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.

在过去的一年中，我研究了Rab 32/Rab 38是否参与了GAS的巨自噬过程，发现自噬核心蛋白ATGs在GAS感染的巨自噬过程中起着重要的作用。已有报道称，自噬体标志物LC 3在巨噬细胞中被募集至GAS。通过CRISPR Cas9系统产生Atg 7 KO或FIP 200 KO巨噬细胞，其中GFP-Rab 32/Rab 38存在表达。我发现即使没有Atg 7或FIP 200，Rab 32和Rab 38在共聚焦显微镜下仍然强烈包围GAS。自噬标志物LC 3在巨噬细胞中仍能募集GAS，表现为LC 3相关的吞噬体，而非巨噬细胞自噬。我认为Rab 32/38介导的含有溶酶体融合的吞噬作用比巨噬细胞的大自噬作用更重要。此外，Rab 32/38双KO细胞显示，GAS-吞噬体是溶酶体（LAMP 1）所必需的，但该吞噬体内部的酸化似乎是有缺陷的。最后，我还进行了EM图像，结果表明，野生型巨噬细胞中存在着密集的溶酶体融合的含GAS的吞噬体，GAS被典型的双层膜包裹。相反，在Rab 32/Rab 38双KO巨噬细胞中主要观察到低强度的含GAS的吞噬体，许多自由或空的空间。我们的结论是Rab 32/Rab 38在酸化过程中起着重要的作用。

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.