Cyto-adherence studies of the adhesin P50 of Mycoplasma hominis.
Recombinant peptides of Mycoplasma hominis adhesin P50 were expressed in Escherichia coli to investigate which regions of the P50 molecule are responsible 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) contained regions of P50 which closely represent the repeat regions A, A+B and A+C. After expression in E. coli, the polyhistidine-tagged recombinant peptides were purified by metal chelation chromatography. The three recombinant peptides were detected in Western blot analysis by a polyclonal antiserum directed against M. hominis FBG and two P50-specific monoclonal antibodies, 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 mol. wt dextran sulphate (MW 500000), indicating that the respective P50 regions bind to sulphatides on the host cell membrane.
Kitzerow
Annette
A
Hadding
Ulrich
U
Henrich
Birgit
B
D018829:Adhesins, Bacterial / Q000235:genetics / Q000378:metabolism*; D001422:Bacterial Adhesion; D001665:Binding Sites; D002462:Cell Membrane / Q000378:metabolism; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D019535:Mycoplasma hominis / Q000472:pathogenicity*; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism; D017422:Sequence Analysis, DNA
D018829:Adhesins, Bacterial; D008565:Membrane Proteins; D010446:Peptide Fragments; D011994:Recombinant Proteins; C118865:adhesin P50, Mycoplasma hominis