梅毒螺旋体外膜蛋白Tp92及其同源蛋白的研究进展

梅毒是由梅毒螺旋体(Tp)引起的一种严重危害人类健康的慢性感染性疾病,由于Tp目前尚不能体外人工培养,从而限制了对Tp致病机制的深入研究。Tp对宿主靶细胞的早期黏附定植是其后续病程发展的关键,而最早与宿主细胞直接接触的Tp外膜及外膜蛋白就成了关注的焦点。随着Tp全基因组序列的解析和分子生物学技术的发展,Tp外膜蛋白的筛选、鉴定及功能研究取得了一定进展。Tp92是首个在感染了梅毒的兔调理素抗毒血清中通过差示免疫筛选方法筛选出来的Tp外膜蛋白,序列高度保守,具有较强的抗原性,与其他螺旋体属及许多革兰阴性菌的外膜蛋白均具有较高的同源性,其在Tp的致病过程及机体的免疫应答中可能发挥着重要作用。就目前Tp外膜蛋白Tp92及其同源蛋白的研究进展进行了综述。     

梅毒螺旋体部分膜蛋白的研究现状

梅毒螺旋体(Tp)目前尚不能体外培养,抗原获取困难,从而影响到Tp致病机制、实验室诊断方法及疫苗的研究。随着分子生物学技术的发展和Tp全基因序列的解析、多种重组Tp膜蛋白的成功表达及其结构功能的日益明确,为Tp发病机理的研究、诊断方法的更新和疫苗的研制奠定了良好的基础。     

梅毒螺旋体遗传物质和致病机制的研究进展

梅毒螺旋体(Treponema pallidum,Tp)是慢性全身性性传播疾病梅毒的病原体。由于Tp不能持续体外培养,阻碍了对Tp结构及其致病机制的深入研究。目前,Tp(Nicholes株)基因组测序的完成以及分子生物学技术的发展,为Tp的研究提供了机遇。就Tp的遗传物质和致病机制的研究进展进行综述。     

梅毒螺旋体免疫相关膜蛋白的研究进展

梅毒是一种由梅毒螺旋体(Treponema.pallidum,Tp)感染所引起的慢性性传播疾病。近年来,其发病率居高不下,引起了全社会广泛的关注。随着分子生物技术的发展和人们的不断探究发现,膜蛋白可能在Tp致病过程中与宿主黏附、宿主免疫炎症反应等方面起着非常重要的作用,可能为Tp的主要致病因子。因此,对Tp膜蛋白的研究是认识其对宿主的致病性和进行致病机制研究的关键,就Tp的几种主要免疫相关膜蛋白的研究进展作了简要综述。     

梅毒螺旋体Tp0751重组蛋白诱导人单核细胞表达前炎症细胞因子的研究

炎症和持续的获得性免疫应答被认为是造成梅毒螺旋体感染后机体病理损伤的主要原因.Tp膜蛋白是介导炎症反应的主要成分,可能为Tp的主要致病因子.因此对Tp膜蛋白的研究是认识其对宿主的致病性和进行致病机制研究的关键.目前国内外类似研究甚少.因此有必要进行Tp膜蛋白的致病性研究.本研究旨在探讨Tp0751重组蛋白潜在的前炎症活性.结果表明,Tp0751重组蛋白可以时间和剂量依赖方式诱导THP-1细胞表达CKs(IL-1β,TNF-α和IL-6).进一步研究表明,Tp0751重组蛋白可以激活NF-κB的表达;TLR2抗体、CD14抗体、MAPKs/p38特异性抑制剂SB203580和NF-κB抑制剂PDTC均可明显抑制NF-κB的激活和CKs的表达.初步结果证实,Tp0751重组蛋白可通过TLR2和CD14途径激活MAPKs/p38和NF-κB诱导THP-1表达CKs,其可能是Tp的一个重要的致病因子.     

梅毒螺旋体TprK膜蛋白的研究现状

梅毒是由梅毒螺旋体(Tp)引起的一种严重危害人类健康的性传播疾病,其中TprK是Tp的一个重要的膜蛋白,从属于TprP重复蛋白家族,具有增强Tp逃避宿主免疫反应和维持慢性感染的能力,可能是Tp有效疫苗的组分之一。现就TprK膜蛋白的研究现状作一综述。     

梅毒螺旋体4种主要膜蛋白分子的研究现状

梅毒螺旋体是梅毒的病原体。因其体外培养至今尚未成功,该螺旋体获取困难,从而制约了其基础研究。但随着基因工程技术的发展,梅毒螺旋体全基因序列已经成功揭示,其主要结构蛋白研究也取得了重大进展,这些都为进一步深入开展研究提供了基础和前提。本文从梅毒螺旋体几种主要外膜蛋白的结构在梅毒致病机制中的作用和功能,以及有关实际应用等几方面进行综述。     

苍白密螺旋体苍白亚种单克隆抗体的制备及应用

苍白密螺旋体苍白亚种,俗称梅毒螺旋体(Treponema pallidum,Tp)是严重危害人类健康的性传播疾病——梅毒的病原体。由于Tp体外培养至今尚未成功,从而制约了Tp的基础研究。自从Tp单克隆抗体(TpMcAb)问世以后,已应用于Tp感染的临床诊断、抗原性质分析等方面的研究,其制备方法的完善和改进将对梅毒的诊断和防治提供新的方法。本文对Tp McAb的制备和应用作一综述,以展示近年来Tp McAb的最新研究进展。     

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。     

梅毒螺旋体硫氧还蛋白的原核表达、纯化及抗氧化活性分析

本研究利用生物信息学方法预测梅毒螺旋体(Treponema pallidum,Tp)Trx蛋白的生物学性质。Trx蛋白无信号肽,定位在细胞质。二级结构无规卷曲占41.9%,β片层为55%,使用枯草芽胞杆菌Trx蛋白结构(PDB:2gzz.1.A)进行同源建模,获得Trx蛋白三级结构。根据TpTrx基因序列设计引物,以TpDNA为模板,PCR扩增得到大小为318 bp的Trx基因,然后将其连接到pET30a构建重组质粒pET30-Trx,将该重组质粒转化大肠杆菌BL21(DE3),得到重组菌大肠杆菌。利用IPTG诱导目的蛋白表达,该质粒编码的重组Trx蛋白分子量约为16 kDa。使用Ni~(2+)亲和层析纯化获得重组Trx蛋白,发现Trx蛋白具有二硫键还原酶活性。利用菌落计数法观察重组大肠杆菌在H_2O_2胁迫下的存活率,发现能表达Trx蛋白的大肠杆菌存活率高于对照。本研究利用生物信息学方法分析了TpTrx蛋白的性质,并发现它具有抗氧化功能,本研究为解析Tp的抗氧化机制提供实验依据。     

Polypeptides of Treponema pallidum: progress toward understanding their structural, functional, and immunologic roles. Treponema Pallidum Polypeptide Research Group.

Treponema pallidum subsp. pallidum, the spirochete that causes syphilis, is unusual in a number of respects, including its small genome size, inability to grow under standard in vitro culture conditions, microaerophilism, apparent paucity of outer membrane proteins, structurally complex periplasmic flagella, and ability to evade the host immune responses and cause disease over a period of years to decades. Many of these attributes are related ultimately to its protein content. Our knowledge of the activities, structure, and immunogenicity of its proteins has been expanded by the application of recombinant DNA, hybridoma, and structural fractionation techniques. The purpose of this monograph is to summarize and correlate this new information by using two-dimensional gel electrophoresis, monoclonal antibody reactivity, sequence data, and other properties as the bases of polypeptide identification. The protein profiles of the T. pallidum subspecies causing syphilis, yaws, and endemic syphilis are virtually indistinguishable but differ considerably from those of other treponemal species. Among the most abundant polypeptides are a group of lipoproteins of unknown function that appear to be important in the immune response during syphilitic infection. The periplasmic flagella of T. pallidum and other spirochetes are unique with regard to their protein content and ultrastructure, as well as their periplasmic location. They are composed of three core proteins (homologous to the other members of the eubacterial flagellin family) and a single, unrelated sheath protein; the functional significance of this arrangement is not understood at present. Although the bacterium contains the chaperonins GroEL and DnaK, these proteins are not under the control of the heat shock regulon as they are in most organisms. Studies of the immunogenicity of T. pallidum proteins indicate that many may be useful for immunodiagnosis and immunoprotection. Future goals in T. pallidum polypeptide research include continued elucidation of their structural locations and functional activities, identification and characterization of the low-abundance outer membrane proteins, further study of the immunoprotective and immunodiagnostic potential of T. pallidum proteins, and clarification of the roles of treponemal proteins in pathogenesis.     

TP0453, a concealed outer membrane protein of Treponema pallidum, enhances membrane permeability

The outer membrane of Treponema pallidum, the non-cultivable agent of venereal syphilis, contains a paucity of protein(s) which has yet to be definitively identified. In contrast, the outer membranes of gram-negative bacteria contain abundant immunogenic membrane-spanning beta-barrel proteins mainly involved in nutrient transport. The absence of orthologs of gram-negative porins and outer membrane nutrient-specific transporters in the T. pallidum genome predicts that nutrient transport across the outer membrane must differ fundamentally in T. pallidum and gram-negative bacteria. Here we describe a T. pallidum outer membrane protein (TP0453) that, in contrast to all integral outer membrane proteins of known structure, lacks extensive beta-sheet structure and does not traverse the outer membrane to become surface exposed. TP0453 is a lipoprotein with an amphiphilic polypeptide containing multiple membrane-inserting, amphipathic alpha-helices. Insertion of the recombinant, non-lipidated protein into artificial membranes results in bilayer destabilization and enhanced permeability. Our findings lead us to hypothesize that TP0453 is a novel type of bacterial outer membrane protein which may render the T. pallidum outer membrane permeable to nutrients while remaining inaccessible to antibody.     

Sequence analysis and recombinant expression of a 28-kilodalton Treponema pallidum subsp. pallidum rare outer membrane protein (Tromp2).

In this study, we report the cloning, sequencing, and expression of the gene encoding a 28-kDa Treponema pallidum subsp. pallidum rare outer membrane protein (TROMP), designated Tromp2. The tromp2 gene encodes a precursor protein of 242 amino acids including a putative signal peptide of 24 amino acids ending in a type I signal peptidase cleavage site of Leu-Ala-Ala. The mature protein of 218 amino acids has a calculated molecular weight of 24,759 and a calculated pI of 7.3. The predicted secondary structure of Tromp2 shows nine transmembrane segments of amphipathic beta-sheets typical of outer membrane proteins. Recombinant Tromp2 (rTromp2) was expressed with its native signal peptide, using a tightly regulated T7 RNA polymerase expression vector. Under high-level expression conditions, rTromp2 fractionated exclusively with the Escherichia coli outer membrane. Antiserum raised against rTromp2 was generated and used to identify native Tromp2 in cellular fractionations. Following Triton X-114 extraction and phase separation of T. pallidum, the 28-kDa Tromp2 protein was detected prominently in the detergent phase. Alkali and high-salt treatment of purified outer membrane from T. pallidum, conditions which remove peripherally associated membrane proteins, demonstrated that Tromp2 is an integral membrane protein. Whole-mount immunoelectron microscopy of E. coli cells expressing rTromp2 showed specific surface antibody binding. These findings demonstrate that Tromp2 is a membrane-spanning outer membrane protein, the second such protein to be identified for T. pallidum.     

Molecular characterization and cellular localization of TpLRR, a processed leucine-rich repeat protein of Treponema pallidum, the syphilis spirochete.

Automated Edman degradation was used to obtain N-terminal and internal amino acid sequences from a 26-kDa protein in isolated Treponema pallidum outer membranes (OMs). The resulting sequences enabled us to PCR amplify from T. pallidum DNA a 275-bp fragment of the corresponding gene. The complete nucleotide sequence of the gene was determined from fragments amplified by long-distance PCR. Primer extension verified the assigned translational start of the open reading frame (ORF) and putative upstream promoter elements. The ORF encoded a highly basic (pI 9.6) 26-kDa protein which contained an N-terminal 25-amino-acid leader peptide terminated by a signal peptidase I cleavage site. The mature protein contained seven tandemly spaced copies (as well as an eighth incomplete copy) of a leucine-rich repeat (LRR), a motif previously identified in a number of prokaryotic and eukaryotic proteins. Accordingly, the polypeptide was designated T. pallidum leucine-rich repeat protein (TpLRR). Although Triton X-114 phase partitioning showed that TpLRR was hydrophilic, cell localization studies showed that most of the antigen was associated with the peptidoglycan-cytoplasmic membrane complex rather than being freely soluble in the periplasmic space. Immunoblot studies showed that syphilis patients develop a weak antibody response to the antigen. Lastly, the lrr(T. pallidum) gene was mapped to a 60-kb SfiI-SpeI fragment of the T. pallidum chromosome which also contains the rrnA and flaA genes. The function(s) of TpLRR is currently unknown; however, protein-protein and/or protein-lipid interactions mediated by its LRR motifs may facilitate interactions between components of the T. pallidum cell envelope.     

Demonstration of rare protein in the outer membrane of Treponema pallidum subsp. pallidum by freeze-fracture analysis.

The surface of Treponema pallidum subsp. pallidum (T. pallidum), the etiologic agent of syphilis, appears antigenically inert and lacks detectable protein, as judged by immunocytochemical and biochemical techniques commonly used to identify the outer membrane (OM) constituents of gram-negative bacteria. We examined T. pallidum by freeze-fracture electron microscopy to visualize the architecture of its OM. Treponema phagedenis biotype Reiter (T. phagedenis Reiter), a nonpathogenic host-associated treponeme, and Spirochaeta aurantia, a free-living spirochete, were studied similarly. Few intramembranous particles interrupted the smooth convex and concave fracture faces of the OM of T. pallidum, demonstrating that the OM of this organism is an unusual, nearly naked lipid bilayer. In contrast, the concave fracture face of the OM of S. aurantia was densely covered with particles, indicating the presence of abundant integral membrane proteins, a feature shared by typical gram-negative organisms. The concentration of particles in the OM concave fracture face of T. phagedenis Reiter was intermediate between those of T. pallidum and S. aurantia. Similar to typical gram-negative bacteria, the OM convex fracture faces of the three spirochetes contained relatively few particles. The unique molecular architecture of the OM of T. pallidum can explain the puzzling in vitro properties of the surface of the organism and may reflect a specific adaptation by which treponemes evade the host immune response.     

Isolation of the outer membranes from Treponema pallidum and Treponema vincentii.

The outer membranes from Treponema pallidum subsp. pallidum and Treponema vincentii were isolated by a novel method. Purified outer membranes from T. pallidum and T. vincentii following sucrose gradient centrifugation banded at 7 and 31% (wt/wt) sucrose, respectively. Freeze fracture electron microscopy of purified membrane vesicles from T. pallidum and T. vincentii revealed an extremely low density of protein particles; the particle density of T. pallidum was approximately six times less than that of T. vincentii. The great majority of T. vincentii lipopolysaccharide was found in the outer membrane preparation. The T. vincentii outer membrane also contained proteins of 55 and 65 kDa. 125I-penicillin V labeling demonstrated that t. pallidum penicillin-binding proteins were found exclusively with the protoplasmic cylinders and were not detectable with purified outer membrane material, indicating the absence of inner membrane contamination. Isolated T. pallidum outer membrane was devoid of the 19-kDa 4D protein and the normally abundant 47-kDa lipoprotein known to be associated with the cytoplasmic membrane; only trace amounts of the periplasmic endoflagella were detected. Proteins associated with the T. pallidum outer membrane were identified by one- and two-dimensional electrophoretic analysis using gold staining and immunoblotting. Small amounts of strongly antigenic 17- and 45-kDa proteins were detected and shown to correspond to previously identified lipoproteins which are found principally with the cytoplasmic membrane. Less antigenic proteins of 65, 31 (acidic pI), 31 (basic pI), and 28 kDa were identified. Compared with whole-organism preparations, the 65- and the more basic 31-kDa proteins were found to be highly enriched in the outer membrane preparation, indicating that they may represent the T. pallidum rare outer membrane proteins. Reconstitution of solubilized T. pallidum outer membrane into lipid bilayer membranes revealed porin activity with two estimated channel diameters of 0.35 and 0.68 nm based on the measured single-channel conductances in 1 M KCl of 0.40 and 0.76 nS, respectively.     

Recombinant Treponema pallidum rare outer membrane protein 1 (Tromp1) expressed in Escherichia coli has porin activity and surface antigenic exposure.

We recently reported the cloning and sequencing of the gene encoding a 31-kDa Treponema pallidum subsp. pallidum rare outer membrane porin protein, designated Tromp1 (D. R. Blanco, C. I. Champion, M. M. Exner, H. Erdjument-Bromage, R. E. W. Hancock, P. Tempst, J. N. Miller, and M. A. Lovett, J. Bacteriol. 177:3556-3562, 1995). Here, we report the stable expression of recombinant Tromp1 (rTromp1) in Escherichia coli. rTromp1 expressed without its signal peptide and containing a 22-residue N-terminal fusion resulted in high-level accumulation of a nonexported soluble protein that was purified to homogeneity by fast protein liquid chromatography (FPLC). Specific antiserum generated to the FPLC-purified rTromp1 fusion identified on immunoblots of T. pallidum the native 31-kDa Tromp1 protein and two higher-molecular-mass oligomeric forms of Tromp1 at 55 and 80 kDa. rTromp1 was also expressed with its native signal peptide by using an inducible T7 promoter. Under these conditions, rTromp1 fractionated predominantly with the E. coli soluble and outer membrane fractions, but not with the inner membrane fraction. rTromp1 isolated from the E. coli outer membrane and reconstituted into planar lipid bilayers showed porin activity based on average single-channel conductances of 0.4 and 0.8 nS in 1 M KCl. Whole-mount immunoelectron microscopy using infection-derived immune serum against T. pallidum indicated that rTromp1 was surface exposed when expressed in E. coli. These findings demonstrate that rTromp1 can be targeted to the E. coli outer membrane, where it has both porin activity and surface antigenic exposure.     

Structural and biochemical basis for polyamine binding to the Tp0655 lipoprotein of Treponema pallidum: putative role for Tp0655 (TpPotD) as a polyamine receptor

Tp0655 of Treponema pallidum, the causative agent of syphilis, is predicted to be a 40 kDa membrane lipoprotein. Previous sequence analysis of Tp0655 noted its homology to polyamine-binding proteins of the bacterial PotD family, which serve as periplasmic ligand-binding proteins of ATP-binding-cassette (ABC) transport systems. Here, the 1.8 A crystal structure of Tp0655 demonstrated structural homology to Escherichia coli PotD and PotF. The latter two proteins preferentially bind spermidine and putrescine, respectively. All of these proteins contain two domains that sandwich the ligand between them. The ligand-binding site of Tp0655 can be occupied by 2-(N-morpholino)ethanesulfanoic acid, a component of the crystallization medium. To discern the polyamine binding preferences of Tp0655, the protein was subjected to isothermal titration calorimetric experiments. The titrations established that Tp0655 binds polyamines avidly, with a marked preference for putrescine (Kd=10 nM) over spermidine (Kd=430 nM), but the related compounds cadaverine and spermine did not bind. Structural comparisons and structure-based sequence analyses provide insights into how polyamine-binding proteins recognize their ligands. In particular, these comparisons allow the derivation of rules that may be used to predict the function of other members of the PotD family. The sequential, structural, and functional homology of Tp0655 to PotD and PotF prompt the conclusion that the former likely is the polyamine-binding component of an ABC-type polyamine transport system in T. pallidum. We thus rename Tp0655 as TpPotD. The ramifications of TpPotD as a polyamine-binding protein to the parasitic strategy of T. pallidum are discussed.     

Biology of Treponema pallidum: correlation of functional activities with genome sequence data

Aspects of the biology of T. pallidum subsp. pallidum, the agent of syphilis, are examined in the context of a century of experimental studies and the recently determined genome sequence. T. pallidum and a group of closely related pathogenic spirochetes have evolved to become highly invasive, persistent pathogens with little toxigenic activity and an inability to survive outside the mammalian host. Analysis of the genome sequence confirms morphologic studies indicating the lack of lipopolysaccharide and lipid biosynthesis mechanisms, as well as a paucity of outer membrane protein candidates. The metabolic capabilities and adaptability of T. pallidum are minimal, and this relative deficiency is reflected by the absence of many pathways, including the tricarboxylic acid cycle, components of oxidative phosphorylation, and most biosynthetic pathways. Although multiplication of T. pallidum has been obtained in a tissue culture system, continuous in vitro culture has not been achieved. The balance of oxygen utilization and toxicity is key to the survival and growth of T. pallidum, and the genome sequence reveals a similarity to lactic acid bacteria that may be useful in understanding this relationship. The identification of relatively few genes potentially involved in pathogenesis reflects our lack of understanding of invasive pathogens relative to toxigenic organisms. The genome sequence will provide useful raw data for additional functional studies on the structure, metabolism, and pathogenesis of this enigmatic organism.