RESUMEN
It has been hypothesized that the neutrophil chemoattractant interleukin-8 (IL-8) forms a gradient in the oral cavity, with the highest concentration of IL-8 produced closest to the bacterial biofilm. In periodontitis, this gradient is disrupted, impairing neutrophil chemotaxis to diseased sites. Treponema denticola is prominently associated with periodontal disease, yet little is known about its ability to modulate the production of inflammatory mediators by epithelial cells. Others have shown that dentilisin, the major outer membrane protease of T. denticola, degrades IL-8 in vitro. We now provide evidence that T. denticola also fails to induce IL-8 production from primary gingival epithelial cells (PGEC). The lack of IL-8 production is not explained by IL-8 degradation, because a protease mutant that does not degrade IL-8 does not induce IL-8 production with these stimuli either. The lack of innate immune mediator production may be a more global phenomenon because T. denticola fails to induce IL-6 or intercellular adhesion molecule 1 production from PGEC. T. denticola also fails to induce transcription of IL-8 and human beta-defensin-2 messenger RNA. The lack of immune mediator production is not explained by the failure of T. denticola to interact with Toll-like receptor 2 (TLR-2), as T. denticola stimulates nuclear factor-kappaB nuclear translocation in TLR-2-transfected HEK293 cells. Not only can T. denticola degrade the IL-8 present in the periodontal lesion, but this organism also fails to induce IL-8 production by PGEC. The lack of an epithelial cell response to T. denticola may contribute to the pathogenesis of periodontitis by failing to trigger chemotaxis of neutrophils into the periodontal pocket.
Asunto(s)
Células Epiteliales/metabolismo , Encía/metabolismo , Interleucina-8/biosíntesis , Treponema denticola/inmunología , Infecciones por Treponema/inmunología , Células Cultivadas , Células Epiteliales/inmunología , Células Epiteliales/microbiología , Encía/inmunología , Encía/microbiología , Humanos , Molécula 1 de Adhesión Intercelular/biosíntesis , Interleucina-6/biosíntesis , Receptor Toll-Like 2/inmunología , Receptor Toll-Like 2/metabolismoRESUMEN
Diagnostic advances do not generally receive the recognition given to prevention and treatment contributions, for the control and management of infectious diseases including sexually transmitted infections (STIs). In order to identify seminal diagnostic contributions over a half century (1950-2000), the Editorial Board of the WHO Sexually Transmitted Diseases Diagnostics Initiative (SDI) Publication Review or "electronic journal club" were asked to nominate their choices of peer-reviewed publications for special recognition. From 43 nominations, 13 were voted by a panel of 25 "experts" as having made the most significant contributions. The 1964 article by Thayer and Martin, which identified a selective media for gonococcal culture, was chosen unanimously by all panel members and is identified as the classic STI diagnostic article for this era.
Asunto(s)
Sistemas de Atención de Punto/normas , Literatura de Revisión como Asunto , Enfermedades de Transmisión Sexual/diagnóstico , Venereología/normas , Predicción , Humanos , Sistemas de Atención de Punto/tendencias , Venereología/tendenciasRESUMEN
We investigated the evolution of 6 genes from the Treponema pallidum repeat (tpr) gene family, which encode potential virulence factors and are assumed to have evolved through gene duplication and gene conversion events. The 6 loci (tprC, D, G, J, I, and K) were sequenced and analyzed in several members of the genus Treponema, including the 3 subspecies of human T. pallidum (T. pallidum subsp. pallidum, pertenue, and endemicum), Treponema paraluiscuniculi (rabbit syphilis), and the unclassified Fribourg-Blanc (simian) isolate. Phylogenetic methods, recombination analysis, and measures of nucleotide diversity were used to investigate the evolutionary history of the tpr genes. Numerous instances of gene conversion were detected by all 3 methods including both homogenizing gene conversion that involved the entire length of the sequence as well as site-specific conversions that affected smaller regions. We determined the relative age and directionality of the gene conversion events whenever possible. Our data are also relevant to a discussion of the evolution of the treponemes themselves. Higher levels of variation exist between the human subspecies than within them, supporting the classification of the human treponemes into 3 subspecies. In contrast to published theories, the divergence and diversity of T. pallidum subsp. pertenue relative to the other subspecies does not support a much older origin of yaws at the emergence of modern human, nor is the level of divergence seen in T. pallidum subsp. pallidum consistent with a very recent (< 500 years) origin of this subspecies. In general, our results demonstrate that intragenomic recombination has played a significant role in the evolution of the studied tpr genes and emphasize that efforts to infer evolutionary history of the treponemes can be complicated if past recombination events are not recognized.
Asunto(s)
Evolución Molecular , Genes Bacterianos , Filogenia , Recombinación Genética , Treponema pallidum/genética , Animales , Duplicación de Gen , Humanos , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Conejos , Alineación de Secuencia , Treponema pallidum/aislamiento & purificaciónRESUMEN
BACKGROUND/AIMS: Oral treponemes are implicated in the pathogenesis of periodontal disease. We have previously shown that Treponema denticola ATCC type strains and strain GM-1 are resistant to killing by human beta-defensins (hbetaD)-1 and -2. We hypothesize that resistance to beta-defensins is a common feature of oral treponemes, which allows colonization and persistence in the oral cavity. In this study, we tested additional isolates of T. denticola, as well as six other species of treponemes, for resistance to hbetaD-1, -2 and -3. We also examined the four ATCC strains of T. denticola and strain GM-1 for resistance to hbetaD-3. METHODS: Resistance was determined by motility and Alamar Blue assays for metabolic activity. RESULTS: All T. denticola strains tested were resistant to hbetaD-1, -2 and -3, with the exception of strain Ambigua, which was sensitive to hbetaD-2 and -3. All other treponemes except Treponema vincentii were resistant to hbetaD-1. Treponema pectinovorum was sensitive to hbetaD-2, while T. vincentii, T. pectinovorum and Treponema maltophilum were sensitive to hbetaD-3. Escherichia coli was used as a control organism and was killed by all three defensins. CONCLUSION: Resistance to the constitutively expressed hbetaD-1 may assist treponemes in initial colonization of epithelial surfaces, while resistance to the inducible hbetaD-2 and -3 would allow some treponemes to survive in active periodontal lesions.
Asunto(s)
Treponema/fisiología , beta-Defensinas/farmacología , Farmacorresistencia Bacteriana , Humanos , Pruebas de Sensibilidad Microbiana , Boca/microbiología , Treponema/efectos de los fármacos , beta-Defensinas/fisiologíaRESUMEN
The presentation of syphilitic aortitis is often atypical and available serological tests are non-specific. The diagnostic gold standard remains direct identification of microorganisms in tissue. We present a case of syphilitic aortitis that presented as a mediastinal mass and report the use of polymerase chain reaction for Treponema pallidum to diagnose syphilitic aortic disease.
Asunto(s)
Aortitis/diagnóstico , Reacción en Cadena de la Polimerasa/métodos , Sífilis Cardiovascular/diagnóstico , Anciano , Errores Diagnósticos , Femenino , Humanos , Angiografía por Resonancia Magnética/métodosRESUMEN
By means of a differential screening technique, a 92-kDa antigen, designated Tp92, was identified from Treponema pallidum subspecies pallidum. This protein is similar in sequence to the protective surface antigens D15 from Haemophilus influenzae and Oma87 from Pasteurella multocida. Amino acid sequence analyses revealed a cleavable N-terminal signal sequence and predicted the outer membrane location for Tp92. In support of this, antiserum raised against recombinant Tp92 promotes opsonization and phagocytosis of T. pallidum by rabbit macrophages, and anti-Tp92 reactivity is absent from washed treponemal preparations presumed to be lacking outer membranes. The Tp92 amino acid sequence is 95.5%-100% conserved among 11 strains representing 4 pathogenic treponemes, and immunization with recombinant Tp92 partially protected rabbits from subsequent T. pallidum challenge. These results demonstrate that Tp92 is an invariant, immunoprotective antigen that may be present on the surface of T. pallidum and may represent a potential vaccine candidate for syphilis.
Asunto(s)
Antígenos de Superficie/química , Antígenos de Superficie/inmunología , Proteínas Bacterianas , Proteínas Opsoninas , Treponema pallidum/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Antibacterianos/biosíntesis , Vacunas Bacterianas/inmunología , Secuencia Conservada , Electroforesis en Gel de Poliacrilamida , Humanos , Datos de Secuencia Molecular , Fagocitosis , Reacción en Cadena de la Polimerasa , Conejos , Propiedades de Superficie , Sífilis/prevención & controlRESUMEN
Two new tprD alleles have been identified in Treponema pallidum: tprD2 is found in 7 of 12 T. pallidum subsp. pallidum isolates and 7 of 8 non-pallidum isolates, and tprD3 is found in one T. pallidum subsp. pertenue isolate. Antibodies against TprD2 are found in persons with syphilis, demonstrating that tprD2 is expressed during infection.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/genética , Genes Bacterianos , Treponema pallidum/genética , Alelos , Secuencia de Aminoácidos , Anticuerpos Antibacterianos/sangre , Proteínas de la Membrana Bacteriana Externa/inmunología , Duplicación de Gen , Humanos , Datos de Secuencia Molecular , Homología de Secuencia de Aminoácido , Sífilis/sangre , Treponema pallidum/inmunologíaRESUMEN
We have previously shown that the TprK antigen of T. pallidum, Nichols strain, is predominantly expressed in treponemes obtained 10 days after infection and that the hydrophilic domain of TprK is a target of opsonic antibodies and confers significant protection against homologous challenge. The T. pallidum genome sequence reported the presence of a single copy of the tprK gene in the Nichols strain. In the present study we demonstrate size heterogeneity in the central portions of the TprK hydrophilic domains of 14 treponemal isolates. Sequence analysis of the central domains and the complete open reading frames (ORFs) of the tprK genes confirms this heterogeneity. Further, multiple tprK sequences were found in the Nichols-defined tprK locus in three isolates (Sea 81-4, Bal 7, and Bal 73-1). In contrast, only a single tprK sequence could be identified in this locus in the Nichols strain. Alignment of the DNA and deduced amino acid sequences of the whole tprK ORFs shows the presence of seven discrete variable domains flanked by highly conserved regions. We hypothesize that these heterogeneous regions may be involved in antigenic heterogeneity and, in particular, evasion of the immune response. The presence of different tprK alleles in the tprK locus strongly suggests the existence of genetically different subpopulations within treponemal isolates.
Asunto(s)
Alelos , Proteínas Bacterianas , Genes Bacterianos , Porinas/genética , Treponema pallidum/genética , Secuencia de Aminoácidos , Animales , Mapeo Cromosómico , Heterogeneidad Genética , Datos de Secuencia Molecular , Porinas/química , Conejos , Treponema pallidum/inmunologíaRESUMEN
Treponema pallidum subsp. pallidum, the causative agent of venereal syphilis, was detected in a 200-year-old skeletal specimen from Easter Island. An initial diagnosis of treponemal infection was confirmed by extensive purification of immunoglobulin that reacted strongly with T. pallidum antigen. Extracted DNA exhibited a single-base polymorphism that distinguished T.p. subsp. pallidum from 4 other human and nonhuman treponemes. Extensive precautions against contamination of the subject matter with modern treponemal DNA were employed, including analysis of archaeological and modern specimens in 2 geographically separate laboratories. Molecular determination of historical disease states by using skeletal material can significantly enhance our understanding of the pathology and spread of infectious diseases.
Asunto(s)
Huesos/microbiología , Sífilis/historia , Treponema pallidum/aislamiento & purificación , Antígenos Bacterianos/inmunología , Secuencia de Bases , Huesos/inmunología , ADN Bacteriano/análisis , ADN Bacteriano/aislamiento & purificación , Ensayo de Inmunoadsorción Enzimática , Historia del Siglo XVIII , Humanos , Inmunoglobulina G/análisis , Inmunoglobulina G/inmunología , Polimorfismo de Longitud del Fragmento de Restricción , Polinesia , Análisis de Secuencia de ADN , Sífilis/diagnóstico , Sífilis/microbiología , Treponema pallidum/clasificación , Treponema pallidum/genética , Treponema pallidum/inmunologíaRESUMEN
In this study we describe the development of the T-cell response to a panel of Treponema pallidum antigens over the course of syphilis infection in the rabbit and determine whether these antigens induce the expression of Th1 cytokines. It was determined that the membrane proteins TpN17 and TpN47, as well as the endoflagellar sheath protein TpN37, induce strong proliferation responses through most of syphilis infection; Tromp1 induced only weak proliferative responses. An unexpected drop in proliferative response to these antigens at day 90 of infection, followed by a dramatic increase in response at day 180, suggests that there may be a secondary dissemination of T. pallidum which induces a recall response. Crude epitope mapping of TpN17 and TpN37 showed that multiple epitopes may be present on both antigens, which is likely a contributing factor in the immunodominance of these antigens. The T-cell response to the TpN37 molecule shows acquisition of newly recognized epitopes during the course of infection. Sonicated T. pallidum was found to induce the expression of interleukin-2 (IL-2) and gamma interferon and not IL-10 mRNA, showing that the general T-cell response to T. pallidum antigens in syphilis infection is biased towards the Th1 phenotype. Of the antigens tested, TpN37 appears to contribute the most to the Th1 cytokine response and therefore may play a key role in the clearance of T. pallidum from lesions.
Asunto(s)
Antígenos Bacterianos/inmunología , Proteínas Bacterianas , Proteínas Portadoras/inmunología , Lipoproteínas/inmunología , Porinas/inmunología , Sífilis/inmunología , Linfocitos T/inmunología , Treponema pallidum/inmunología , Animales , Antígenos Bacterianos/genética , Proteínas Portadoras/genética , Células Cultivadas , Modelos Animales de Enfermedad , Mapeo Epitopo , Expresión Génica , Interferón gamma/genética , Interleucina-10/genética , Interleucina-2/genética , Lipoproteínas/genética , Masculino , Porinas/genética , Conejos , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Bazo/citología , Bazo/inmunología , Linfocitos T/citologíaRESUMEN
Previous investigations have demonstrated that immunization with Treponema pallidum subsp. pallidum glycerophosphodiester phosphodiesterase significantly protects rabbits from subsequent treponeme challenge. In this report, we show that the glycerophosphodiester phosphodiesterase amino acid sequence is conserved among 12 strains from a total of five pathogenic treponemes. The invariant nature of this immunoprotective antigen makes it an attractive candidate for inclusion in a universal subunit vaccine against T. pallidum infection. In addition, these studies show a silent nucleotide substitution at position 579 of the gpd open reading frame which is consistently observed in the non-T. pallidum subsp. pallidum strains. This sequence alteration introduces a PleI restriction site in the nonsyphilis strains and thus allows genetic differentiation from T. pallidum subsp. pallidum strains.
Asunto(s)
Secuencia Conservada , Hidrolasas Diéster Fosfóricas/genética , Treponema pallidum/enzimología , Animales , Secuencia de Bases , ADN Bacteriano , Amplificación de Genes , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Polimorfismo de Longitud del Fragmento de Restricción , Conejos , Análisis de Secuencia de ADN , Treponema pallidum/genéticaRESUMEN
We previously reported that Macaca fascicularis immunized with formalin-killed Porphyromonas gingivalis were protected against the bone loss of periodontitis. To examine mechanisms of protection, we determined specific immunoglobulin G (IgG), IgM and IgA titers and opsonic capacities of sera from immunized and control animals. Serum IgG and IgA titers to P. gingivalis appeared early and persisted throughout the 36-week observation period. IgM titers were elevated until 6 to 12 weeks and then decreased through week 36. A significant association was observed between peak IgM titers prior to ligature placement and protection against bone loss (measured at week 30). In control monkeys, no significant IgG, IgA or IgM titers were seen. In sera from immunized animals, significant opsonic capacity was seen by 6-12 weeks and persisted throughout the study. In contrast, control sera showed only low opsonization capacity. Anti P. gingivalis antibody titers in purified IgG, IgA and IgM fractions were determined by enzyme-linked immunosorbent assay, and opsonic activity was demonstrated only in the IgG fraction.
Asunto(s)
Pérdida de Hueso Alveolar/inmunología , Anticuerpos Antibacterianos/sangre , Isotipos de Inmunoglobulinas/biosíntesis , Porphyromonas gingivalis/inmunología , Pérdida de Hueso Alveolar/prevención & control , Animales , Anticuerpos Antibacterianos/biosíntesis , Inmunización , Isotipos de Inmunoglobulinas/sangre , Macaca fascicularis , Proteínas Opsoninas/inmunología , Periodontitis/inmunología , Periodontitis/microbiología , Periodontitis/prevención & control , Estadísticas no ParamétricasRESUMEN
We have identified a family of genes that code for targets for opsonic antibody and protective immunity in T. pallidum subspecies pallidum using two different approaches, subtraction hybridization and differential immunologic screening of a T. pallidum genomic library. Both approaches led to the identification of a polymorphic multicopy gene family with predicted amino acid homology to the major sheath protein of Treponema denticola. One of the members of this gene family, tpr K, codes for a protein that is predicted to have a cleavable signal peptide and be located in the outer membrane of the bacterium. Reverse transcription polymerase chain reaction analysis of T. pallidum reveals that Tpr K is preferentially transcribed in the Nichols strain of T. pallidum. Antibodies directed to purified recombinant variable domain of Tpr K can opsonize T. pallidum, Nichols strain, for phagocytosis, supporting the hypothesis that this portion of the protein is exposed at the surface of the treponeme. Immunization of rabbits with the purified recombinant variable domain of Tpr K provides significant protection against infection with the Nichols strain of T. pallidum. This gene family is hypothesized to be central to pathogenesis and immunity during syphilis infection.
Asunto(s)
Anticuerpos Antiprotozoarios/inmunología , Proteínas Bacterianas , Proteínas Opsoninas/inmunología , Porinas/inmunología , Treponema pallidum/inmunología , Secuencia de Aminoácidos , Animales , Variación Antigénica/genética , Genes Protozoarios , Inmunización , Datos de Secuencia Molecular , Porinas/genética , Señales de Clasificación de Proteína/genética , Conejos , Proteínas Recombinantes/inmunología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Técnica de Sustracción , Treponema pallidum/genéticaRESUMEN
Infectious syphilis, caused by the spirochete bacterium Treponema pallidum subsp. pallidum, remains a public health concern worldwide. The immune-response evasion mechanisms employed by T. pallidum are poorly understood, and prior attempts to identify immunoprotective antigens for subsequent vaccine design have been unsuccessful. Previous investigations conducted in our laboratory identified the T. pallidum glycerophosphodiester phosphodiesterase as a potential immunoprotective antigen by using a differential immunologic expression library screen. In studies reported here, heterologous expression of the T. pallidum glycerophosphodiester phosphodiesterase in Escherichia coli yielded a full-length, enzymatically active protein. Characterization of the recombinant molecule showed it to be bifunctional, in that it exhibited specific binding to human immunoglobulin A (IgA), IgD, and IgG in addition to possessing enzymatic activity. IgG fractionation studies revealed specific binding of the recombinant enzyme to the Fc fragment of human IgG, a characteristic that may play a role in enabling the syphilis spirochete to evade the host immune response. In further investigations, immunization with the recombinant enzyme significantly protected rabbits from subsequent T. pallidum challenge, altering lesion development at the sites of challenge. In all cases, animals immunized with the recombinant molecule developed atypical pale, flat, slightly indurated, and nonulcerative reactions at the challenge sites that resolved before lesions appeared in the control animals. Although protection in the immunized rabbits was incomplete, as demonstrated by the presence of T. pallidum in the rabbit infectivity test, glycerophosphodiester phosphodiesterase nevertheless represents a significantly immunoprotective T. pallidum antigen and thus may be useful for inclusion in an antigen cocktail vaccine for syphilis.
Asunto(s)
Antígenos Bacterianos/uso terapéutico , Proteínas de la Membrana Bacteriana Externa/uso terapéutico , Hidrolasas Diéster Fosfóricas/uso terapéutico , Sífilis/prevención & control , Vacunación , Animales , Antígenos Bacterianos/genética , Antígenos Bacterianos/inmunología , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas de la Membrana Bacteriana Externa/inmunología , Escherichia coli/genética , Inmunoglobulinas/metabolismo , Proteínas Opsoninas , Hidrolasas Diéster Fosfóricas/genética , Hidrolasas Diéster Fosfóricas/inmunología , Conejos , Proteínas Recombinantes/uso terapéutico , Treponema pallidum/enzimología , Treponema pallidum/inmunologíaRESUMEN
OBJECTIVES: To establish a model of CNS invasion by Treponema pallidum and to use it to investigate the immune mechanisms responsible for clearance. METHODS: Four macaques were intrathecally inoculated with 0.6 to 2.1 x 10(8) T. pallidum and underwent clinical examinations and blood and CSF collections every 1 to 2 weeks for 12 to 13 weeks. The following were determined: serum Venereal Disease Research Laboratory (VDRL) and microhemagglutination-T. pallidum reactivities, CSF-VDRL, CSF white blood cell (WBC) count, and the presence of viable T. pallidum in CSF by the rabbit infectivity test (all animals), as well as the presence of T. pallidum in CSF by reverse transcriptase (RT)-PCR, WBC phenotype by fluorescence-activated cell sorter, WBC cytokine production by RT-PCR, and brain MRI at 10 weeks (two animals). RESULTS: All animals became systemically infected and developed CSF pleocytosis that resolved after 8 weeks. CSF T. pallidum was detected from 2 to 8 weeks. CSF T lymphocytes were predominantly CD4+. Interferon-gamma (IFN-gamma) mRNA was consistently detected in CSF WBCs, but interleukin (IL)-4 and IL-5 were not. All animals remained clinically well. MRIs were normal. CONCLUSIONS: In this model, T. pallidum is cleared from the CNS just as in most humans with early syphilis. Local production of IFN-gamma likely participates in this process. This model could be used to clarify the effect of retrovirus-induced immunodeficiency on clearance of T. pallidum from the CNS and on the local CNS immune response.
Asunto(s)
Neurosífilis , Treponema pallidum/inmunología , Animales , Anticuerpos Antibacterianos/líquido cefalorraquídeo , Líquido Cefalorraquídeo/citología , Líquido Cefalorraquídeo/inmunología , Líquido Cefalorraquídeo/microbiología , Modelos Animales de Enfermedad , Interferón gamma/análisis , Interleucina-10/análisis , Interleucina-4/análisis , Interleucina-5/análisis , Recuento de Leucocitos , Macaca mulatta , Macaca nemestrina , Masculino , Neurosífilis/líquido cefalorraquídeo , Neurosífilis/inmunología , Neurosífilis/microbiología , ARN Bacteriano/análisis , ARN Mensajero/análisis , Conejos , Treponema pallidum/genética , Treponema pallidum/aislamiento & purificaciónRESUMEN
The species Treponema pallidum includes three subspecies (pallidum, pertenue, and endemicum) that cause syphilis, yaws, and bejel, respectively. A closely related species, Treponema paraluiscuniculi, is the etiologic agent of venereal syphilis in rabbits but does not infect humans. Although these treponemes cause distinct diseases, no laboratory method for differentiation has been reported. Genetic signatures were defined in the 5' and 3' flanking regions of the 15-kDa lipoprotein gene (tpp15) that distinguish the human pathogens and T. paraluiscuniculi, as well as distinguishing T. pallidum subsp. pallidum from the causes of human nonvenereal treponematoses. A single Eco47III restriction site in the 5' flanking region differentiates T. pallidum subsp. pallidum from the other subspecies and species, and an XcmI site in the 3' flanking region differentiates T. paraluiscuniculi from the human pathogens. Polymerase chain reaction methods and restriction polymorphism were used to analyze 27 strains of pathogenic Treponema species.
Asunto(s)
ADN Bacteriano/análisis , Lipoproteínas/genética , Treponema/clasificación , Treponema/genética , Infecciones por Treponema/genética , Animales , Clonación Molecular , ADN Bacteriano/genética , Genes Bacterianos , Humanos , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Polimorfismo de Longitud del Fragmento de Restricción , Conejos , Análisis de Secuencia de ADN , Treponema/patogenicidad , Treponema pallidum/genética , Treponema pallidum/patogenicidadRESUMEN
To identify potential opsonic targets of Treponema pallidum subsp. pallidum, a treponemal genomic expression library was constructed and differentially screened with opsonic and non-opsonic T. pallidum antisera. This method identified an immunoreactive clone containing an open reading frame encoding a 356 residue protein. Nucleotide sequence analysis demonstrated the translated protein to be a homologue of glycerophosphodiester phosphodiesterase, a glycerol metabolizing enzyme previously identified in Haemophilus influenzae, Escherichia coli, Bacillus subtilis and Borrelia hermsii. Sequence alignment analyses revealed the T. pallidum and H. influenzae enzymes share a high degree of amino acid sequence similarity (72%), suggesting that in T. pallidum this molecule may be surface exposed and involved in IgD binding as is the case with its counterpart in H. influenzae.
Asunto(s)
Hidrolasas Diéster Fosfóricas/aislamiento & purificación , Treponema pallidum/enzimología , Secuencia de Aminoácidos , Secuencia de Bases , Datos de Secuencia Molecular , Hidrolasas Diéster Fosfóricas/químicaRESUMEN
Syphilis is diagnosed by serologic testing or by identification of the causative agent, Treponema pallidum. The bacterium has historically been detected in clinical specimens by dark-field microscopy, immunostaining with polyclonal or monoclonal antibodies, or the rabbit inoculation test (RIT). RIT is considered to be very sensitive and specific, although it is available only in research settings and is not clinically useful due to the length of time required to obtain a result. In recent years, several PCR methods have been developed for the detection of T. pallidum, but none of these has shown a clear advantage in sensitivity over RIT. We have developed a specific and highly sensitive reverse transcriptase PCR (RT-PCR) that targets a 366 bp region of the 16S rRNA of T. pallidum. This RT-PCR can detect a single organism by Southern analysis when whole organisms are diluted and 10(-2) to 10(-3) T. pallidum organisms when RNA equivalents are used to make cDNA. The test was demonstrated to detect 10(-2) T. pallidum RNA equivalents in cerebrospinal fluid. Twenty different strains of T. pallidum, isolated from cerebrospinal fluids, aqueous humor, blood, and chancres, were shown to be detectable by this test. This efficient and sensitive technique could be more useful than existing methods for detecting very low numbers of organisms in clinical samples.
Asunto(s)
Reacción en Cadena de la Polimerasa/métodos , ARN Bacteriano/análisis , ADN Polimerasa Dirigida por ARN , Sífilis/diagnóstico , Treponema pallidum/aislamiento & purificación , ADN Bacteriano/análisis , Genes Bacterianos/genética , Humanos , Datos de Secuencia Molecular , ARN Bacteriano/sangre , ARN Bacteriano/líquido cefalorraquídeo , ARN Ribosómico 16S/genética , Treponema pallidum/genéticaRESUMEN
The 15-kDa lipoprotein of Treponema pallidum is a major immunogen during natural syphilis infection in humans and experimental infection in other hosts. The humoral and cellular immune responses to this molecule appear late in infection as resistance to reinfection is developing. One therefore might hypothesize that this antigen is important for protective immunity. This possibility is explored by using both genetic and antigenic approaches. Limited or no cross-protection has been demonstrated between the T. pallidum subspecies and strains or between Treponema species. We therefore hypothesized that if the 15-kDa antigen was of major importance in protective immunity, it might be a likely site of antigenic diversity. To explore this possibility, the sequences of the open reading frames of the 15-kDa gene have been determined for Treponema pallidum subsp. pallidum (Nichols and Bal-3 strains), T. pallidum subsp. pertenue (Gauthier strain), T. pallidum subsp. endemicum (Bosnia strain), Treponema paraluiscuniculi (Cuniculi A, H, and K strains), and a little-characterized simian isolate of Treponema sp. (Fribourg-Blanc strain). No significant differences in DNA sequences of the genes for the coding region of the 15-kDa antigen were found among the different species and subspecies studied. In addition, all organisms showed expression of the 15-kDa antigen as determined by monoclonal antibody staining. The role of the 15-kDa antigen in protection against homologous infection with T. pallidum subsp. pallidum Nichols was examined in rabbits immunized with a purified recombinant 15-kDa fusion protein. No alteration in chancre development was observed in immunized, compared to unimmunized, rabbits, and the antisera induced by the immunization failed to enhance phagocytosis of T. pallidum subsp. pallidum by macrophages in vitro. These results do not support a major role for this antigen in protection against syphilis infection.
Asunto(s)
Antígenos Bacterianos/genética , Proteínas Bacterianas/genética , Lipoproteínas/genética , Treponema pallidum/genética , Animales , Antígenos Bacterianos/inmunología , Proteínas Bacterianas/inmunología , Secuencia de Bases , Secuencia Conservada , Evolución Molecular , Humanos , Lipoproteínas/inmunología , Datos de Secuencia Molecular , Conejos , Alineación de Secuencia , Treponema pallidum/inmunologíaRESUMEN
To determine the prevalence of cerebrospinal fluid abnormalities in Southeast Asian refugees with reactive serologic tests for syphilis, we evaluated 65 patients, 36 prospectively and 29 retrospectively, in a primary care clinic. Information was collected on history of treponemal infections, neurologic symptoms and signs, and total protein concentration, leukocyte count, and the VDRL test in the cerebrospinal fluid. Neurologic symptoms were reported by all patients for whom data were available. Abnormal neurologic signs were found or noted in medical records in 15 (42%) prospectively evaluated patients and 9 (64%) of 14 retrospectively evaluated patients for whom data were available. No patient had evidence of congenital or non-neurologic sequelae such as cutaneous or cardiovascular manifestations of syphilis. No patient had a positive cerebrospinal fluid VDRL test, 1 had more than 5 x 10(6) leukocytes per liter (5 leukocytes per mm3), and 6 (9%) had elevated total protein levels in the cerebrospinal fluid. Previous therapy for syphilis was not associated with lower serum VDRL reactions, neurologic symptoms and signs, or cerebrospinal fluid findings. In the absence of other indications, routine examination of the cerebrospinal fluid in seropositive Southeast Asian refugees who have nonspecific neurologic symptoms has a low yield, perhaps because of the high prevalence of yaws in this population, and may not be warranted.