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，我调查了Rab32/Rab38是否参与了针对GAS的巨型自噬。自噬核心蛋白ATGs在GAS感染下对巨型自噬的诱导是重要的。有报道称，自噬体标记物LC3在巨噬细胞内募集到GAS。我用CRISPR Cas9系统产生了Atg7KO或FIP200KO巨噬细胞，其中GFP-Rab32/Rab38表达。共聚焦显微镜观察发现，即使在没有ATG7或FIP200的情况下，Rab32和Rab38仍然强烈包围气体。巨噬细胞中的自噬标记物Lc3仍募集到GAS，提示为Lc3相关的吞噬小体，而不是巨噬细胞。我认为Rab32/38介导的LAP在巨噬细胞中比巨噬细胞自噬更重要。此外，Rab32/38双KO细胞显示溶酶体需要气体吞噬小体(LAMP1)，但吞噬体内似乎存在酸化缺陷。最后，我还进行了EM图像，结果显示，在野生型巨噬细胞中，融合了气体的溶酶体吞噬小体有浓缩的强度，并有典型的双层膜包裹气体。相反，在Rab32/Rab38双KO巨噬细胞中，主要观察到低强度的含气体吞噬小体，有许多空隙或空隙。因此，Rab32/Rab38在LAP途径，尤其是在酸化过程中起着非常重要的作用。

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.