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


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

Presented by

Ria Achong


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Contents

  • Introduction

    • Brief introduction on Helicobacter pylori

    • What is CagA?

    • Rationale

  • Methods

  • Results

  • Conclusion


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What Is Helicobacter pylori?

  • Gram negative

  • Microaerophilic

  • Spiral

  • Motile

  • Colonizes gastric epithelium


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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


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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


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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


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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


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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


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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


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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


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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


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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


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    Methods

    • Antibodies used:-

      • Anti-PY – Monoclonal anti-phosphotyrosine antibody

      • Anti-Hp – Polyclonal Hp antibody

      • Anti-CagA polyclonal – CagA antibody

      • LDS56 – Monoclonal CagA antibody


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    Results


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    PTYR is a bacterial protein


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    PTYR recognition with antibodies


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    Overlapping of PTYR and CagA

    Patterns on a 2-D gel


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    Correlation in size of CagA and PTYR molecules


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    Type IV secretion system is required for CagA translocation


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    Type IV secretion system is required for CagA translocation


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    CagA is only phosphorylated in the host cell


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    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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