| Project/Area Number |
23K14170
|
|---|
| Research Category |
Grant-in-Aid for Early-Career Scientists
|
| Allocation Type | Multi-year Fund |
|---|
| Review Section |
Basic Section 43060:System genome science-related
|
|---|
| Research Institution | Osaka University |
|---|
Principal Investigator |
ALKADI MOHAMAD 大阪大学, 免疫学フロンティア研究センター, 特任研究員(常勤) (70963883)
|
|---|
| Project Period (FY) |
2023-04-01 – 2025-03-31
|
| Project Status |
Granted (Fiscal Year 2023)
|
| Budget Amount *help |
¥4,680,000 (Direct Cost: ¥3,600,000、Indirect Cost: ¥1,080,000)
Fiscal Year 2024: ¥1,690,000 (Direct Cost: ¥1,300,000、Indirect Cost: ¥390,000)
Fiscal Year 2023: ¥2,990,000 (Direct Cost: ¥2,300,000、Indirect Cost: ¥690,000)
|
|---|
| Keywords | Infection / Organoids / Pathogenesis / T3SS / scRNA-seq / Single-cell sequencing |
|---|
| Outline of Research at the Start |
iPSC-derived organoids will be used to study the heterogeneity of different cell responses to Vibrio secretion system using single-cell RNA-seq. We will also address the coordination of different bacterial effectors to rewire the host pathways and validate the results experimentally.
|
| Outline of Annual Research Achievements |
iPSC-derived organoids were infected with a (WT) Vibrio parahaemolyticus strain and three T3SS mutant strains: dN1, dN2, and dN1dN2. Cells were collected at 3 hours post-infection (PI) and washed to remove non-adherent bacteria. Bacterial adherence was detected using 16S primers.
We successfully detected adhered bacteria. WT and dN1 samples showed significantly higher expression, highlighting the importance of T3SS2 in adherence to our model cells. scRNA-seq analysis demonstrated the full spectrum of enterocyte development, similar to the in vivo model.
Vibrio showed a preference for adhering to progenitor cells in a T3SS2-dependent manner. T3SS2 also induced a more significant inflammatory response. Overall, our model and analysis show promise in elucidating the pathogenesis in humans.
|
| Current Status of Research Progress |
Current Status of Research Progress
3: Progress in research has been slightly delayed.
Reason
Due to the unexpected lack of secretory cells in our model, we are establishing a new organoid model. This model will be infected and compared with our current model. Depending on the results, further investigation will be conducted on one or both of these models.
|
| Strategy for Future Research Activity |
Due to the lack of secretory cells in iPSC model, I want to use a different model, intestinal stem cells derived organoids. This model is now almost established and we are seeking ethical approval for infection experiment.
Using this model, we can evaluate iPSC model and validate our results such as adherence and targeted cells. T3SS2 is expected to show similar importance in pathogenesis. These results will further be validated with in vitro experiment in one of both models. Deletion mutants of the T3SS2 effectors candidates will be created which can further elucidate the T3SS2-dependent pathogenesis.
|
