2022 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, 大学院歯学研究科, 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 |
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| Outline of Annual Research Achievements |
In the pass year, I investigated whether Rab32/Rab38 is involved in macroautophagy against GAS.
Autophagic core proteins, ATGs, were important for macroautophagy induction even under GAS infection. It has been reported that LC3, an autophagosome marker, recruited to GAS in macrophage. I generated Atg7KO or FIP200KO macrophages generated by CRISPR Cas9 system, in which GFP-Rab32/Rab38 consist expressing. I found even without Atg7 or FIP200, Rab32 and Rab38 still strongly surrounding GAS under confocal microscope observation. LC3, an autophagy marker still recruit to GAS in macrophage, which indicated as an LC3 associated phagosome, instead of macroautophagy. I assume Rab32/38-mediated LAP, which containing lysosome fusion, is much important than macroautophagy in macrophage.
Furthermore, Rab32/38 double KO cells showed that GAS-phagosome are required with lysosome (LAMP1), however, acidification seems defective inside this phagosome.
Finally, I also performed EM images, as results showed that there are condensed intensity of lysosome-fused GAS-containing phagosome and a classical double membrane surrounding GAS in Wild-type macrophage. In contrast, a low intensity of GAS-containing phagosome, many free or empty space, was majorly observed in Rab32/Rab38 double KO macrophage. We conclude that Rab32/Rab38 is very important for LAP pathway, especially in acidification.
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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
In the pass year, the experiments were progressing under try and error, and the results came out there is some difference between wild-type and Rab32/Rab38 double DO cells.
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| Strategy for Future Research Activity |
In the new year, I aim to investigate whether microautophagy target GAS and whether Rab32/Rab38 is involved in this lysosome wrapping mechanism (LWM). This phenotype will required more clear EM images analysis. Lysosome has potential to change into a long-shapes in macrophage. This active movement, named lysosome protrusion, is able to surround an organelle bigger than lysosome itself. I plan to find out whether cytosol GAS can be surrounded by lysosome directly. The structure of lysosome protrusion along GAS will also be confirmed by correlative light and electron microscope (CLEM). I hope to show a direct evidence of microautophagy against bacteria.
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