Helicobacter exploits integrin for type IV secretion and
Helicobacter exploits integrin for type IV secretion and kinase activation Nature 2007 Oct 18; 449(7164): 862 -6 學生: 張維倫 指導老師: 臨床: 許博翔 教授 基礎: 吳俊忠 教授
Outline n n n Introduction Hypothesis Specific Aims Aim 1: ¨ Aim 2: ¨ Results (experiment design and data) n Summary n Inspiration and future works n Your comments for the paper n
H. Pylori and gastric cancer (Introduction) Presence of cag island H. pylori Normal gastric mucosa superficial gastritis Atrophic gastritis Intestinal metaplasia Dysplasia Gastric carcinoma Nature Reviews Cancer (2002) Nature (1997) n Cag pathogenicity island ¨ Encode oncoprotein – cag. A ¨ Encode type IV secretion system
Hypothesis n Integrins are exploits by H. pylori for injection of cag. A into gastric epithelial cells
Specific Aims 1. Integrin α 5β 1 is involved in type-IV secretion 2. Cag. L is expressed on type-IV secretion pili 3. Cag. L is essential for type-IV secretion 4. Cag. L binds integrin α 5β 1 5. Cag. L activates FAK and Src tyrosine kinases
Step 1. Integrin α 5β 1 is involved in type-IV secretion of Cag. A Co-localization of phosphorylated Cag. A with vinculin during H. pylori infection n β 1 is required for injection of Cag. A into target cells n α 5 is required for injection of Cag. A into target cells n
Co-localization of phosphorylated Cag. A (Cag. A-p. Y) with vinculin during H. pylori infection n Study design Cell culture AGS cell 2 h Stained with anti-Cag. A-p. Y Stained with anti-vinculin Overlay n Result
Step 2. Cag. L is expressed on type. IV secretion pili n n Cag. L is the only gene product that contains RGD motif Cag. L is expressed on the surface of H. pylori Cag. L & Cag. A signals are present at the tips of H. pylori’s pili Pilus formation is Cag. L- and RGD-dependent
Cag. L is the only gene product that contains RGD motif n Method ¨ Sequence n Result analysis of cag. PAI gene
Step 3. Cag. L and its RGD motif are essential for type-IV secretion n Study design 1. cag. L(WT) Co-cultured with AGS cell 2. △cag. L Cag. A-p Cytosolic Cag. A 3. △cag. L/cag. L(WT) Cell morphology 4. △cag. L/cag. L(RGA) Immunoblots Microscopy
n Results
Step 4: Cag. L binds integrin α 5β 1 in an RGD-dependent manner n Study design bead Cag. L (WT/RGD) AGS bead Cag. L (RGA) + AGS△β 1 bead BSA Number of beads bound
Step 5: Cag. L activates FAK and Src tyrosine kinases n Cag. L (wt) strain can activate FAK and Src, while Cag. L (RGA) can not n Recombinant Cag. L (RGD) can activate FAK and Src, while Cag. L (RAD) can not
n Results
summary T 4 SS Integrin cag. A FAK-p Src-p cag. A-p
Cag. L (WT/RGD) Cag. L (RGA) Cag. L (WT) bead AGS AGS△β 1
Inspiration and future works n n Thinking process and study design Cag. L may be a novel vaccination target to decrease the bacterial virulence Regulation of cell surface integrin may be potential to prevent carcinogenesis How to inhibit interaction between Cag. L and integrin by immune regulation or other tools ?
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