The autophagic response to <i>Staphylococcus aureus</i> provides an intracellular niche in neutrophils

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