Abstract
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 β-barrel and POTRA domains of TP0326, the spirochete’s BamA ortholog.
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Acknowledgements
This work was partially supported by NIH grant AI-26756 (J.D.R.) and the Department of Research, Connecticut Children’s Medical Center.
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Luthra, A., Anand, A., Radolf, J.D. (2015). Treponema pallidum in Gel Microdroplets: A Method for Topological Analysis of BamA (TP0326) and Localization of Rare Outer Membrane Proteins. In: Buchanan, S., Noinaj, N. (eds) The BAM Complex. Methods in Molecular Biology, vol 1329. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2871-2_6
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DOI: https://doi.org/10.1007/978-1-4939-2871-2_6
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-2870-5
Online ISBN: 978-1-4939-2871-2
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