Macrophages target Salmonella by Lc3-associated phagocytosis in a systemic infection model.

ABSTRACT

Innate immune defense against intracellular pathogens, like Salmonella, relies heavily on the autophagy machinery of the host. This response is studied intensively in epithelial cells, the target of Salmonella during gastrointestinal infections. However, little is known of the role that autophagy plays in macrophages, the predominant carriers of this pathogen during systemic disease. Here we utilize a zebrafish embryo model to study the interaction of S. enterica serovar Typhimurium with the macroautophagy/autophagy machinery of macrophages in vivo. We show that phagocytosis of live but not heat-killed Salmonella triggers recruitment of the autophagy marker GFP-Lc3 in a variety of patterns labeling tight or spacious bacteria-containing compartments, also revealed by electron microscopy. Neutrophils display similar GFP-Lc3 associations, but genetic modulation of the neutrophil/macrophage balance and ablation experiments show that macrophages are critical for the defense response. Deficiency of atg5 reduces GFP-Lc3 recruitment and impairs host resistance, in contrast to atg13 deficiency, indicating that Lc3-Salmonella association at this stage is independent of the autophagy preinitiation complex and that macrophages target Salmonella by Lc3-associated phagocytosis (LAP). In agreement, GFP-Lc3 recruitment and host resistance are impaired by deficiency of Rubcn/Rubicon, known as a negative regulator of canonical autophagy and an inducer of LAP. We also found strict dependency on NADPH oxidase, another essential factor for LAP. Both Rubcn and NADPH oxidase are required to activate a Salmonella biosensor for reactive oxygen species inside infected macrophages. These results identify LAP as the major host protective autophagy-related pathway responsible for macrophage defense against Salmonella during systemic infection. Abbreviations ATG autophagy related gene; BECN1 Beclin 1; CFU colony forming units; CYBA/P22PHOX cytochrome b-245, alpha chain; CYBB/NOX2 cytochrome b-245 beta chain; dpf days post fertilization; EGFP enhanced green fluorescent protein; GFP green fluorescent protein; hfp hours post fertilization; hpi hours post infection; IRF8 interferon regulatory factor 8; Lcp1/L-plastin lymphocyte cytosolic protein 1; LAP LC3-associated phagocytosis; MAP1LC3/LC3 microtubule-associated protein 1A/1B-light chain 3; mCherry red fluorescent protein; mpeg1 macrophage expressed gene 1; mpx myeloid specific peroxidase; NADPH oxidase nicotinamide adenine dinucleotide phosphate oxidase; NCF4/P40PHOX neutrophil cytosolic factor 4; NTR-mCherry nitroreductase-mCherry fusion; PTU phenylthiourea; PtdIns3K class III phosphatidylinositol 3-kinase; PtdIns3P phosphatidylinositol 3-phosphate; RB1CC1/FIP200 RB-1 inducible coiled coin 1; ROS reactive oxygen species; RT-PCR reverse transcriptase polymerase chain reaction; RUBCN/RUBICON RUN and cysteine rich domain containing BECN1-interacting protein; SCV Salmonella-containing vacuole; S. Typhimurium/S.T Salmonella enterica serovar Typhimurium; TEM transmission electron microscopy; Tg transgenic; TSA tyramide signal amplification; ULK1/2 unc-51-like autophagy activating kinase 1/2; UVRAG UVRAG UV radiation resistance associated; wt wild type.