Membrane topology and cellular location of the Treponema pallidum glycerophosphodiester phosphodiesterase (GlpQ) ortholog

Recent reports that isolated Treponema pallidum outer membranes contain an ortholog for glycerophosphodiester phosphodiesterase (GlpQ) (D. V. Shevchen ko, 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 op sonic 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, 19 97) prompted a more detailed investigation of its physicochemical propertie s and cellular location. [C-14]palmitate radiolabeling studies of a GlpQ-al kaline phosphatase fusion expressed in Escherichia coli confirmed the predi ction 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 polypep tide is associated with the T. pallidum peptidoglycan sacculus, Three diffe rent approaches, i.e., (i) proteinase K treatment of intact treponemes, (ii ) indirect immunofluorescence analysis of treponemes encapsulated in agaros e beads, and (iii) opsonophagocytosis of treponemes incubated with antiseru m against recombinant GlpQ by rabbit peritoneal marrophages, confirmed that GlpQ is entirely subsurface in T. pallidum. Moreover, rabbits hyperimmuniz ed 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, Final ly, GlpQ was not radiolabeled when T. pallidum outer membranes were incubat ed with 3-(trifluoromethyl)-3-(m-[I-125]iodophenyl)-diazarene, a photoactiv atable, lipophilic probe which promiscuously labels both proteins and lipid s within phospholipid bilayers, Taken as a whole, these studies indicate th at the T. pallidum GlpQ ortholog: is a periplasmic protein associated predo minantly with the spirochete's peptidoglycan-cytoplasmic membrane complex.