Complete genome sequence of Treponema pallidum, the syphilis spirochete.

The complete genome sequence of Treponema pallidum was determined and shown to be 1,138,006 base pairs containing 1041 predicted coding sequences (open reading frames). Systems for DNA replication, transcription, translation, and repair are intact, but catabolic and biosynthetic activities are minimized. The number of identifiable transporters is small, and no phosphoenolpyruvate:phosphotransferase carbohydrate transporters were found. Potential virulence factors include a family of 12 potential membrane proteins and several putative hemolysins. Comparison of the T. pallidum genome sequence with that of another pathogenic spirochete, Borrelia burgdorferi, the agent of Lyme disease, identified unique and common genes and substantiates the considerable diversity observed among pathogenic spirochetes.

The genome of Treponema pallidum: new light on the agent of syphilis.

Treponema pallidum subsp, pallidum, the causative agent of the sexually transmitted disease syphilis, is a fastidious, microaerophilic obligate parasite of humans. This bacterium is one of the few prominent infectious agents that has not been cultured continuously in vitro and consequently relatively little is known about its virulence mechanisms at the molecular level. T. pallidum therefore represented an attractive candidate for genomic sequencing. The complete genome sequence of T. pallidum has now been completed and comprises 1,138,006 base pairs containing 1041 predicted protein coding sequences. An important goal of this project is to identify possible virulence factors. Analysis of the genome indicates a number of potential virulence factors including a family of 12 proteins related to the Msp protein of Treponema denticola, a number of putative hemolysins, as well as several other classes of proteins of interest. The results of this analysis are reviewed in this article and indicate the value of whole genome sequences for rapidly advancing knowledge of infectious agents.

Specific Th1 cell lines that confer protective immunity against experimental Borrelia burgdorferi infection in mice.

Although humoral responses to Borrelia burgdorferi (Bb) have been shown to be protective in some animal models of Lyme disease, the role of T cells in this disease is less well understood. This work describes three Bb-specific T cell lines that prevent disease progression in syngeneic mice. The T cell lines were generated in C3H mice immunized with Bb in complete Freund's adjuvant. All lines were Bb-specific, CD4+, TCRalphabeta+, and they proliferated and produced interferon-gamma and interleukin-2 on stimulation with Bb. Injection of the cell lines into naive C3H recipients significantly reduced the number of organisms recoverable from the blood and tissues of infected mice and protected them from developing Bb-induced periarthritis. These studies demonstrated that Th1 cells can confer resistance to Bb infection in susceptible mice and suggested that the timing of this T cell response may be critical for determining disease outcome.

Identification and transcriptional analysis of a Treponema pallidum operon encoding a putative ABC transport system, an iron-activated repressor protein homolog, and a glycolytic pathway enzyme homolog.

We have characterized a 5.2-kilobase (kb) putative transport related operon (tro) locus of Treponema pallidum subsp. pallidum (Nichols strain) (Tp) encoding six proteins: TroA, TroB, TroC, TroD, TroR and Phosphoglycerate mutase (Pgm). Four of these gene products (TroA-TroD) are homologous to members of the ATP-Binding Cassette (ABC) superfamily of bacterial transport proteins. TroA (previously identified as Tromp1) has significant sequence similarity to a family of Gram-negative periplasmic substrate-binding proteins and to a family of streptococcal proteins that may have dual roles as substrate binding proteins and adhesins. TroB is homologous to the ATP-binding protein component, whereas TroC and TroD are related to the hydrophobic membrane protein components of ABC transport systems. TroR is similar to Gram-positive iron-activated repressor proteins (DesR, DtxR, IdeR, and SirR). The last open reading frame (ORF) of the tro operon encodes a protein that is highly homologous to the glycolytic pathway enzyme, Pgm. Primer extension results demonstrated that the tro operon is transcribed from a sigma 70-type promoter element. Northern analysis and reverse transcriptase-polymerase chain reactions provided evidence for the presence of a primary 1-kb troA transcript and a secondary, less abundant, troA-pgm transcript. The tro operon is flanked by a Holliday structure DNA helicase homolog (upstream) and two ORFs representing a purine nucleoside phosphorylase homolog and tpp15, a previously characterized gene encoding a membrane lipoprotein (downstream). The presence of a complex operon containing a putative ABC transport system and a DtxR homolog indicates a possible linkage between transport and gene regulation in Tp.

From microbial genome sequence to applications.

Whole genome sequences of microbial pathogens present new opportunities for clinical applications. Chief among these are development of antimicrobials, diagnostics, and vaccines. While antimicrobial development is a more difficult, long-term prospect, new diagnostics and vaccines are likely to be the first products of microbial genomics. To take advantage of whole genome sequences, methods for production of gene products in surrogate hosts (heterologous expression) are required that will work for large-scale, high-throughput gene expression. This will allow genomic information from even the most experimentally difficult pathogens to be mined for applications. In addition, screening methods to test gene products for their potential as vaccine candidates are needed for large-scale screening. These areas for technological development should be stimulated by the potential for converting genomic sequence information into applications.

Genome structure of spirochetes.

The genome structures of several pathogenic spirochetes have recently been determined. The genomes of Borrelia species consist of a linear chromosome of approximately one million base pairs (Mb) and various linear and circular plasmids. Analysis of restriction fragment length polymorphisms and 16S ribosomal RNA sequence data indicate the division of Borrelia burgdorferi into at least three distinct genetic groups. Leptospira interrogans has a circular chromosome 5 Mb in size and a 0.35 Mb extrachromosomal element. Repetitive sequence elements similar to insertion sequences have been identified in the Leptospira interrogans genome. The chromosome of Treponema pallidum subsp. pallidum is circular and has a size of approximately one Mb. Genetic studies conducted to date indicate that B. burgdorferi and L. interrogans have a high degree of genetic diversity, whereas remarkably few genetic differences have been observed among the pathogenic Treponema. Knowledge of the genomic structure of these organisms will serve as a basis for future genetic studies.

Demonstration of Treponema pallidum in a cutaneous gumma by indirect immunofluorescence.

Treponema pallidum was demonstrated in a cutaneous, tertiary syphilitic lesion by indirect immunofluorescence microscopy, but not by darkfield microscopy illumination or silver stain. The numerous organisms observed by this method may help explain the histologically vigorous tissue reaction in tertiary syphilis, despite the scarcity of organisms demonstrable by other methods.

OptiSol corneal storage medium and transmission of Treponema pallidum.

This study was conducted to provide experimental information on the probability of syphilis transmission resulting from corneal transplantation. To determine the effects of commonly employed corneal storage conditions on the survival and infectivity of Treponema pallidum, T. pallidum subsp. pallidum (Nichols) was inoculated into OptiSol storage medium or a T. pallidum survival medium at a concentration of 10(6)/ml and incubated in cornea viewing chambers for 24 h at 4 degrees C. When inoculated intradermally into rabbits (0.1 ml per site), none of the 10 sites developed lesions from suspensions incubated in OptiSol in the presence or absence of 100 microgram/ml gentamicin; T. pallidum incubated in the survival medium yielded lesions at one of 10 sites, whereas freshly extracted organisms produced lesions at all 10 sites. In another set of experiments, the infectivity of corneal tissue from rabbits inoculated intratesticularly with 2 x 10(7) T. pallidum 10 days earlier was determined. Corneas from five T. pallidum-infected rabbits were excised, extracted, and tested for infectivity either immediately after removal or after 24-h storage in OptiSol. Recipient rabbits developed lesions at five of 50 intradermal sites when the corneas were neither stored in OptiSol nor rinsed before extraction. Corneas from 10 donor rabbits that were rinsed with phosphate-buffered saline to remove blood and aqueous humor before extraction did not yield lesions at any of 200 sites in the recipient animals. The results of this study indicate that retention of T. pallidum infectivity is poor under typical corneal storage conditions and that rabbit corneal tissue contains few, if any, infectious T. pallidum organisms under the experimental conditions employed.

Vaccination of cattle and sheep with a combined Clostridium perfringens types C and D toxoid.

Cattle and sheep (30 each) were vaccinated with a combined Clostridium perfringens type C and C perfringens type D toxoid. Vaccination and blood sample collections were made every 2 weeks over a period of 8 weeks. Increases in antitoxin titers occurred after the 2nd administration of the 2.0-ml combined product. Highest titers were recorded when the 2nd vaccinal dose was given 2 weeks after the first. This confirms reports that response to clostridial antigens is greater when the 2nd immunizing dose is delayed 2 to 6 weeks after the initial dose.

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.

In vitro cultivation of Treponema pallidum: independent confirmation.

In vitro cultivation of the virulent Nichols strain of Treponema pallidum was achieved in a tissue culture system as described by A. H. Fieldsteel, D. L. Cox, and R. A. Moeckli (Infect. Immun. 32:908-915, 1981). In 7 of 8 experiments, 8.9- to 26.2-fold increases in the number of T. pallidum were observed over a 12- to 12-day period of incubation.

Correlation between plasmid content and infectivity in Borrelia burgdorferi.

Infectivity-associated plasmids were identified in Borrelia burgdorferi B31 by using PCR to detect each of the plasmids in a panel of 19 clonal isolates. The clones exhibited high-, low-, and intermediate-infectivity phenotypes based on their frequency of isolation from needle-inoculated C3H/HeN mice. Presence or absence of 21 of the 22 plasmids was determined in each of the clones by using PCR primers specific for regions unique to each plasmid, as identified in the recently available genome sequence. Southern blot hybridization results were used to confirm the PCR results in some cases. Plasmid lp25 exhibited a direct correlation with infectivity in that it was consistently present in all clones of high or intermediate infectivity and was absent in all low-infectivity clones. lp28-1, containing the vmp-like sequence locus, also correlated with infectivity; all clones that lacked lp28-1 but contained lp25 had an intermediate infectivity phenotype, in which infection was primarily restricted to the joints. Plasmids cp9, cp32-3, lp21, lp28-2, lp28-4, and lp56 apparently are not required for infection in this model, because clones lacking these plasmids exhibited a high-infectivity phenotype. Plasmids cp26, cp32-1, cp32-2 and/or cp32-7, cp32-4, cp32-6, cp32-8, cp32-9, lp17, lp28-3, lp36, lp38, and lp54 were consistently present in all clones examined. On the basis of these results, lp25 and lp28-1 appear to encode virulence factors important in the pathogenesis of B. burgdorferi B31.

Factors affecting the multiplication and subculture of Treponema pallidum subsp. pallidum in a tissue culture system.

Limited multiplication of Treponema pallidum subsp. pallidum (Nichols strain) can be obtained in the presence of Sf1Ep rabbit epithelial cell cultures, but continuous culture has not yet been achieved. In the system currently employed, growth is exponential for the first 10 to 15 days of culturing, after which multiplication and the percentage of motile organisms decrease. In an effort to identify culture conditions which may adversely affect treponemal viability and growth, eight culture parameters were monitored over a 12-day period of incubation. Several of these parameters, including pH, redox potential, dissolved oxygen concentration, and glucose levels were found to change dramatically during the course of incubation, indicating that they may be responsible for the cessation of treponemal multiplication. The feasibility of extending the period of growth by subculturing was also investigated. In preparation for planned serial subcultivation experiments, several subculture procedures were tested and found to be effective in allowing the transfer of T. pallidum from 3-day-old primary cultures to secondary cultures without loss of motility or growth potential. Increases of up to 55-fold were observed in secondary cultures, but increased growth due to subculturing was not a consistent finding. Use of subculture intervals of greater than or equal to 6 days resulted in a progressive decrease in treponemal multiplication in secondary cultures, although retention of motility was extended in the subcultures compared with motility in the primary cultures. These results indicate that the lack of continued multiplication of T. pallidum in subcultures is not due to damage to the treponemes during subculture. Prolonged multiplication of T. pallidum may be obtained through the stabilization of culture conditions by either performing subcultures at regular intervals or by medium replacement techniques. It was also found that primary T. pallidum cultures could be established by using as the inoculum treponemes that had been stored at -70 degrees C in a medium containing 15% glycerol.

Serum requirement for the multiplication of Treponema pallidum in a tissue-culture system: association of growth-promoting activity with the protein fraction.

The nature of the serum requirement of Treponema pallidum subspecies pallidum (Nichols strain) was examined in a culture system utilizing Sf1Ep cottontail rabbit cells. In this system, significant multiplication of treponemes occurs in the presence of select lots of fetal bovine serum (FBS) or calf serum (CS) at concentrations of greater than or equal to 5% (vol/vol). Heat-inactivation of the serum greatly enhances treponemal multiplication, and normal human serum was found to be as effective as FBS in supporting the growth of T. pallidum. The protein fraction of FBS obtained by membrane ultrafiltration was capable of supporting the multiplication of T. pallidum when added to the basal tissue culture medium; an average increase of 23-fold was observed in these cultures, as compared with a mean increase of 25-fold in the 20% FBS controls. In contrast, the ultrafiltrate fraction of FBS (consisting of compounds with molecular weights of less than 10,000 daltons) did not support either growth or the retention of motility. Proteins precipitable with 25% (wt/vol) polyethylene glycol (i.e., albumin, transferrin, ceruloplasmin, and other proteins) also promoted the growth of T. pallidum. This observation provides further evidence that the required serum components are associated with the protein fraction.

In vitro culture system to determine MICs and MBCs of antimicrobial agents against Treponema pallidum subsp. pallidum (Nichols strain).

A new procedure for determining the susceptibility of Treponema pallidum subsp. pallidum to antimicrobial agents was developed, utilizing a tissue culture system which promotes the in vitro multiplication of this organism. In the absence of antibiotics, T. pallidum (Nichols virulent strain) multiplied an average of 10-fold when incubated for 7 days in the presence of Sf1Ep cottontail rabbit epithelial cell cultures. Varied concentrations of penicillin G, tetracycline, erythromycin, and spectinomycin were added to triplicate cultures to determine their effects on treponemal multiplication, motility, and virulence. The MIC of each antibiotic was defined as the lowest concentration which prevented treponemal multiplication, whereas the MBC was defined as the lowest concentration which abrogated the ability of the cultured treponemes to multiply and cause lesions in rabbits. The in vitro culture technique provided highly reproducible MICs and (in parentheses) MBCs of each of the antibiotics tested: aqueous penicillin G, 0.0005 (0.0025) microgram/ml; tetracycline, 0.2 (0.5) microgram/ml; erythromycin, 0.005 (0.005) microgram/ml; and spectinomycin, 0.5 (0.5) microgram/ml. The significance of these results in light of the in vivo activities and the previous in vitro evaluations of these antibiotics is discussed. The T. pallidum in vitro cultivation system shows promise as a method for studying the interaction between T. pallidum and antimicrobial agents and for screening new antibiotics for syphilis therapy.

Antigenic complexity of Treponema pallidum: antigenicity and surface localization of major polypeptides.

The protein structure of Treponema pallidum was characterized by two-dimensional electrophoresis (2DE), consisting of isoelectric focusing (IEF, pH 5 to 7) in the first dimension and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in the second dimension. Up to 85 major polypeptide species could be detected in the organisms in 2DE gels by Coomassie Blue staining. The antigenicity of the individual polypeptides was determined by transferring the 2DE pattern to nitrocellulose paper and utilizing a sensitive immunoperoxidase procedure to demonstrate the reactivity of immunoglobulins in sera obtained from rabbits infected intratesticularly at least 6 mo previously. The infected rabbit serum reacted with virtually every major polypeptide detectable by protein staining techniques, indicating that infected rabbits produce antibodies against nearly all major T. pallidum proteins at the time when the animals exhibit systemic resistance to reinfection. Surface radioiodination of freshly purified T. pallidum by an Iodogen procedure yielded preferential labeling of a major polypeptide with an apparent m.w. of 39,000. The results of this study indicate that the antigenic complexity of T. pallidum is much greater than described previously. The 39-kd polypeptide appears to be a major surface constituent of T. pallidum and as such may play an important role in the induction of immunity to syphilis.

Kinetics and in vivo induction of genetic variation of vlsE in Borrelia burgdorferi.

The Lyme disease agent, Borrelia burgdorferi, is able to persistently infect humans and animals for months or years in the presence of an active immune response. It is not known how the organisms survive immune attack in the mammalian host. vlsE, a gene localized near one end of linear plasmid lp28-1 and encoding a surface-exposed lipoprotein in B. burgdorferi B31, was shown recently to undergo extensive genetic and antigenic variation within 28 days of initial infection in C3H/HeN mice. In this study, we examined the kinetics of vlsE sequence variation in C3H/HeN mice at 4, 7, 14, 21, and 28 days and at 7 and 12 months postinfection. Sequence changes were detected by PCR amplification and sequence analysis as early as 4 days postinfection and accumulated progressively in both C3H/HeN and CB-17 severe combined immunodeficient (SCID) mice throughout the course of infection. The sequence changes were consistent with sequential recombination of segments from multiple silent vls cassette sites into the vlsE expression site. No vlsE sequence changes were detected in organisms cultured in vitro for up to 84 days. These results indicate that vlsE recombination is induced by a factor(s) present in the mammalian host, independent of adaptive immune responses. The possible inducing conditions appear to be present in various tissue sites because isolates from multiple tissues showed similar degrees of sequence variation. The rate of accumulation of predicted amino acid changes was higher in the immunologically intact C3H/HeN mice than in SCID mice, a finding consistent with immune selection of VlsE variants.

Genetic variation of the Borrelia burgdorferi gene vlsE involves cassette-specific, segmental gene conversion.

The Lyme disease spirochete Borrelia burgdorferi possesses 15 silent vls cassettes and a vls expression site (vlsE) encoding a surface-exposed lipoprotein. Segments of the silent vls cassettes have been shown to recombine with the vlsE cassette region in the mammalian host, resulting in combinatorial antigenic variation. Despite promiscuous recombination within the vlsE cassette region, the 5' and 3' coding sequences of vlsE that flank the cassette region are not subject to sequence variation during these recombination events. The segments of the silent vls cassettes recombine in the vlsE cassette region through a unidirectional process such that the sequence and organization of the silent vls loci are not affected. As a result of recombination, the previously expressed segments are replaced by incoming segments and apparently degraded. These results provide evidence for a gene conversion mechanism in VlsE antigenic variation.

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.