title: Treponema pallidum in Gel Microdroplets: A Method for Topological Analysis of BamA (TP0326) and Localization of Rare Outer Membrane Proteins.
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.
authors.affiliation: Department of Medicine, University of Connecticut Health, Farmington, CT, 06030, USA.
authors.forename: Amit
authors.initials: A
authors.lastname: Luthra
authors.affiliation: Department of Medicine, University of Connecticut Health, Farmington, CT, 06030, USA.
authors.forename: Arvind
authors.initials: A
authors.lastname: Anand
authors.affiliation: Department of Medicine, University of Connecticut Health, Farmington, CT, 06030, USA. JRadolf@uchc.edu.|Department of Pediatrics, University of Connecticut Health, Farmington, CT, 06030, USA. JRadolf@uchc.edu.|Department of Genetics and Genomics Sciences, University of Connecticut Health, Farmington, CT, 06030, USA. JRadolf@uchc.edu.|Department of Immunology, University of Connecticut Health, Farmington, CT, 06030, USA. JRadolf@uchc.edu.|Department of Molecular Biology and Biophysics, University of Connecticut Health, Farmington, CT, 06030, USA. JRadolf@uchc.edu.
authors.forename: Justin D
authors.initials: JD
authors.lastname: Radolf
mesh_terms: D000818:Animals; D001425:Bacterial Outer Membrane Proteins / Q000378:metabolism*; D005782:Gels; D055616:Microtechnology / Q000379:methods*; D021381:Protein Transport; D014210:Treponema pallidum / Q000166:cytology*
chemical_list: D001425:Bacterial Outer Membrane Proteins; D005782:Gels
keywords: BamA (TP0326); Gel microdroplets; Rare outer membrane protein; Surface immunolabeling; Syphilis; Treponema pallidum