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.

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.

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.

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.

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.

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.

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.

Morphology of Treponema pallidum.

In recent years many investigations have been carried out on the morphology of Treponema pallidum by means of the electron microscope, and the use of ultra-thin sections has shown up a number of structural details. However, there is still need for much more evidence before the internal structure of treponemes can be elucidated fully and the functions of the structures interpreted. To provide such evidence, the authors have examined under the electron microscope negative-stained treponemes and ultra-thin sections, using both cultivated strains and treponemes obtained direct from syphilids in people suffering from fresh secondary syphilis. It has been shown that treponemes have a complex structure. T. pallidum has a two-layered outer wall, a cytoplasmic membrane proper, cytoplasm and a bunch of fibrils following a different path in different places on the treponeme. The sites of insertion of the fibrils (the basal granules) were investigated; structures similar to mesosomes and nucleoids were found. Cysts and granular forms are described.

Outer envelope of virulent Treponema pallidum.

Ultrathin sections of virulent Treponema pallidum (Nichols strain) were examined with the electron microscope, and the presence of an outer cell envelope was documented.

Shape of Treponema pallidum.

Treponema pallidum was found to be not helical, but a flat wave twisted into one to five different planes per cell.