2021 Fiscal Year Research-status Report
Study the mechanism how Rab32/Rab38 positive lysosome related organelle is involved in bacteria suppression through macroautophagy and microautophagy in macrophage and mouse infectious model.
| Project/Area Number |
20K15789
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| Research Institution | Osaka University |
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Principal Investigator |
LU SHIOULING 大阪大学, 歯学研究科, 助教 (80830083)
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| Project Period (FY) |
2020-04-01 – 2024-03-31
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| Keywords | Group A Streptococcus / Macrophage / Rab32 / Rab38 / microautophagy |
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| Outline of Annual Research Achievements |
Macrophage is a professional phagocyte to engulf bacteria into lysosome for digestion. To reach interaction with lysosome, the intracellular bacteria could be transported by phagocytosis and macroautophagy. However, only a part of GAS was surrounded by autophagosome, the majority was by lysosome directly. In macrophage, lysosome membrane is more dynamic and could change shape along with a big cargo directly: lysosome protrusion1. This action also recognized as the lysosome wrapping mechanism (LWM) of microautophagy.
In this year, I found microautophagy was related to GAS killing by lysosome in macrophage, while GAS growth was only slightly affected in macroautophagy defective macrophage, ATG7KO and FIP200KO cells, generated during this year. Using the identified two small GTPase proteins in our group, we had found Rab32 and Rab38 are involved in lysosome vesicle transport. I found Rab32/Rab38 highly recruited to GAS-containing lysosome in macrophage. Lost of Rab32/38 decreased macrophage bacterial clearance. Notice, Rab32 and Rab38 positive ring could not be fixed well by regular formaldehyde. Rab32 or Rab38 have to be observed in life cell conditions, according to our GFP-Rab32 or GFP-Rab38 fusion protein expressing cell observation. As, an innate immune professional cells, macrophage activating response to GAS engulfment was analyed. I found that the inflammatory cytokines, including TNFalpha, IL6 and ROS, were decreased in GAS-infected DKO cells, comparing to WT cells.
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| Current Status of Research Progress |
Current Status of Research Progress
2: Research has progressed on the whole more than it was originally planned.
Reason
Rab32/rab38 double knockout macrophage is stably kept in regular maintenance and experiments cycles were running frequently. Therefore, I had continually collecting the data from planed experiments.
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| Strategy for Future Research Activity |
In this year, I had planed to performed GAS infection model in WT and Rab32/38 double knock out mice. We will observe the survival rate and detect the bacterial number in local tissue or disseminate into organs or blood in local infectious model or systemic infectious model. Pro-inflammatory cytokines will analyze by ELISA detection kit to observed the inflammatory response. The tissue injury tissue section with HE stain will performed for observation.
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