Membrane Topology and Cellular Location of the Treponema pallidum Glycerophosphodiester Phosphodiesterase (GlpQ) Ortholog

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American Society for Microbiology

RESUMO

Recent reports that isolated Treponema pallidum outer membranes contain an ortholog for glycerophosphodiester phosphodiesterase (GlpQ) (D. V. Shevchenko, D. R. Akins, E. J. Robinson, M. Li, O. V. Shevchenko, and J. D. Radolf, Infect. Immun. 65:4179–4189, 1997) and that this protein is a potential opsonic target for T. pallidum (C. E. Stebeck, J. M. Shaffer, T. W. Arroll, S. A. Lukehart, and W. C. Van Voorhis, FEMS Microbiol. Lett. 154:303–310, 1997) prompted a more detailed investigation of its physicochemical properties and cellular location. [14C]palmitate radiolabeling studies of a GlpQ-alkaline phosphatase fusion expressed in Escherichia coli confirmed the prediction from DNA sequencing that the protein is lipid modified. Studies using Triton X-114 phase partitioning revealed that the protein’s amphiphilicity is due to lipid modification and that a substantial portion of the polypeptide is associated with the T. pallidum peptidoglycan sacculus. Three different approaches, i.e., (i) proteinase K treatment of intact treponemes, (ii) indirect immunofluorescence analysis of treponemes encapsulated in agarose beads, and (iii) opsonophagocytosis of treponemes incubated with antiserum against recombinant GlpQ by rabbit peritoneal macrophages, confirmed that GlpQ is entirely subsurface in T. pallidum. Moreover, rabbits hyperimmunized with GlpQ were not protected against intradermal challenge with virulent treponemes. Circular dichroism spectroscopy confirmed that the recombinant form of the polypeptide lacked discernible evidence of denaturation. Finally, GlpQ was not radiolabeled when T. pallidum outer membranes were incubated with 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)-diazarene, a photoactivatable, lipophilic probe which promiscuously labels both proteins and lipids within phospholipid bilayers. Taken as a whole, these studies indicate that the T. pallidum GlpQ ortholog is a periplasmic protein associated predominantly with the spirochete’s peptidoglycan-cytoplasmic membrane complex.

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