ABSTRACT

Staphylococcus aureus is a major human pathogen causing multiple pathologies, from cutaneous lesions to life-threatening sepsis. Although neutrophils contribute to immunity against S. aureus, multiple lines of evidence suggest that these phagocytes can provide an intracellular niche for staphylococcal dissemination. However, the mechanism of neutrophil subversion by intracellular S. aureus 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 S. aureus-infected hosts remains controversial. Here, using larval zebrafish, we showed that the autophagy marker Lc3 rapidly decorates S. aureus 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 cyba/p22phox knockdown and diphenyleneiodonium (DPI) treatment inhibited Lc3 decoration of phagosomes. Importantly, NADPH oxidase inhibition diverted neutrophil S. aureus 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 S. aureus 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 S. aureus-infected neutrophils may explain the conflicting reports relating to anti-staphylococcal autophagy and provide new insights for therapeutic strategies against antimicrobial-resistant Staphylococci.

Abbreviations: 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.

摘要

金黄色葡萄球菌是一种主要的人类病原体，可引起多种病变，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的免疫力，但多种证据表明这些吞噬细胞可以为葡萄球菌传播提供细胞内生态位。然而，细胞内金黄色葡萄球菌破坏中性粒细胞的机制仍不清楚。通过巨自噬/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，但自噬对于金黄色葡萄球菌感染的宿主是否有益或有害仍存在争议。在这里，我们利用斑马鱼幼虫，发现自噬标记物 Lc3 在被巨噬细胞和中性粒细胞吞噬后迅速装饰金黄色葡萄球菌。中性粒细胞吞噬后，形成 Lc3 阳性、非酸化的宽敞吞噬体。这种反应依赖于吞噬细胞 NADPH 氧化酶，因为cyba/p22phox敲低和二亚苯基碘 (DPI) 处理都会抑制吞噬体的 Lc3 修饰。重要的是，NADPH 氧化酶抑制将中性粒细胞金黄色葡萄球菌加工成紧密的酸化囊泡，从而导致宿主对感染的抵抗力增强。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，Sqstm1 的丢失会损害宿主防御。总之，我们发现中性粒细胞对金黄色葡萄球菌的细胞内处理最好是通过 Lc3 相关的吞噬作用 (LAP) 来解释，LAP 似乎为细菌发病机制提供了细胞内生态位，而选择性自噬受体 Sqstm1 具有宿主保护作用。LAP 和 Sqstm1 介导的途径在金黄色葡萄球菌感染的中性粒细胞中的拮抗作用可能解释与抗葡萄球菌自噬相关的相互矛盾的报道，并为针对抗菌药物耐药葡萄球菌的治疗策略提供新的见解。

缩写： ATG：自噬相关；CFU：菌落形成单位；CMV：巨细胞病毒；Cyba/P22phox：细胞色素b-245，α多肽；DMSO：二甲亚砜；DPI：二亚苯基碘鎓；EGFP：增强型绿色荧光蛋白；GFP：绿色荧光蛋白；hpf：受精后小时数；hpi：感染后数小时；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；RFP：红色荧光蛋白；ROS：活性氧；RT-PCR：逆转录聚合酶链式反应；Sqstm1/p62: 隔离体 1; Tg：转基因；TSA：酪酰胺信号放大。