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Tyrosine Phosphorylation of the Helicobacter pylori CagA Antigen after Cag- driven Host Cell Translocation.

Tyrosine Phosphorylation of the Helicobacter pylori CagA Antigen after Cag- driven Host Cell Translocation. Presented by Ria Achong. Contents. Introduction Brief introduction on Helicobacter pylori What is CagA ? Rationale Methods Results Conclusion .

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Tyrosine Phosphorylation of the Helicobacter pylori CagA Antigen after Cag- driven Host Cell Translocation.

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  1. Tyrosine Phosphorylation of the Helicobacter pylori CagA Antigen after Cag-driven Host Cell Translocation. Presented by Ria Achong

  2. Contents • Introduction • Brief introduction on Helicobacter pylori • What is CagA? • Rationale • Methods • Results • Conclusion

  3. What Is Helicobacter pylori? • Gram negative • Microaerophilic • Spiral • Motile • Colonizes gastric epithelium

  4. Helicobacter pylori as an etiologic agent Helicobacter pylori causes • Gastric ulcers • Duodenal ulcers • Adenocarcinomas of the distal stomach • Gastric musosa associated lymphoid tissue (MALT) lymphomas

  5. Mode of Action • Passes through mucous lining in stomach • Attaches to gastric epithelial cells and enters • Causes destruction of gastric mucosa • Survives gastric acidity by production of urease

  6. Genomic basis for pathogenesis Some genes associated with Hp activity • VacA –pore forming, vacuolating cytotoxin • BabA – Lewisbantigen adhesin • CagPAI – pathogenicity island for Type IV secretion system

  7. What is CagA? • Part of the CagPAI • Codes for CagA - immunodominant antigen of size 128-146 kDa • Highly associated with virulence and ulceration • Used to distinguish between Type I and Type II Helicobacter pylori • Function not known

  8. What happen when Helicobacter pylori binds to host cells • Bacteria binds to host cell • Cytoskeletal rearrangements occur • Pedestals form • Unidentified 145kDa protein is tyrosine phosphorylated at base of pedestal

  9. Rationale • To provide evidence that • the tyrosine phosphorylated target in the cell membrane of the host cells is the cagA protein inserted by the bacterial cell • the cag-A protein is phoshorylated after insertion

  10. Materials Helicobacter pylori (Hp)strains:- • Wild types – G27, 87A300, 342 • Mutants- • Lacking Type IV secretion system • G27DvirB9, G27DvirB10, G27DvirB11, G27DvirD4 • Lacking respective Cag gene • G27DcagA, G27DcagM, G27DcagE, G27DcagI, 342DcagA Host cells:– AGS cells

  11. Methods • Hp strain 87A300 was labeled with 35S • AGS cells were infected with the various Hp strains • Infected AGS cells were washed and prepared to produce pellets called cell lysates • Cell lysates were then immunoprecipitated using RIPA-soluble buffer

  12. Methods • This produced two samples:- • imp – precipitated proteins • post imp – RIPA-soluble proteins • Samples are tested using gel electrophoresis and various antibodies to isolate and identify two target proteins:- • PTYR – phosphorylated tyrosine target • CagA protein

  13. Methods • Antibodies used:- • Anti-PY – Monoclonal anti-phosphotyrosine antibody • Anti-Hp – Polyclonal Hp antibody • Anti-CagA polyclonal – CagA antibody • LDS56 – Monoclonal CagA antibody

  14. Results

  15. PTYR is a bacterial protein

  16. PTYR recognition with antibodies

  17. Overlapping of PTYR and CagA Patterns on a 2-D gel

  18. Correlation in size of CagA and PTYR molecules

  19. Type IV secretion system is required for CagA translocation

  20. Type IV secretion system is required for CagA translocation

  21. CagA is only phosphorylated in the host cell

  22. Conclusion • The target of tyrosine phosphorylated after adhesion of bacteria to host cell is translocated CagA • CagA is only phosphorylated in the host cell • Function of CagA is still not known

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