Helicobacter exploits integrin for type IV secretion and kinase activation 指導老師 : 臨床 : 許博翔 教授 基礎 : 吳俊忠 教授 學生 : 張維倫 Nature 2007 Oct 18; 449(7164): 862-6

Outline Introduction Hypothesis Specific Aims  Aim1:  Aim2: Results (experiment design and data) Summary Inspiration and future works Your comments for the paper

H. Pylori and gastric cancer (Introduction) Cag pathogenicity island  Encode oncoprotein – cagA  Encode type IV secretion system H. pylori Normal gastric mucosa superficial gastritis Atrophic gastritis Intestinal metaplasia Dysplasia Gastric carcinoma Presence of cag island Nature Reviews Cancer (2002) Nature (1997)

Hypothesis Integrins are exploits by H. pylori for injection of cagA into gastric epithelial cells

Specific Aims 1. Integrin α 5 β 1 is involved in type-IV secretion 2. CagL is expressed on type-IV secretion pili 3. CagL is essential for type-IV secretion 4. CagL binds integrin α 5 β 1 5. CagL activates FAK and Src tyrosine kinases

Step 1. Integrin α 5 β 1 is involved in type-IV secretion of CagA Co-localization of phosphorylated CagA with vinculin during H. pylori infection β 1 is required for injection of CagA into target cells α 5 is required for injection of CagA into target cells

Co-localization of phosphorylated CagA (CagA-pY) with vinculin during H. pylori infection Study design Result AGS cell 2 h Stained with anti-CagA-pY Stained with anti-vinculin Cell culture Overlay

Step 2. CagL is expressed on type- IV secretion pili CagL is the only gene product that contains RGD motif CagL is expressed on the surface of H. pylori CagL & CagA signals are present at the tips of H. pylori’s pili Pilus formation is CagL- and RGD-dependent

CagL is the only gene product that contains RGD motif Method  Sequence analysis of cagPAI gene Result

Step 3. CagL and its RGD motif are essential for type-IV secretion Study design AGS cell 1. cagL(WT) 4. △ cagL/cagL(RGA) Co-cultured with 2. △ cagL 3. △ cagL/cagL(WT) CagA-p Cytosolic CagA Cell morphology Immunoblots Microscopy

Results

Step 4: CagL binds integrin α 5 β 1 in an RGD-dependent manner Study design bead CagL (WT/RGD) CagL (RGA) BSA AGS AGS △ β1 Number of beads bound ＋

Step 5: CagL activates FAK and Src tyrosine kinases CagL (wt) strain can activate FAK and Src, while CagL (RGA) can not Recombinant CagL (RGD) can activate FAK and Src, while CagL (RAD) can not

Results

cagA cagA-p FAK-p Src-p Integrin summary T4SS

CagL (WT/RGD) bead AGS CagL (RGA) bead AGS AGS △ β1 bead CagL (WT)

Inspiration and future works Thinking process and study design CagL 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 CagL and integrin by immune regulation or other tools ?

comments α 5 β 1 integrin 在胃部中的表現量其實不一, 在 antrum 表現強, 在 fundus 表現弱. 所以胃癌好發 於 antrum. 其次, 在有 IM 的地方, α 5 β 1 integrin 表現更強. 我們可以調出有胃癌的菌株跟沒有胃癌的菌株, 看 cagL 表現的強弱有沒有不同, 或其上帶有的 RGD sequence 有無不同 Kd 多少表示 binding 是強的