Sheila A. Lukehart

SUMMARY The agents of human treponematoses include four closely related members of the genus Treponema: three subspecies of Treponema pallidum plus Treponema carateum. T. pallidum subsp. pallidum causes venereal syphilis, while T. pallidum subsp. pertenue, T. pallidum subsp. endemicum, and T. carateum are the agents of the endemic treponematoses yaws, bejel (or endemic syphilis), and pinta, respectively. All human treponematoses share remarkable similarities in pathogenesis and clinical manifestations, consistent with the high genetic and antigenic relatedness of their etiological agents. Distinctive features have been identified in terms of age of acquisition, most common mode of transmission, and capacity for invasion of the central nervous system and fetus, although the accuracy of these purported differences is debated among investigators and no biological basis for these differences has been identified to date. In 2012, the World Health Organization (WHO) officially set a goal for yaws eradication by 2020. This challenging but potentially feasible endeavor is favored by the adoption of oral azithromycin for mass treatment and the currently focused distribution of yaws and endemic treponematoses and has revived global interest in these fascinating diseases and their causative agents.

Background Although azithromycin promised to be a safe and effective single-dose oral treatment of early syphilis, azithromycin treatment failure has been documented and is associated with mutations in the 23S rDNA of corresponding Treponema pallidum strains. The prevalence of strains harboring these mutations varies throughout the United States and the world. We examined T. pallidum strains circulating in Seattle, Washington, from 2001 to 2010 to determine the prevalence of 2 mutations associated with macrolide resistance and to determine whether these mutations were associated with certain T. pallidum strain types. Methods Subjects were enrolled in a separate ongoing study of cerebrospinal fluid abnormalities in patients with syphilis. T. pallidum DNA purified from blood and T. pallidum strains isolated from blood or cerebrospinal fluid were analyzed for two 23S rDNA mutations and for the molecular targets used in an enhanced molecular stain typing system. Results Nine molecular strain types of T. pallidum were identified in Seattle from 2001 to 2010. Both macrolide resistance mutations were identified in Seattle strains, and the prevalence of resistant T. pallidum exceeded 80% in 2005 and increased through 2010. Resistance mutations were associated with discrete molecular strain types of T. pallidum. Conclusions Macrolide-resistant T. pallidum strains are highly prevalent in Seattle, and each mutation is associated with discrete strain types. Macrolides should not be considered for treatment of syphilis in regions where prevalence of the mutations is high. Combining the resistance mutations with molecular strain typing permits a finer analysis of the epidemiology of syphilis in a community.

Syphilis is a fascinating and perplexing infection, with protean clinical manifestations and both diagnostic and management ambiguities. Treponema pallidum subsp. pallidum, the agent of syphilis, is challenging to study in part because it cannot be cultured or genetically manipulated. Here, we review recent progress in the application of modern molecular techniques to understanding the biological basis of this multistage disease and to the development of new tools for diagnosis, for predicting efficacy of treatment with alternative antibiotics, and for studying the transmission of infection through population networks.

Pathogens that cause chronic infections often employ antigenic variation to evade the immune response and persist in the host. In Treponema pallidum (T. pallidum), the causative agent of syphilis, the TprK Ag undergoes variation of seven V regions (V1–V7) by nonreciprocal recombination of silent donor cassettes with the tprK expression site. These V regions are the targets of the host humoral immune response during experimental infection. The present study addresses the causal role of the acquired immune response in the selection of TprK variants in two ways: 1) by investigating TprK variants arising in immunocompetent versus immunosuppressed hosts; and 2) by investigating the effect of prior specific immunization on selection of TprK variants during infection. V region diversity, particularly in V6, accumulates more rapidly in immunocompetent rabbits than in pharmacologically immunosuppressed rabbits (treated with weekly injections of methylprednisolone acetate). In a complementary experiment, rabbits preimmunized with V6 region synthetic peptides had more rapid accumulation of V6 variant treponemes than control rabbits. These studies demonstrate that the host immune response selects against specific TprK epitopes expressed on T. pallidum, resulting in immune selection of new TprK variants during infection, confirming a role for antigenic variation in syphilis.

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 β‐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‐κB 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.

The clearance ofTreponema pallidumsubsp.pallidumfrom early syphilis lesions involves infiltration of a large number of mononuclear cells and is characteristic of a cell-mediated immune response. In the present study, we sought to determine the relative abundance of different T-lymphocyte populations and Th1/Th2-associated cytokines present in testicular lesions following experimental infection with the Chicago strain ofT. pallidum. Using flow cytometry, we examined the proportion of CD4⁺and CD8⁺T cells present throughout the progression and resolution of primary syphilis in the rabbit model. We related these findings to the results of real-time reverse transcription-PCR quantification of treponemal and cytokine mRNA levels. Treponemal mRNA levels reached peak values on day 18 postinfection, coincident with an initial peak in the level of T cells, which were primarily CD4⁺T cells. T-cell levels increased again during resolution of orchitis, and there was an increased proportion of CD8⁺T cells. The maximum gamma interferon (IFN-γ) and interleukin-10 (IL-10) mRNA levels were observed on days 11 and 18, respectively, while only negligible amounts of IL-4 and IL-2 were detected throughout the infection. In addition to showing the temporal relationship between treponemal burden and T-cell responses during lesion progression, our results also demonstrate that the composition of the T-cell population changes during lesion resolution. The presence of the mRNA for IFN-γ, but not IL-4, is consistent with cytokine expression in human syphilis and provides further support for the hypothesis that there is a Th1 predominance during the early immune response toT. pallidum.

Treponema denticola, a periodontal pathogen, is relatively resistant to human beta-defensins, which are small cationic antimicrobial peptides produced by a number of cells, including the gingival epithelium. Using two independent methods, we previously demonstrated thatT. denticolaproteases are not responsible for decreased vulnerability to defensins. In this study, we confirmed that the major outer membrane protease, dentilisin, is not responsible forT. denticolainsensitivity to defensins and examined several other possible mechanisms, including reduced binding to the bacterial surface and efflux pump activity. It has been suggested that some bacteria mask their surfaces with serum proteins.T. denticolagrown in a serum-free medium did not exhibit increased susceptibility to human beta-defensin 2 and 3 (hβD-2 and hβD-3, respectively), suggesting that cloaking of the outer surface with host proteins is not involved in defensin resistance. Nonetheless, we demonstrated thatT. denticolabinds significantly less hβD-2 and -3 than susceptible organisms bind, suggesting that the unusual outer membrane composition ofT. denticolamay discourage cationic peptide binding. Efflux pumps have been shown to mediate resistance to antibiotics and cationic peptides in other bacteria, and their role inT. denticola's relative resistance to β-defensins was investigated. Three inhibitors of bacterial ATP-binding cassette (ABC) efflux pumps had no effect onT. denticola's susceptibility to hβD-2 or -3. In contrast, a proton motive force inhibitor, carbonyl cyanide 3-chlorophenylhydrazone, increased the susceptibility ofT. denticolato killing by hβD-3, demonstrating a potential role for efflux pumps (other than ABC pumps) in resistance to this peptide. Our data suggest that the combination of decreased defensin binding and efflux of any peptide which enters the cytoplasm may explainT. denticola's relative resistance to human beta-defensins.