Treponema pallidum in gel microdroplets: a novel strategy for investigation of treponemal molecular architecture

Abstract

Controversy exists regarding the constituents and antigenic properties of the Treponema pallidum outer membrane; a major point of contention concerns the cellular location(s) of the spirochaete's lipoprotein immunogens. To address these issues and circumvent problems associated with prior efforts to localize treponemal surface antigens, we developed a novel strategy for investigating T. pallidum molecular architecture. Virulent treponemes were encapsulated in porous agarose beads (gel microdroplets) and then probed in the presence or absence of Triton X-100. Intact, encapsulated treponemes were not labelled by monospecific antisera directed against four major T. pallidum lipoproteins or a candidate T. pallidum outer membrane protein (TpN50) with C-terminal sequence homology to Escherichia coli OmpA or by human or rabbit syphilitic serum. Each of these immunologic reagents, however, labelled encapsulated treponemes co-incubated with detergent. In contrast, antibodies generated against isolated T. pallidum outer membranes labelled intact organisms and the pattern of fluorescence was consistent with the distribution of rare outer membrane proteins visualized by freeze-fracture electron microscopy. In addition to providing strong evidence that the protein portions of treponemal lipoproteins are located within the periplasmic space, these studies have extended our understanding of the topographical relationships among T. pallidum cell envelope constituents. They also demonstrate the feasibility of generating antibodies against rare outer membrane proteins and detecting them on the surfaces of virulent treponemes.

Crystal Structure of the Tp34 (TP0971) Lipoprotein of Treponema pallidum

“…This transient disruption of outer membrane integrity, however, is not thought to be sufficient to allow large proteins, such as antibodies, to traverse the outer membrane. On the other hand, such a mechanism could be more forgiving within the human host, where extracellular matrix or other host proteins within blood or interstitium might contribute to outer membrane integrity, much the way that soft agarose can stabilize the T. pallidum outer membrane in gel microdroplet assays (42). If so, then other larger nutrients, including lactoferrin, may be able to enter the periplasm of the organism, whereby binding to cognate receptors could occur.…”

“…Encapsulation of spirochetes in agarose gel microdroplets protects the fragile T. pallidum outer membrane from physical disruption (during reactivity with antibodies) by providing a supporting matrix but one that is sufficiently porous to allow reagent (e.g. antibody probes, secondary antibody conjugates) access and removal (42,61). The location of proteins beneath the surface is inferred by observing immunofluorescence of the bacteria upon treatment of encapsulated T. pallidum with low levels of Triton X-100 (0.15%) (which partially disrupts outer membrane integrity) in the presence of the primary antibody probes (42,61).…”

“…A previous study by Cox et al (42) using polyclonal antibodies provided initial evidence that Tp34 was not located on the surface of the T. pallidum outer membrane. In that study (42), an agarose gel microdroplet method, the current reference method for assessing mem- brane surface topology in the pathogenic spirochetes T. pallidum and Borrelia burgdorferi (61) was utilized. Encapsulation of spirochetes in agarose gel microdroplets protects the fragile T. pallidum outer membrane from physical disruption (during reactivity with antibodies) by providing a supporting matrix but one that is sufficiently porous to allow reagent (e.g.…”

“…antibody probes, secondary antibody conjugates) access and removal (42,61). The location of proteins beneath the surface is inferred by observing immunofluorescence of the bacteria upon treatment of encapsulated T. pallidum with low levels of Triton X-100 (0.15%) (which partially disrupts outer membrane integrity) in the presence of the primary antibody probes (42,61). In our study the anti-Tp34 monoclonal antibody (4A4) was used to further investigate Tp34 membrane topology using the gel microdroplet technique.…”

“…Spirochetes were harvested in T. pallidum medium (41) Indirect Immunofluorescence Detection of Antigens in T. pallidum-The agarose gel microdroplet method (42) was utilized to assess whether lipoproteins were exposed on the surface of T. pallidum. T. pallidum was encapsulated within agarose gel microdroplets as previously described (42). The encapsulation method stabilizes the fragile outer membrane of T. pallidum during antibody exposure and washing treatments.…”