ABSTRACT

Macroautophagy/autophagy functions to degrade cellular components and intracellular pathogens. Autophagy receptors, including SQSTM1/p62, target intracellular pathogens. Staphylococcus aureus is a significant pathogen of humans, especially in immunocompromise. S. aureus may use neutrophils as a proliferative niche, but their intracellular fate following phagocytosis has not been analyzed in vivo. In vitro, SQSTM1 can colocalize with intracellular Staphylococcus aureus, but whether SQSTM1 is beneficial or detrimental in host defense against S. aureus in vivo is unknown. Here we determine the fate and location of S. aureus within neutrophils throughout zebrafish infection. We show Lc3 and Sqstm1 recruitment to phagocytosed S. aureus is altered depending on the bacterial location within the neutrophil and that Lc3 marking of bacterial phagosomes within neutrophils may precede bacterial degradation. Finally, we show Sqstm1 is important for controlling cytosolic bacteria, demonstrating for the first time a key role of Sqstm1 in autophagic control of S. aureus in neutrophils.

Abbreviations: AR: autophagy receptor; CFU: colony-forming unit; CHT: caudal hematopoietic tissue; GFP: green fluorescent protein; hpf: hours post-fertilization; hpi: hours post-infection; LWT: london wild-type: lyz: lysozyme; Map1lc3/Lc3: microtubule-associated protein 1 light chain 3; RFP: red fluorescent protein; Sqstm1/p62: sequestosome 1; Tg: transgenic; TSA: tyramide signal amplification; UBD: ubiquitin binding domain.

摘要

巨自噬/自噬具有降解细胞成分和细胞内病原体的功能。自噬受体，包括 SQSTM1/p62，靶向细胞内病原体。金黄色葡萄球菌是人类的重要病原体，尤其是在免疫功能低下时。金黄色葡萄球菌可能使用中性粒细胞作为增殖生态位，但尚未在体内分析吞噬作用后的细胞内命运。在体外，SQSTM1 可以与细胞内的金黄色葡萄球菌共定位，但 SQSTM1在体内对抗金黄色葡萄球菌的宿主防御中是有益还是有害尚不清楚。在这里我们确定了金黄色葡萄球菌的命运和位置在整个斑马鱼感染的中性粒细胞内。我们显示 Lc3 和 Sqstm1 对吞噬的金黄色葡萄球菌的募集根据嗜中性粒细胞内细菌的位置而改变，并且嗜中性粒细胞内细菌吞噬体的 Lc3 标记可能先于细菌降解。最后，我们表明 Sqstm1 对于控制胞质细菌很重要，首次证明了 Sqstm1在中性粒细胞中金黄色葡萄球菌的自噬控制中的关键作用。

缩写： AR：自噬受体；CFU：菌落形成单位；CHT：尾部造血组织；GFP：绿色荧光蛋白；hpf：受精后的小时数；hpi：感染后的小时数；LWT：伦敦野生型：lyz：溶菌酶；Map1lc3/Lc3：微管相关蛋白1轻链3；RFP：红色荧光蛋白；Sqstm1/p62: sequestosome 1; Tg：转基因；TSA：酪胺信号放大；UBD：泛素结合域。