Bateson Centre and Department of Infection, Immunity and Cardiovascular Disease, University of Sheffield, Sheffield, UK.
Institute of Biology Leiden, Faculty of Science, Leiden University, Leiden, The Netherlands.
Autophagy. 2021 Apr;17(4):888-902. doi: 10.1080/15548627.2020.1739443. Epub 2020 Mar 15.
is a major human pathogen causing multiple pathologies, from cutaneous lesions to life-threatening sepsis. Although neutrophils contribute to immunity against , multiple lines of evidence suggest that these phagocytes can provide an intracellular niche for staphylococcal dissemination. However, the mechanism of neutrophil subversion by intracellular remains unknown. Targeting of intracellular pathogens by macroautophagy/autophagy is recognized as an important component of host innate immunity, but whether autophagy is beneficial or detrimental to -infected hosts remains controversial. Here, using larval zebrafish, we showed that the autophagy marker Lc3 rapidly decorates following engulfment by macrophages and neutrophils. Upon phagocytosis by neutrophils, Lc3-positive, non-acidified spacious phagosomes are formed. This response is dependent on phagocyte NADPH oxidase as both knockdown and diphenyleneiodonium (DPI) treatment inhibited Lc3 decoration of phagosomes. Importantly, NADPH oxidase inhibition diverted neutrophil processing into tight acidified vesicles, which resulted in increased host resistance to the infection. Some intracellular bacteria within neutrophils were also tagged by Sqstm1/p62-GFP fusion protein and loss of Sqstm1 impaired host defense. Together, we have shown that intracellular handling of by neutrophils is best explained by Lc3-associated phagocytosis (LAP), which appears to provide an intracellular niche for bacterial pathogenesis, while the selective autophagy receptor Sqstm1 is host-protective. The antagonistic roles of LAP and Sqstm1-mediated pathways in -infected neutrophils may explain the conflicting reports relating to anti-staphylococcal autophagy and provide new insights for therapeutic strategies against antimicrobial-resistant . ATG: autophagy related; CFU: colony-forming units; CMV: cytomegalovirus; Cyba/P22phox: cytochrome b-245, alpha polypeptide; DMSO: dimethyl sulfoxide; DPI: diphenyleneiodonium; EGFP: enhanced green fluorescent protein; GFP: green fluorescent protein; hpf: hours post-fertilization; hpi: hours post-infection; Irf8: interferon regulatory factor 8; LAP: LC3-associated phagocytosis; lyz: lysozyme; LWT: london wild type; Map1lc3/Lc3: microtubule-associated protein 1 light chain 3; NADPH oxidase: nicotinamide adenine dinucleotide phosphate oxidase; RFP: red fluorescent protein; ROS: reactive oxygen species; RT-PCR: reverse transcriptase polymerase chain reaction; Sqstm1/p62: sequestosome 1; Tg: transgenic; TSA: tyramide signal amplification.
金黄色葡萄球菌是一种主要的人类病原体，可导致多种疾病，从皮肤损伤到危及生命的败血症。尽管中性粒细胞有助于抵抗金黄色葡萄球菌，但有多项证据表明，这些吞噬细胞可为金黄色葡萄球菌的传播提供细胞内小生境。然而，细胞内金黄色葡萄球菌对中性粒细胞的颠覆机制尚不清楚。巨自噬/自噬靶向细胞内病原体被认为是宿主先天免疫的重要组成部分，但自噬对感染宿主是有益还是有害仍存在争议。在这里，我们使用幼虫斑马鱼表明，自噬标记物 Lc3 在被巨噬细胞和中性粒细胞吞噬后迅速修饰。在被中性粒细胞吞噬后，形成 Lc3 阳性、非酸化的大吞噬体。这种反应依赖于吞噬细胞的 NADPH 氧化酶，因为敲低 NADPH 氧化酶和二苯基碘（DPI）处理均抑制了吞噬体中 Lc3 的修饰。重要的是，NADPH 氧化酶抑制将中性粒细胞中金黄色葡萄球菌的处理转移到紧密酸化的小泡中，从而导致宿主对感染的抵抗力增加。中性粒细胞内的一些细胞内细菌也被 Sqstm1/p62-GFP 融合蛋白标记，并且 Sqstm1 的缺失会损害宿主防御。总之，我们已经表明，中性粒细胞对金黄色葡萄球菌的细胞内处理最好通过 Lc3 相关的吞噬作用（LAP）来解释，这似乎为细菌发病机制提供了一个细胞内小生境，而选择性自噬受体 Sqstm1 对宿主具有保护作用。LAP 和 Sqstm1 介导的途径在感染中性粒细胞中的拮抗作用可能解释了与抗金黄色葡萄球菌自噬相关的相互矛盾的报告，并为对抗抗微生物耐药性金黄色葡萄球菌提供了新的见解。ATG：自噬相关；CFU：集落形成单位；CMV：巨细胞病毒；Cyba/P22phox：细胞色素 b-245，α 多肽；DMSO：二甲亚砜；DPI：二苯基碘；EGFP：增强型绿色荧光蛋白；GFP：绿色荧光蛋白；hpf：受精后小时；hpi：感染后小时；Irf8：干扰素调节因子 8；LAP：LC3 相关的吞噬作用；lyz：溶菌酶；LWT：伦敦野生型；Map1lc3/Lc3：微管相关蛋白 1 轻链 3；NADPH 氧化酶：烟酰胺腺嘌呤二核苷酸磷酸氧化酶；RT-PCR：逆转录聚合酶链反应；Sqstm1/p62：自噬相关蛋白 1；Tg：转基因；TSA：辣根过氧化物酶信号放大。
