RESUMEN
The gene encoding p50, an adhesin of Mycoplasma hominis, was identified, cloned, and sequenced. Comparison of the derived amino acid sequence with the N-terminal amino acids sequenced by the Edman reaction of the native protein revealed that p50 is expressed as a 467-amino-acid precursor. Posttranslational modification leads to a 441-amino-acid lipoprotein with an extended, predominantly helical structure and a leucine zipper. Computer analysis of the amino acid sequence identified a threefold-repetitive sequence motif comprising approximately three-quarters of the total protein. Different regions of the p50 polypeptide chain were expressed in Escherichia coli. Western blot (immunoblot) analysis of the E. coli lysates revealed that the epitopes of four p50-specific monoclonal antibodies were localized in the middle and C-terminal part of the protein. Epitope mapping by exonuclease III digestion showed that all of the four monoclonal antibodies bound within the same region of the threefold-repetitive amino acid sequence motif. The repeats, which were highly homologous but not identical in structure, could be differentiated by the monoclonal antibodies.
Asunto(s)
Adhesinas Bacterianas/química , Anticuerpos Monoclonales/inmunología , Mycoplasma hominis/química , Adhesinas Bacterianas/genética , Adhesinas Bacterianas/inmunología , Secuencia de Aminoácidos , Secuencia de Bases , Mapeo Epitopo , Datos de Secuencia Molecular , Transcripción GenéticaRESUMEN
The mechanism of Mycoplasma hominis adherence to host cells of the urogenital tract was investigated with monoclonal antibodies (MAbs) directed against antigenic surface-localized polypeptides P50, P60, P80, and P100 of cytoadherent M. hominis FBG. A cell enzyme-linked immunosorbent assay was established allowing quantification of cytoadherent mycoplasmas detected by one of the following MAbs: four MAbs directed against P100 (molecular weight, about 100,000), three MAbs against P80, one MAb against P60, and three MAbs against P50. MAb binding to one of the surface proteins resulted in a decrease of mycoplasmal adherence to HeLa cells. To exclude the thesis that this is caused by nonspecific blocking of adherence, P100 and P50 were purified by affinity chromatography and tested instead of intact mycoplasmas in the cell enzyme-linked immunosorbent assay for cytoadherence. Both proteins bound to the surface of the eukaryotic cells. MAb binding to single epitopes of these proteins resulted in inhibition of protein adherence. These experiments strongly suggest that of the four surface-localized proteins at least P100 and P50 are adhesins of M. hominis FBG.
Asunto(s)
Adhesión Bacteriana , Proteínas Bacterianas/análisis , Proteínas de la Membrana/análisis , Mycoplasma/química , Animales , Anticuerpos Monoclonales/inmunología , Antígenos Bacterianos/análisis , Proteínas Bacterianas/inmunología , Células HeLa , Humanos , Proteínas de la Membrana/inmunología , Ratones , Mycoplasma/fisiologíaRESUMEN
Monoclonal antibodies (MAbs) were generated against lysates of clinical Mycoplasma hominis isolates. Three of these, designated BG2, BA10, and FE6, recognized an integral membrane protein of M. hominis with an apparent molecular weight of 50,000 (p50). Electron microscopy studies demonstrated that this protein is distributed evenly over the cell surface. These anti-p50 MAbs were species specific for M. hominis; they reacted with 42% of 126 tested clinical M. hominis isolates and showed no reactivity to heterologous mycoplasma species. Immunoblot analysis after limited proteolysis of purified p50 demonstrated that the three MAbs reacted with different epitopes of the protein. Unlike BA10 and FE6, MAb BG2 induced a decrease in arginine metabolism and a reduction of CFU in metabolic inhibition tests. F(ab)2 fragments of MAb BG2 showed the same inhibitory effect as the intact MAb molecule, while Fab and Fc fragments had no influence on vital functions. Preincubation of the mycoplasmas with MAb BG2 followed by trypsin treatment yielded the same amount of CFU as the control without antibodies. In conclusion, the cell aggregates were resolved by the trypsin treatment. These experiments and tests with the antibody fragments led to the conclusion that only the intact MAb structure or the F(ab)2 structure had metabolic inhibition potential and that the observed metabolism inhibition as well as the apparent decrease in viability were a result of agglutination by MAb BG2.