Cyto-adherence studies of the adhesin P50 of Mycoplasma hominis

Recombinant peptides of Mycoplasma hominis adhesin P50 were expressed in Es cherichia coli to investigate which regions of the P50 molecule are respons ible for cyto-adherence, The respective DNA fragments were obtained by PCR amplification of the p50 gene with the use of mutating oligonucleotides to change the TGA codons of mycoplasma to TGG codons, which are translated in E, coli as tryptophan, The resulting three clones (I, I + II and I + III) c ontained regions of P50 which closely represent the repeat regions A, A + B and A + C, After expression in E, coli, the polyhistidine-tagged recombina nt peptides were purified by metal chelation chromatography, The three reco mbinant peptides were detected in Western blot analysis by a polyclonal ant iserum directed against M. hominis FBG and two P50-specific monoclonal anti bodies, BA10 and BG2, Each of the three recombinant peptides I, I + II and I + III was able to adhere to immobilised HeLa cells in an adhesion assay. The cyto-adhesion of the peptides could be inhibited by pre-incubation with the appropriate antibody. Therefore, it is suggested that adherence may be mediated by all regions of the P50 molecule. Attachment of the recombinant peptides to immobilised HeLa cells was inhibited by high mel. wt dextran s ulphate (MW 500 000), indicating that the respective P50 regions bind to su lphatides on the host cell membrane.