The Treponema pallidum Outer Membrane.

The outer membrane (OM) of Treponema pallidum, the uncultivatable agent of venereal syphilis, has long been the subject of misconceptions and controversy. Decades ago, researchers postulated that T. pallidum's poor surface antigenicity is the basis for its ability to cause persistent infection, but they mistakenly attributed this enigmatic property to the presence of a protective outer coat of serum proteins and mucopolysaccharides. Subsequent studies revealed that the OM is the barrier to antibody binding, that it contains a paucity of integral membrane proteins, and that the preponderance of the spirochete's immunogenic lipoproteins is periplasmic. Since the advent of recombinant DNA technology, the fragility of the OM, its low protein content, and the lack of sequence relatedness between T. pallidum and Gram-negative outer membrane proteins (OMPs) have complicated efforts to characterize molecules residing at the host-pathogen interface. We have overcome these hurdles using the genomic sequence in concert with computational tools to identify proteins predicted to form ß-barrels, the hallmark conformation of OMPs in double-membrane organisms and evolutionarily related eukaryotic organelles. We also have employed diverse methodologies to confirm that some candidate OMPs do, in fact, form amphiphilic ß-barrels and are surface-exposed in T. pallidum. These studies have led to a structural homology model for BamA and established the bipartite topology of the T. pallidum repeat (Tpr) family of proteins. Recent bioinformatics has identified several structural orthologs for well-characterized Gram-negative OMPs, suggesting that the T. pallidum OMP repertoire is more Gram-negative-like than previously supposed. Lipoprotein adhesins and proteases on the spirochete surface also may contribute to disease pathogenesis and protective immunity.

Treponema pallidum in Gel Microdroplets: A Method for Topological Analysis of BamA (TP0326) and Localization of Rare Outer Membrane Proteins.

The noncultivable spirochete Treponema pallidum subspecies pallidum (T. pallidum) is the etiological agent of venereal syphilis. In contrast to the outer membranes (OMs) of gram-negative bacteria, the OM of T. pallidum lacks lipopolysaccharide, contains a paucity of integral membrane proteins, and is extremely labile. The lability of the T. pallidum OM greatly hinders efforts to localize the bacterium's rare outer membrane proteins (OMPs). To circumvent this problem, we developed the gel microdroplet method in which treponemes are encapsulated in porous agarose beads and then probed with specific antibodies in the absence or presence of low concentrations of the non-ionic detergent Triton X-100. To demonstrate the general utility of this method for surface localization of any T. pallidum antigen, herein we describe a protocol for immunolabeling of encapsulated treponemes using antibodies directed against the ß-barrel and POTRA domains of TP0326, the spirochete's BamA ortholog.

Viscous dynamics of Lyme disease and syphilis spirochetes reveal flagellar torque and drag.

The spirochetes that cause Lyme disease (Borrelia burgdorferi) and syphilis (Treponema pallidum) swim through viscous fluids, such as blood and interstitial fluid, by undulating their bodies as traveling, planar waves. These undulations are driven by rotation of the flagella within the periplasmic space, the narrow (∼20-40 nm in width) compartment between the inner and outer membranes. We show here that the swimming speeds of B. burgdorferi and T. pallidum decrease with increases in viscosity of the external aqueous milieu, even though the flagella are entirely intracellular. We then use mathematical modeling to show that the measured changes in speed are consistent with the exertion of constant torque by the spirochetal flagellar motors. Comparison of simulations, experiments, and a simple model for power dissipation allows us to estimate the torque and resistive drag that act on the flagella of these major spirochetal pathogens.

Aportación de las pruebas serológicas automatizadas en el diagnóstico de sífilis: comparación de 2 inmunoensayos quimioluminiscentes con el Treponema pallidum hemagglutination assay / Contribution of automated serological tests in the diagnosis of syphilis: Comparison of 2 chemiluminescence immunoassays with Treponema pallidum hemagglutination assay

La aparición de pruebas treponémicas automatizadas ha conllevado un cambio en el algoritmo diagnóstico de la sífilis. Tras comparar el algoritmo automatizado con el tradicional, evaluamos 2 inmunoensayos quimioluminiscentes (CLIA). Sobre 94 sueros se realizó RPR, TPHA y la técnica Architect Syphil TP (STP). En una segunda fase en 100 muestras se comparó el STP frente a Immulite Syphilis screen (ISS). En la primera fase hubo un falso positivo del STP respecto a 8 del RPR. En la segunda fase hubo una diferencia de especificidad estadísticamente significativa a favor del ISS. El uso de CLIA disminuye los errores analíticos y el tiempo de personal técnico frente a las pruebas no treponémicas. Ambos CLIA son útiles como prueba de cribado (AU)

The introduction of automated treponemal chemiluminescence assay (CLIA) has led to a change in the diagnostic algorithm of syphilis. The objective of the work was to compare the traditional algorithm with the automated one and evaluating two CLIA assays. A total of 94 sera were tested for rapid plasma reagin (RPR), Treponema pallidum hemagglutination (TPHA) and Architect Syphil TP (Syphilis Treponema pallidum, STP). In a second phase, 100 samples were compared, STP against Immulite Syphilis screen (ISS). In the first phase, 8 false positive RPR were found with just 1 STP. In the second phase, a statistically significant difference was found in the specificity in favour of the ISS. The use of CLIA reduces analytical errors and staff time spent on the nontreponemal test. Both CLIA are useful as screening tests (AU)

Syphilitic and lymphogranuloma venereum (LGV) proctocolitis: clues to a frequently missed diagnosis.

A rising incidence of syphilis and lymphogranuloma venereum (LGV) underscores the importance of recognizing these sexually transmitted infections (STI) in routine anocolonic biopsies. To increase awareness of their morphologic manifestations, we undertook a clinicopathologic study of our experience: syphilis (7 patients, 7 specimens), LGV (2 patients, 4 specimens), and syphilis/LGV (1 patient, 3 specimens). The diagnoses of all study specimens were confirmed with pertinent clinical studies. All study patients were human immunodeficiency virus positive, and all 9 with available history were men who have sex with men. The majority presented with bleeding (9), pain (6), and tenesmus (4). Ulcerations were the most common endoscopic abnormality (7), whereas mass lesions were confined to the syphilis group (4). None of the initial impressions included LGV, and syphilis was prospectively suggested only by pathologists (6 of 8) without the knowledge of clinical information and on the basis of morphology. Alternative impressions included condyloma acuminatum (3), inflammatory bowel disease (3), and malignancy (2), among others. All study specimens shared the following histologic core features: an intense lymphohistiocytic infiltrate with prominent plasma cells and lymphoid aggregates, only mild to moderate acute inflammation, minimal basal plasmacytosis and crypt distortion, and only rare granulomas and Paneth cell metaplasia. The spirochetes were focally demonstrated on a Treponema pallidum immunohistochemical stain (1) but not on silver stains (3). All patients with available follow-up data showed resolution of symptoms and imaging abnormalities after STI therapy (6). In summary, we report a unique pattern of STI proctocolitis consistently identified in patients with serologically confirmed syphilis and/or LGV infection; pertinent STI therapy leads to resolution of clinical abnormalities. This histologic pattern is important to recognize for timely treatment, for prevention of onward STI transmission, and to avoid the diagnostic pitfalls of inflammatory bowel disease or malignancy.

TprC/D (Tp0117/131), a trimeric, pore-forming rare outer membrane protein of Treponema pallidum, has a bipartite domain structure.

Identification of Treponema pallidum rare outer membrane proteins (OMPs) has been a longstanding objective of syphilis researchers. We recently developed a consensus computational framework that employs a battery of cellular localization and topological prediction tools to generate ranked clusters of candidate rare OMPs (D. L. Cox et al., Infect. Immun. 78:5178-5194, 2010). TP0117/TP0131 (TprC/D), a member of the T. pallidum repeat (Tpr) family, was a highly ranked candidate. Circular dichroism, heat modifiability by SDS-PAGE, Triton X-114 phase partitioning, and liposome incorporation confirmed that full-length, recombinant TprC (TprC(Fl)) forms a ß-barrel capable of integrating into lipid bilayers. Moreover, TprC(Fl) increased efflux of terbium-dipicolinic acid complex from large unilamellar vesicles and migrated as a trimer by blue-native PAGE. We found that in T. pallidum, TprC is heat modifiable, trimeric, expressed in low abundance, and, based on proteinase K accessibility and opsonophagocytosis assays, surface exposed. From these collective data, we conclude that TprC is a bona fide rare OMP as well as a functional ortholog of Escherichia coli OmpF. We also discovered that TprC has a bipartite architecture consisting of a soluble N-terminal portion (TprC(N)), presumably periplasmic and bound directly or indirectly to peptidoglycan, and a C-terminal ß-barrel (TprC(C)). Syphilitic rabbits generate antibodies exclusively against TprC(C), while secondary syphilis patients fail to mount a detectable antibody response against either domain. The syphilis spirochete appears to have resolved a fundamental dilemma arising from its extracellular lifestyle, namely, how to enhance OM permeability without increasing its vulnerability to the antibody-mediated defenses of its natural human host.

Syphilis presenting as isolated cervical lymphadenopathy: two related cases.

Two young adult brothers, with no apparent risk for sexually transmitted infections (STI), presented with unilateral cervical lymphadenitis. Syphilis was diagnosed by fine-needle aspiration cytology in one case, and subsequent serology and revision of a resected lymph node in the second case. Clinicians should have a high index of suspicion and a low diagnostic threshold in patients with unexplained lymphadenopathy, even in the absence of a history of primary syphilis, or obvious risk for STI.

Antigenic variation of TprK V regions abrogates specific antibody binding in syphilis.

The tprK gene in the syphilis spirochete, Treponema pallidum subsp. pallidum, undergoes antigenic variation in seven variable (V) regions. tprK is highly variable within T. pallidum strains, and a method has been developed to derive clones of T. pallidum that express a single, unique tprK sequence. Rabbits were infected with three different T. pallidum clones or the parent strain from which the clones were derived, and their sera were examined by immunoassay for antibody reactivity against synthetic peptides representing the TprK V regions from each clone. The parent strain expresses many different V region sequences, and infection with this strain induced antibody responses against a wide variety of V regions. In rabbits infected with the Chicago C clone, antibodies developed against all of the V regions except V1, while antibodies developed against only V5, V6, and V7 in Chicago A-infected rabbits. During Chicago B infection, antibodies developed against all of the V regions except V1 and V3. Antibodies were highly specific for the V regions of the infecting clone, and cross-reactivity was rare. The demonstration that the V regions elicit a variant-specific antibody response supports the hypothesis that TprK variants may help organisms to avoid the developing immune response in infected individuals, contributing to the ability of T. pallidum to establish chronic infection.

Studies on the lysozyme independence of immune immobilisation of Treponema pallidum and the frequency of lysozyme autoantibodies in syphilitic sera.

The role of lysozyme in the immune immobilisation of Treponema pallidum is not yet fully understood. The T. pallidum immobilisation assay was used to demonstrate that the immobilisation and lysis of T. pallidum in vitro by antibodies (serum, IgG fraction or IgM fraction) and complement proceed in a lysozyme-independent mode. In the presence of lysozyme the rate of immobilisation increased. In contrast with its effect on Escherichia coli, the effect of lysozyme on T. pallidum was governed exclusively by its enzymic activity rather than by the cationic protein nature of the molecule. Lysozyme, released from stimulated phagocytes, induced formation of lysozyme antibodies in 59.6% of syphilis patients as determined by lysozyme antibody ELISA. The highest frequency was found in patients with untreated secondary syphilis, whereas untreated primary syphilis was only rarely accompanied by the presence of lysozyme antibodies. Cross-reactivities between lysozyme and treponemal antigens were excluded by immunoblotting. The autoantibodies did not influence the lysozyme activity. It was concluded that the formation of lysozyme antibodies is only an epiphenomenon in the host defence against treponemal infection.

[The functional properties of the cellular structures of cultured Treponema pallidum]. / Funktsional'nye svoistva kletochnykh struktur kul'tural'nykh Treponema pallidum.

New data on the functional properties of the cell structures of cultured T. pallidum were obtained, which permitted the construction of new more effective preparations for the diagnostics of syphilis. T. pallidum cell walls were used as antigen in the complement fixation test (CFT) and the development of a new enzyme immunoassay system for the determination of IgM and IgG antibodies to T. pallidum with the visual evaluation of results, while T. pallidum cytoplasm was used as sorbent in the immunofluorescence test (IFT-abs) for the serodiagnosis of syphilis. The immunization of rabbits with T. pallidum cell walls increased the titers of positive control sera used in CFT for diagnosing syphilis.

Comparison of molecular and microscopic techniques for detection of Treponema pallidum in genital ulcers.

We compared the ability of direct immunofluorescent staining (DFA) and the PCR to detect Treponema pallidum in specimens from patients with genital ulcer disease. Touch preparations from 156 patients with genital lesions were fixed in acetone and stained with a fluorescein-labeled monoclonal antibody specific for the 37-kDa antigen of T. pallidum. After microscopic examination, the smear was removed from the slide with a swab. DNA was extracted with phenol-chloroform and precipitated with isopropanol. Ten microliters of the extracted DNA was amplified by PCR using primers for the gene encoding the 47-kDa protein of T. pallidum and hybridized to an internal probe. Twenty-two of 156 specimens were positive for T. pallidum by DFA and PCR, while 127 were negative by both methods, yielding a concordance of 95.5% (kappa = 0.84). Four specimens were positive by PCR and negative by DFA, while three specimens were negative by PCR and positive by DFA. The DFA-negative, PCR-positive specimens may have resulted from the presence of large numbers of leukocytes on the slides, obscuring visualization of treponemes. The DFA-positive, PCR-negative results were not due to inhibition of the PCR since purified T. pallidum DNA was amplified when added to aliquots of these specimens. Negative results in these specimens were most likely due to inefficient recovery of their DNA. These data suggest that DFA and PCR are equivalent methods for detection of T. pallidum on touch preparations of genital lesions. Further refinements of the PCR assay are necessary for it to significantly improve the detection of T. pallidum in genital lesions.

Enumeration of Treponema pallidum cells cultivated in vitro by an enzyme-linked immunosorbent assay.

An enzyme-linked immunosorbent assay was developed to enumerate Treponema pallidum cells. The assay could detect from 2 X 10(7) to 4 X 10(8) treponemes per ml. Reactive rabbit serum and goat anti-rabbit immunoglobulin G (peroxidase conjugate) were used in the assay. Optimum results were obtained when 2,2'-azino-di(ethylbenzthiazolinesulfonic acid) was used as the dye for the enzyme reaction and the reactions were allowed to run for 45 min. Interestingly, assays in which in vivo-cultivated T. pallidum was used produced lower absorbance values than those in which T. pallidum was cultivated in vitro.

Cultivation of virulent Treponema pallidum in tissue culture.

In a series of seven experiments, the virulent Nichols strain of Treponema pallidum was shown to attach and replicate on the surface of tissue culture cells of cottontail rabbit epithelium (Sf1Ep) growing in conventional monolayer cultures under an atmosphere of 1.5% oxygen. Five days after inoculation of 10(6)T. pallidum, the number of treponemes had increased to between 8 x 10(6) and 2.59 x 10(7). The viability of harvested organisms ranged from 86 to 97%. The number of T. pallidum continued to increase, generally reaching a plateau between days 9 and 12 of incubation, with increases ranging up to 100-fold and averaging 49-fold. There appeared to be a ceiling of multiplication of about 2 x 10(8) irrespective of the inoculum, which ranged from 10(6) to 10(8)T. pallidum. Concurrent deoxyribonucleic acid assays were performed on the cultures containing T. pallidum to obtain further evidence of replication. Significant increases in treponemal deoxyribonucleic acid were observed when the inocula ranged from 10(6) to 10(7), with the greatest increases, as might be expected, being in the former group. There was also excellent correlation in the amount of deoxyribonucleic acid per treponeme; the averages for the 10(6), 2.5 x 10(6), and 10(7) groups were 3.46 x 10(-14), 3.28 x 10(-14), and 2.79 x 10(-14) g per treponeme, respectively (3.14 +/- 0.72 x 10(-14) g per treponeme). In each experiment, organisms were harvested from the group inoculated with 10(6)T. pallidum after 7 days of incubation to test for virulence. In all instances, the organisms were virulent; erythematous, indurated, treponeme-containing lesions were produced from an average of six to seven organisms. Scanning electron microscopy revealed that during the course of replication many microcolonies of treponemes formed on the surface of the cells.

Helical conformation of Treponema pallidum (Nichols strain), Treponema paraluis-cuniculi, Treponema denticola, Borrelia turicatae, and unidentified oral spirochetes.

Borrelia turicatae (mouse virulent) and Treponema denticola, a small oral treponeme, formed right-handed helices as determined by scanning electron microscopy. Treponema pallidum (Nichols strain), Treponema paraluis-cuniculi, and two unidentified oral spirochetes displayed left-handed helices.

[Syphilis. Current physiobiological data. I. The bacteriological problem]. / La syphilis. Actualités physio-biologiques. I. Le problème bactériologique.

For lack of being able to grow Treponema pallidum, the only method which allows us to study the biology of this germ and the physiopathology of this infection lies in researches in experimental syphilis. After pointing out the different aspects of Treponema pallidum, either with light microscopy or electron microscopy, the authors review the different kinds of reproduction suggested by syphiligraphs, the recent trials to cultivate the treponema, and the processes of elimination. Then, they examine the biological properties and the antigenic structure of T.p. as it has been established by comparison with cultivable spirochetes. To end with, the authors show that both the TPI test and the FTA test are two very specific reactions; these tests mean nothing but the fact that the patient has been in contact with the antigens of Treponema pallidum and the quantitative tests cannot be considered as expressing the infectious potential capacity.

Examination of various cell culture techniques for co-incubation of virulent Treponema pallidum (Nichols I strain) under anaerobic conditions.

Treponema pallidum (Nichols virulent) was incubated with and without cells in cell culture medium reduced to -275 mV Ecal, pH 7.3, under deoxygenated conditions. Five to ten percent of the treponemes attached to cells and remained motile for at least 120 h in cell-treponeme systems of co-incubation. Virulent treponemes could be detected after 120 to 144 h in the supernatant fluids of cell-treponeme co-incubation cultures and in cell-free tubes containing medium harvested from aerobically cultivated mammalian cells. Medium supplemented with ox serum ultrafiltrate, pyruvate, and sodium thioglycolate and gas mixtures containing H2 and CO2 enhanced treponemal survival. Increases in treponemal numbers were observed using dark-field microscopy but were not substantiated using the rabbit lesion test. Continuous passage of the treponeme was not achieved in vitro.