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.

在过去的一年里，我研究了Rab32/Rab38是否参与了对GAS的巨噬。自噬核心蛋白ATGs在诱导巨噬过程中发挥着重要作用。据报道，LC3是一种自噬体标记物，在巨噬细胞中被招募到GAS。我生成了CRISPR Cas9系统生成的Atg7KO或FIP200KO巨噬细胞，其中GFP-Rab32/Rab38存在表达。我发现在共聚焦显微镜下，即使没有Atg7或FIP200， Rab32和Rab38仍然强烈地围绕着GAS。自噬标志物LC3在巨噬细胞中仍然招募到GAS，表明LC3相关的吞噬体，而不是巨噬细胞。我认为rab32 /38介导的LAP，包含溶酶体融合，在巨噬细胞中比巨噬更重要。此外，Rab32/38双KO细胞显示，溶酶体（LAMP1）需要gas吞噬体，但该吞噬体内部的酸化似乎存在缺陷。最后，我也做了EM图像，结果显示在野生型巨噬细胞中存在溶酶体融合的含有GAS的吞噬体的浓缩强度和典型的双膜包围GAS。而在Rab32/Rab38双KO巨噬细胞中，主要观察到低强度的含gas吞噬体，大量的自由或空腔。我们认为Rab32/Rab38在LAP途径中非常重要，特别是在酸化过程中。

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.