More than a century after the discovery of Treponema pallidum (Tp), syphilis remains a major threat to global public health. Our limited understanding of this sexually transmitted disease reflects the many peculiarities of its etiologic agent, most notably an inability to replicate in vitro, a labile outer membrane (OM), and an extremely narrow host range. We showed during prior funding intervals that the molecular architecture of Tp differs radically from that of Gram-negative bacteria. The spirochete's highly unusual OM is the ultrastructural basis for its designation as the "stealth pathogen" and the prime determinant of its capacity to evade innate and adaptive immune responses. The experimental data derived from our in silico mining of the Tp genome during the current funding interval have yielded compelling evidence that the particles visualized in the Tp OM by freeze-fracture EM more than 20 years ago are integral OMPs and canonical ?-barrels. Three developments now enable us to carry this work forward to an integrated structural, functional, and immunological investigation of Tp rare OMPs. One is the characterization of TP0326/BamA, an ortholog for the central component of the molecular machine that chaperones precursor OMPs from the periplasm into the OM. Because of its potential to illuminate both the biogenesis and composition of the Tp OM, we liken TP0326 to the treponemal equivalent of the Rosetta Stone. The second is the demonstration that TprC/D is a trimeric, channel-forming protein and functional E. coli OmpF ortholog. We now hypothesize that the molecule's bipartite domain structure represents a potential topological-functional paradigm for other members of the Tpr family. The third is the generation of a robust list of candidate OMPs that, when more fully investigated, will yield a comprehensive picture of the surface weaponry Tp deploy to establish and maintain persistent infection. Our long-term objective is to continue to refine our model for the molecular architecture of the Tp cell envelope as a conceptual framework for elucidating the complex and shifting balance between pathogen and host that characterizes human syphilis. We have formulated Specific Aims to maintain our momentum towards this overarching goal: (i) further characterization of TP0326/BamA;(ii) further characterization of TprC and its closely related Tpr family members, TprI and TprF;and (iii) using our roster of candidate OMPs to expand our knowledge of Tp's OMP repertoire and the contributions of novel OMPs to host-pathogen interactions during syphilitic infection.
Syphilis is a chronic sexually transmitted disease caused by the noncultivatable spirochete Treponema pallidum (Tp). In a number of previously published studies, we have demonstrated that the outer membrane (OM) of Tp differs markedly from those of Gram-negative bacteria, such as Escherichia coli, most notably with respect to its paucity of integral membrane proteins. The experiments in this proposal will identify the repertoire of surface-exposed OM proteins Tp uses to establish and maintain persistent infection in its obligate human host.
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|Luthra, Amit; Anand, Arvind; Radolf, Justin D (2015) Treponema pallidum in Gel Microdroplets: A Method for Topological Analysis of BamA (TP0326) and Localization of Rare Outer Membrane Proteins. Methods Mol Biol 1329:67-75|
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|Kenedy, Melisha R; Luthra, Amit; Anand, Arvind et al. (2014) Structural modeling and physicochemical characterization provide evidence that P66 forms a *-barrel in the Borrelia burgdorferi outer membrane. J Bacteriol 196:859-72|
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