Lipid modification of the 17-kilodalton membrane immunogen of Treponema pallidum determines macrophage activation as well as amphiphilicity.
A murine monoclonal antibody specific for a 17-kDa major membrane immunogen of Treponema pallidum was used to select recombinant Escherichia coli clones expressing the molecule from a T. pallidum genomic library. Sequence analysis of the structural gene for the immunogen (designated tpp17) revealed a 468-bp open reading frame encoding a polypeptide of 156 amino acids with a calculated molecular mass of 16,441 Da. The deduced amino acid sequence included a putative leader peptide terminated by a consensus tetrapeptide for the modification and processing of prokaryotic lipoproteins. Immunoprecipitation of the cloned immunogen radiolabeled with [3H]palmitate confirmed that it was a lipoprotein. The amino acid sequence also predicted that the mature protein contains four cysteine residues in addition to the lipid-modified cysteine of the N terminus. The existence of disulfide-bonded multimeric forms of the native immunogen was demonstrated by immunoblotting T. pallidum solubilized in the presence and absence of 2-mercaptoethanol. Triton X-114 phase partitioning of a nonlipidated form of the 17-kDa immunogen cleaved from a glutathione S-transferase fusion protein demonstrated that lipid modification is responsible for the immunogen's hydrophobic character. The same nonlipidated form of the immunogen also was used to demonstrate that lipid modification is essential for the molecule's ability to stimulate production of tumor necrosis factor alpha by murine macrophages. We conclude that covalently attached fatty acids not only anchor T. pallidum lipoproteins to spirochetal membranes but also confer upon these molecules the ability to activate immune effector cells.
Akins
D R
DR
Department of Microbiology, University of Texas Southwestern Medical Center, Dallas 75235.
Purcell
B K
BK
Mitra
M M
MM
Norgard
M V
MV
Radolf
J D
JD
D000595:Amino Acid Sequence; D000818:Animals; D000942:Antigens, Bacterial / Q000737:chemistry*; D000954:Antigens, Surface / Q000737:chemistry*; D001426:Bacterial Proteins; D001483:Base Sequence; D003001:Cloning, Molecular; D004252:DNA Mutational Analysis; D004220:Disulfides / Q000737:chemistry; D005798:Genes, Bacterial; D006801:Humans; D066298:In Vitro Techniques; D008074:Lipoproteins / Q000737:chemistry / Q000235:genetics* / Q000276:immunology*; D008262:Macrophage Activation; D008264:Macrophages / Q000276:immunology; D051379:Mice; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D011993:Recombinant Fusion Proteins / Q000276:immunology; D015183:Restriction Mapping; D017384:Sequence Deletion; D012995:Solubility; D014210:Treponema pallidum / Q000276:immunology*
D000942:Antigens, Bacterial; D000954:Antigens, Surface; D001426:Bacterial Proteins; D004220:Disulfides; D008074:Lipoproteins; D009838:Oligodeoxyribonucleotides; D011993:Recombinant Fusion Proteins; C080366:17 kDa immunogen, Treponema pallidum