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Cyto-adherence studies of the adhesin P50 of Mycoplasma hominis
Abstract:
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.
Insights
Recombinant peptides of Mycoplasma hominis adhesin P50 were expressed in E. coli to identify cyto-adherence regions. All tested P50 regions mediated HeLa cell attachment, suggesting broad involvement in adherence.
Area of Science:
- Microbiology
- Molecular Biology
- Cell Adhesion
Background:
- Mycoplasma hominis adhesin P50 plays a role in bacterial cyto-adherence.
- Understanding the specific regions responsible for P50-mediated adherence is crucial for elucidating pathogenic mechanisms.
Purpose of the Study:
- To investigate which regions of the Mycoplasma hominis P50 adhesin are responsible for cyto-adherence.
- To characterize the interaction of P50 recombinant peptides with host cells.
Main Methods:
- Expression of recombinant P50 peptides in Escherichia coli using PCR amplification and mutating oligonucleotides.
- Purification of polyhistidine-tagged peptides via metal chelation chromatography.
- Detection using Western blot analysis and adhesion assays with HeLa cells.
Main Results:
- Three recombinant peptides representing P50 regions A, A+B, and A+C were successfully expressed and purified.
- All three recombinant peptides demonstrated adherence to immobilized HeLa cells.
- Adherence was inhibited by specific antibodies and high molecular weight dextran sulfate, indicating binding to host cell sulphatides.
Conclusions:
- All tested regions of the Mycoplasma hominis P50 adhesin molecule appear to mediate cyto-adherence.
- The P50 adhesin likely binds to sulphatides on the host cell membrane.