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.

2020 Fiscal Year Research-status Report

• Project/Area Number: 20K15789
• Research Institution: Osaka University
• Principal Investigator: LU SHIOULING 大阪大学, 歯学研究科, 特任助教 (80830083)
• Project Period (FY): 2020-04-01 – 2024-03-31
• Keywords: Group A Streptococcus / Macrophage / Rab32 / Rab38

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. Previous reports showed Group A Streptococcus (GAS), a human infectious bacteria, evaded into cytoplasm and were captured by macroautophagy. However, among those presented results, only part of GAS was surrounded by autophagosome, and even though most population of GAS was suppressed in cells. Although macroautophagy is required, it should not be the sole procedure for GAS clearance. 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.
This year, I detected microautophagy (LC3) is only partially involved in GAS killing in macrophage. I found most bacteria was traped in lysosome piror to LC3 recruitment. And I identified the new roles of small GTPase proteins, Rab32 and Rab38, involved in macrophage against GAS. Our laboratory had established Rab32/Rab38 single KO or double KO mouse. I had preliminary data showing that Rab32 and Rab38 are recruited to GAS. And double knock macrophage had a lower ability to suppress GAS. This project will further study whether Rab32 or/and Rab38 is involved in lysosome protrusion alone GAS (microautophagy) in macrophage.

Current Status of Research Progress

2: Research has progressed on the whole more than it was originally planned.

Reason

Because the Rab32 and Rab38 knockout mouse were generated in the first year of this grand, I can isolated macrophage cells from wild type and Rab32/rab38 double knockout mice for my experiments.

Strategy for Future Research Activity

The goal of this project is to clarify the mechanism how Rab32/Rab38 positive lysosome related organelle is involved in GAS suppression through macroautophagy and microautophagy in macrophage. In the first year, I had completed the specific aim 1, which Rab32/Rab38 double knockout macrophage were partially defective in bacteria clearance.
Next plan, I will performed second specific aims: to study the location between Rab32/Rab38, organelles and GAS in macrophage. Once Rab32 and Rab38 is involved in intracellular bacterial killing, I would like to know which pathway of Rab32 and Rab38 involved in and I will analyze the distributions of Rab32 and Rab38 inside macrophage. Combined with ER, Golgi, mitochondrial, endosome and lysosome markers, the identity of main organelle where Rab32 and Rab38 located in macrophage will be observed under confocal microscope. Furthermore, under GAS infection, I will confirm whether Rab32 and Rab38 are recruited to GAS in macrophage. It is expected that Rab32 and Rab38 will be localized on lysosome and recruited to GAS, similar to the preliminary results shown in Hela cells. The location of Rab32 and Rab38 is also expected on ER, since they may be involved in autophagy pathway. The macrophage used here would be isolated from bone marrow macrophage from WT mouse. Both endogenous and exogenous Rab32 and Rab38 will be observed by well-established staining protocol.