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

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slide1

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

Presented by

Ria Achong

contents
Contents
  • Introduction
    • Brief introduction on Helicobacter pylori
    • What is CagA?
    • Rationale
  • Methods
  • Results
  • Conclusion
what is helicobacter pylori
What Is Helicobacter pylori?
  • Gram negative
  • Microaerophilic
  • Spiral
  • Motile
  • Colonizes gastric epithelium
helicobacter pylori as an etiologic agent
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
mode of action
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
genomic basis for pathogenesis
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
what is caga
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
what happen when helicobacter pylori binds to host cells
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
rationale
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
materials
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

methods
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
methods12
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
methods13
Methods
  • Antibodies used:-
    • Anti-PY – Monoclonal anti-phosphotyrosine antibody
    • Anti-Hp – Polyclonal Hp antibody
    • Anti-CagA polyclonal – CagA antibody
    • LDS56 – Monoclonal CagA antibody
overlapping of ptyr and caga
Overlapping of PTYR and CagA

Patterns on a 2-D gel

conclusion
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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