Identification of lactoferrin-binding proteins from Treponema pallidum subspecies pallidum and Treponema denticola

Lactoferrin-binding or -associated proteins were identified in Treponema pallidum subspecies pallidum and Treponema denticola by affinity column chromatography using human lactoferrin and detergent-solubilized, radiolabelled spirochaetes. Two discrete polypeptides of T. pallidum with masses of 45 and 40kDa and a broad band from 29-34 kDa exhibited association with human apo- and partially ferrated lactoferrin. T. denticola produced two proteins that associated with a lactoferrin affinity matrix (50 and 35 kDa). T. pallidum and T. denticola did not associate with soluble, human transferrin in parallel experiments. Soluble human lactoferrin competed with all lactoferrin-associated proteins from T. pallidum and T. denticola in competitive-binding assays. However, the T. denticola proteins dissociated from a lacto-ferrin-affinity matrix in the presence of differing concentrations of unlabelled, soluble lactoferrin competitor. Treatment with phospholipase D altered migration of the diffuse 29-34 kDa band of T. pallidum suggesting that the polypeptide was lipid-modified. Each of the lactoferrin-binding proteins from T. pallidum and T. denticola reacted with pooled rabbit syphilitic antisera. The lactoferrin-binding proteins of T. pallidum reacted with human sera from patients at all stages of syphilis. In addition, a monoclonal antibody generated against the 45 kDa polypeptide of T. pallidum crossreacted with the 29–34 kDa protein.     

Physical map of the genome of Treponema pallidum subsp. pallidum (Nichols).

A physical map of the chromosome of Treponema pallidum subsp. pallidum (Nichols), the causative agent of syphilis, was constructed from restriction fragments produced by NotI, SfiI, and SrfI. These rare-cutting restriction endonucleases cleaved the T. pallidum genome into 16, 8, and 15 fragments, respectively. Summation of the physical lengths of the fragments indicates that the chromosome of T. pallidum subsp. pallidum is approximately 1,030 to 1,080 kbp in size. The physical map was constructed by hybridizing a variety of probes to Southern blots of single and double digests of T. pallidum genomic DNA separated by contour-clamped homogeneous electric field electrophoresis. Probes included cosmid clones constructed from T. pallidum subsp. pallidum genomic DNA, restriction fragments excised from gels, and selected genes. Physical mapping confirmed that the chromosome of T. pallidum subsp. pallidum is circular, as the SfiI and SrfI maps formed complete circles. A total of 13 genes, including those encoding five membrane lipoproteins (tpn47, tpn41, tpn29-35, tpn17, and tpn15), a putative outer membrane porin (tpn50), the flagellar sheath and hook proteins (flaA and flgE), the cytoplasmic filament protein (cfpA), 16S rRNA (rrnA), a major sigma factor (rpoD), and a homolog of cysteinyl-tRNA synthetase (cysS), have been localized in the physical map as a first step toward studying the genetic organization of this noncultivable pathogen.     

Restriction analysis of DNA from Treponema pallidum, the causative agent of syphilis

When purified DNA from pathogenic Treponema pallidum is digested with restriction endonucleases it results in the formation of discrete DNA fragments which range between 2.5 to 10 Kilobase pairs. No such precise fragmentation occurs with DNA isolated from nonpathogenic T. pallidum. The appearance of the discrete restriction fragments from the pathogenic T. pallidum DNA does not represent a contamination of satellite DNA from rabbit, the host in which the organism was propagated, but rather represents the presence of redundant DNA or DNA of less complexity in the pathogenic T. pallidum DNA preparation.     

Whole genome analyses of transporters in spirochetes: Borrelia burgdorferi and Treponema pallidum

The completely sequenced genomes of two spirochetes, Borrelia burgdorferi(Bbu) and Treponema pallidum (Tpa) were analyzed for the distribution of transporter types. Both organisms exhibited fewer proteins with >7 alpha-helical transmembrane spanners (TMSs), and fewer identified transport systems per megabase pair of DNA than most other prokaryotes analyzed. Each organism exhibits one recognizable ion channel protein of the MscS family. Tpa has twice as many primary carriers as Bbu but lacks PTS permeases that are plentiful in Bbu. Tpa is the only prokaryote so far sequenced which has two F-type ATPases. Large families of secondary nutrient uptake carriers (MFS and APC) that are prevalent in other organisms are essentially lacking in Spirochetes. The largest Spirochete secondary carrier families consist of efflux systems. While both Bbu and Tpa exhibit an unusual degree of transporter diversity, major differences in specificity exist between these two organisms.     

Electron microscope study of the plasmid DNA from various strains of Treponema pallidum

Electron microscopic investigation and electrophoretic analysis of DNA from two cultural strains of pale treponemes (Treponema pallidum VIII and Treponema pallidum Reiter) allowed us to identify the circular molecules of plasmid DNA their size evaluated as 7 to 8 MD. Side by side with the molecules of this size the electron microscopic pictures of the plasmid DNA of both strains of pale treponemes show small superspiralized molecules of plasmid DNA. According to electrophoretic data the size of this DNA is evaluated as two thousand base pairs.     

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.     

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.     

Ultrastructure of Lipopolysaccharide Isolated from Treponema pallidum

A lipopolysaccharide (LPS) fraction was extracted from Nichols, nonpathogenic Treponema pallidum by the hot, phenol-water procedure. The LPS was freed of nucleic acids and water-soluble proteins by successive exposures to ribonuclease, deoxyribonuclease, and Pronase. Purified LPS responded positively in a colorimetric assay for lipopolysaccharide. Electron microscope examination of the LPS both before and after purification demonstrated a heterogeneous mixture of forms including spheres, doughnuts, and ribbons. The trilaminar nature of the ribbon forms was observed by both negative staining and thin sectioning. Lyophilization of the LPS caused an increase in the number and length of ribbon forms seen. Results suggest that the surface layers of treponemes are similar to those of gram-negative bacteria.     

Complete genome sequence of Treponema pallidum ssp. pallidum strain SS14 determined with oligonucleotide arrays

Background

Probiotics such as bifidobacteria have been shown to maintain a healthy intestinal microbial balance and help protect against infections. However, despite these benefits, bifidobacteria still remain poorly understood at the biochemical, physiological and especially the genetic level. Herein we describe, for the first time, the development of a non-invasive luciferase-based reporter system for real-time tracking of Bifidobacterium species in vivo.

Results

The reporter vector pLuxMC1 is based on the recently described theta-type plasmid pBC1 from B. catenatulatum [1] and the luxABCDE operon from pPL2lux [2]. Derivatives of pLuxMC1, harbouring a bifidobacterial promoter (pLuxMC2) as well as a synthetically derived promoter (pLuxMC3) [3] placed upstream of luxABCDE, were constructed and found to stably replicate in B. breve UCC2003. The subsequent analysis of these strains allowed us to assess the functionality of pLuxMC1 both in vitro and in vivo.

Conclusion

Our results demonstrate the potential of pLuxMC1 as a real-time, non-invasive reporter system for Bifidobacterium. It has also allowed us, for the first time, to track the colonisation potential and persistence of this probiotic species in real time. An interesting and significant outcome of the study is the identification of the caecum as a niche environment for B. breve UCC2003 within the mouse gastrointestinal tract (GI) tract.     

Footprint of positive selection in Treponema pallidum subsp. pallidum genome sequences suggests adaptive microevolution of the syphilis pathogen

In the rabbit model of syphilis, infection phenotypes associated with the Nichols and Chicago strains of Treponema pallidum (T. pallidum), though similar, are not identical. Between these strains, significant differences are found in expression of, and antibody responses to some candidate virulence factors, suggesting the existence of functional genetic differences between isolates. The Chicago strain genome was therefore sequenced and compared to the Nichols genome, available since 1998. Initial comparative analysis suggested the presence of 44 single nucleotide polymorphisms (SNPs), 103 small (≤3 nucleotides) indels, and 1 large (1204 bp) insertion in the Chicago genome with respect to the Nichols genome. To confirm the above findings, Sanger sequencing was performed on most loci carrying differences using DNA from Chicago and the Nichols strain used in the original T. pallidum genome project. A majority of the previously identified differences were found to be due to errors in the published Nichols genome, while the accuracy of the Chicago genome was confirmed. However, 20 SNPs were confirmed between the two genomes, and 16 (80.0%) were found in coding regions, with all being of non-synonymous nature, strongly indicating action of positive selection. Sequencing of 16 genomic loci harboring SNPs in 12 additional T. pallidum strains, (SS14, Bal 3, Bal 7, Bal 9, Sea 81-3, Sea 81-8, Sea 86-1, Sea 87-1, Mexico A, UW231B, UW236B, and UW249C), was used to identify "Chicago-" or "Nichols -specific" differences. All but one of the 16 SNPs were "Nichols-specific", with Chicago having identical sequences at these positions to almost all of the additional strains examined. These mutations could reflect differential adaptation of the Nichols strain to the rabbit host or pathoadaptive mutations acquired during human infection. Our findings indicate that SNPs among T. pallidum strains emerge under positive selection and, therefore, are likely to be functional in nature.     

Pulse radiolysis studies on superoxide reductase from Treponema pallidum

Superoxide reductases (SORs) are small metalloenzymes, which catalyze reduction of O2*- to H2O2. The reaction of the enzyme from Treponema pallidum with superoxide was studied by pulse radiolysis methods. The first step is an extremely fast bi-molecular reaction of the ferrous center with O2, with a rate constant of 6 x 10 (8) M(-1) s(-1). A first intermediate is formed which is converted to a second one with a slower rate constant of 4800 s(-1). This latter value is 10 times higher than the corresponding one previously reported in the case of SOR from Desulfoarculus baarsii. The reconstituted spectra for the two intermediates are consistent with formation of transient iron-peroxide species.     

Assessment of the immune responses to Treponema pallidum Gpd DNA vaccine adjuvanted with IL-2 and chitosan nanoparticles before and after Treponema pallidum challenge in rabbits

Syphilis is a multistage,sexually transmitted disease caused by the spirochete,Treponema pallidum(Tp).A significantly high incidence of syphilis has been reported in several countries,including China,and there is an urgent need for the development of efficacious vaccines against syphilis.DNA vaccines are a major breakthrough in the field of vaccination with several advantages over traditional vaccines.Animal model studies of Tp DNA vaccines have not been reported elsewhere but our previous reports describe the development of a single-gene Tp DNA vaccine and preclinical immunization study.In this study,chitosan(CS) nanoparticles were used as a vector and an interleukin-2 expression plasmid(pIL-2) as an adjuvant to enhance a TpGpd DNA vaccine candidate(pTpGpd) in a rabbit Tp skin challenge model.At week 8 after the first immunization,three rabbits from each group were used to determine cytokine measurements and spleen lymphocyte proliferation assay.pTpGpd in combination with pIL-2 wrapped with CS led to the greatest enhancement of anti-TpGpd antibodies and T-cell proliferation.During infection,levels of anti-TpGpd antibodies and T-cell proliferation were measured.Both the serum special IgG and IL-2,interferon-γ were significantly increased by the co-injection of the IL-2 plasmid compared with the injection of TpGpd DNA alone(P<0.05).Furthermore,IL-2 plasmid coinjection efficiently enhanced the antigen-specific lymphocyte proliferation response.Additionally,the ratios of positive skin lesions and ulcer lesions in groups immunized with pTpGpd were significantly lower than those of the pIL-2,CS or pIL-2 mixed with CS control groups(P<0.001).CS vectored and pIL-2 adjuvanted pTpGpd immunized animals exhibited the lowest rates of positive skin tests(8.33%) and ulcer lesions(4.17%) and the fastest recovery(42 d).These experiments indicate that co-injection of a pIL-2 plasmid with pTpGpd DNA vaccine wrapped with CS can significantly strengthen the long-term stability of immune response during infection,efficiently improve the protective effect against T.pallidum spirochetes infection and attenuate syphilitic lesion development.     

Match probability calculations for multi-locus DNA profiles

The paper considers aspects of the match probability calculation for multi-locus DNA profiles and a related calculation which aims to assess the probability that a pair of profiles is concordant for the presence and absence of bands. It is suggested that levels of allelism and linkage for multi-locus profiles may be higher than reported in previous studies, and that comparison of bandsharing values between different studies is problematic. Our view is that the independence assumptions which underpin the calculations have not been established. The effect of ignoring (local) heterogeneities in band frequencies may be non-conservative. Concerns thus raised about the match probability calculation could be important in practical casework. The speculative nature of some aspects of the concordance probability calculation would seem to render it inappropriate for use in court.Editor's commentsThe author continues the investigation of multilocus probes begun introduced by Curnow in this volume. He contrasts bandsharing estimates from Jeffreyset al. (1991) and the UK Forensic Science Service. Much of the difference is attributable to the greater resolving power of Jeffreys' gels (J.S. Buckleton, personal communication). Tests for independence of multilocus probe bands, and studies of the joint possession of subsets of bands within populations, were conducted by the UK Forensic Science Service, but not published (J.S. Buckleton, personal communication). In spite of the complexities of multilocus probe profiles, it is interesting to note that, just as when several single-locus probes are used, the same profiles are not found for unrelated people in large populations.     

Separation of Treponema pallidum from Tissue Debris Through Continuous-Particle Electrophoresis

Treponema pallidum can only be cultured in living animal tissue, such as rabbit testes. However, the extract of these organisms from the testicular material leaves the T. pallidum contaminated with tissue debris. This paper describes the separation of T. pallidum from the debris by continuous-particle electrophoresis. The importance of equilibration time before electrophoresis is discussed.     

Genetics of Treponema: relationship between Treponema pallidum and five cultivable treponemes.

Three genetically distinct groups of treponemes have been identified by saturation reassociation assays using 125I-labeled treponemal DNAs. The three groups are (i) virulent Treponema pallidum (Nichols strain), (ii) T. phagedenis and its biotypes Reiter and Kazan 5, and (iii) T. refringens biotypes Nichols and Noguchi. There is no detectable DNA sequence homology (less than 5%) among the three groups. The groups have distinct guanine + cytosine contents: 52.4 to 53.7% for T. pallidum, 41.5% for T. refringens, and 38 to 39% for T. phagedenis.     

Organization of the ribosomal RNA genes in Treponema phagedenis and Treponema pallidum.

The genomic DNA fragment which contains ribosomal RNA (rRNA) genes for Treponema phagedenis was cloned into bacteriophage vector lambda EMBL3. A restriction map of the fragment was constructed and the organization of the rRNA genes was determined. The fragment contained at least one copy of the 16S, 23S and 5S sequences and the genes are arranged in the order 16S-23S-5S. Southern hybridization using radiolabeled rRNA gene probes to genomic DNA from T. phagedenis strain Reiter and T. pallidum strain Nichols showed that these organisms have two radioactive fragments which hybridize to the probes in their genome. These results suggest that both pathogenic and non-pathogenic strains of Treponema may carry at least two sets of rRNA genes on their chromosomes.     

Comparative modeling of class 1 lysyl tRNA synthetase from Treponema pallidum

Lysyl tRNA synthetases facilitate amino acylation and play a crucial role in the essential cellular process of translation. They are grouped into two distinct classes (class I and class II). Class I lysyl tRNA synthetase is considered as a drug target for syphilis caused by Treponema pallidum. Comparative genome analysis shows the absence of its sequence homolog in eukaryotes. The structure of class I lysyl tRNA synthetase from Treponema pallidum is unknown and the difficulties in the in vitro culturing of Treponema makes it non-trivial. We used the structural template of class I lysyl tRNA synthetase from the archaea Pyrococcus horikoshii for modeling the Treponema pallidum lysyl tRNA synthetase structure. Thus, we propose the usefulness of the modeled class I lysyl tRNA synthetase for the design of suitable inhibitors towards the treatment of syphilis.     

梅毒螺旋体感染实验室诊断方法研究进展

梅毒是一种性传播疾病,病程长、危害大、临床表现复杂,容易造成漏诊和误诊.梅毒的早期诊断对控制梅毒传播具有重要意义.本文对当前梅毒螺旋体的病原学、血清学及分子生物学等检测方法及研究进展作一综述.