[PMC free article] [PubMed] [Google Scholar] 3. peptidoglycan-cytoplasmic membrane complex. Syphilis, a sexually transmitted disease caused by the spirochetal pathogen subsp. on artificial medium and the syphilis spirochetes unusual outer membrane ultrastructure (50). In recent years, the quest for outer membrane (OM) proteins of as potential Azaperone virulence determinants and vaccine candidates has become a major focus of syphilis research (50). In Azaperone this regard, we recently reported that OMs isolated from by using a plasmolysis-based procedure contain a 38.5-kDa putative lipoprotein with sequence relatedness to glycerophosphodiester phosphodiesterase (GlpQ) (56), an enzyme which hydrolyzes deacylated phospholipids to alcohol plus glycerol-3-phosphate CD197 (37, 39). Although GlpQ is usually periplasmic in (37), the ortholog is usually surface uncovered and capable of inducing bactericidal antibodies (29, 55). Consistent with this, Stebeck and coworkers Azaperone reported that this treponemal ortholog is usually a potential opsonic target for motile (60). These findings prompted a detailed investigation of the physicochemical properties and cellular location of this protein. Here we report that, as with GlpQ of GlpQ protein is usually lipid modified but that, unlike its counterpart, the treponemal polypeptide has a subsurface location. Similar to other treponemal lipoprotein immunogens, GlpQ appears to be associated predominantly with the peptidoglycan-cytoplasmic membrane (CM) complex. Moreover, contrary to the recent report by Cameron et al. (16) demonstrating attenuated lesion development when GlpQ-immunized rabbits were intradermally inoculated with virulent (Nichols) was propagated by intratesticular inoculation of adult New Zealand White rabbits as previously described (52). Spirochetes were separated from testicular tissue debris by low-speed centrifugation (350 for 10 min) and, when necessary, purified by Percoll density gradient centrifugation (30). For opsonophagocytosis assays, organisms were extracted from infected testes in medium 199 (M199) (Mediatech, Herndon, Va.) supplemented with 20% heat-inactivated fetal bovine serum (HIFBS) (heated for 30 min at 56C) (Mediatech) and gassed with 3% O2C5% CO2 overnight at 37C. Spirochetes were enumerated by dark-field microscopy with a Petroff-Hausser counting chamber (Hausser Scientific Company, Horsham, Pa.). DH5 was the recipient strain for all those recombinant constructs and was grown in Luria-Bertani broth with appropriate antibiotic supplementation. Production and purification of a recombinant, nonlipidated GlpQ (rGlpQ). The portion of the gene encoding the mature (i.e., processed) protein was PCR amplified from genomic DNA by using the forward and reverse primers 5-GCGGGATCCTGTGCGTCCGAACGTATGATAGTTG-3 (cell supernatant on an Ni-nitrilotriacetic acid agarose matrix (Qiagen, Azaperone Inc., Santa Clarita, Calif.) according to the manufacturers instructions. The His tag was removed by digestion with recombinant tobacco etch virus protease (Gibco BRL) as described by the manufacturer. Immunologic reagents. Immune rabbit sera (IRS) were obtained approximately 10 months following intratesticular inoculation of rabbits with motile alkaline phosphatase (PhoA) was obtained from Caltag Laboratories (Burlingame, Calif.). Rabbit antisera directed against endoflagella (TpEf) (34) and native 47-kDa lipoprotein (Tp47) (22) were described previously. To affinity purify anti-GlpQ antibodies from IRS, 80 g of rGlpQ was coupled to 100 l of 1 1,1-carbonyldiimidazole-activated 6% cross-linked beaded agarose (Reacti-Gel 6; Pierce, Rockford, Ill.) according to the manufacturers instructions. The Reacti-Gel matrix was equilibrated with 250 mM Tris (pH 7.4) and incubated for 2 h at 4C with 250 l of IRS. The adsorbed anti-GlpQ antibodies were eluted from the matrix in 200-l fractions with 0.5 M acetic acid and neutralized with 100 l of 1 1 M Tris base. Both the anti-GlpQ and the resulting IRS depleted of anti-GlpQ antibodies were tested by Western blot analysis for their ability to recognize the native GlpQ (nGlpQ) protein in lysates as described above. To quantitatively remove immunoglobulin G (IgG) antibodies from IRS, IRS was exceeded over a GammaBind G Sepharose matrix (Pharmacia Biotech, Alameda, Calif.). The adsorbed IgG antibodies were eluted in 500-l fractions with 0.5 M acetic acid and neutralized with 0.25 ml of 1 1 M Tris base. The protein concentrations of the affinity-purified anti-GlpQ antibodies and total IgG fractions were determined with the Bio-Rad protein assay (Bio-Rad Laboratories, Hercules, Calif.). SDS-polyacrylamide gel electrophoresis (SDS-PAGE), immunoblot analysis, and two-dimensional gel electrophoresis. Samples were boiled for 5.
