal within 10 min. The rapidity of penicillin binding contrasted with the observation that only 40% of virulent treponemes became immobilized during prolonged incubation in vitro with a much higher concentration (1 mM) of unlabeled penicillin. Two lines of evidence indicated that most, if not all, of the PBPs are integral cytoplasmic membrane proteins: (i) preincubation of organisms in 0.1% Triton X-100 solubilized nearly all of the outer membranes but did not affect radiolabeling of PBPs, and (ii) except for the 80-kDa protein, the PBPs partitioned into the detergent phase following extraction with the nonionic detergent Triton X-114. The presence of peptidoglycan in T. pallidum was confirmed by the detection of muramic acid in the sodium dodecyl sulfate-insoluble, proteinase K-resistant residue obtained from Triton X-114-extracted organisms.