The autophagic response to Staphylococcus aureus provides an intracellular niche in neutrophils

Tomasz K Prajsnar,Justyna J Serba,Bernice M Dekker,Josie F Gibson,Samrah Masud,Angeleen Fleming,Simon A Johnston,Stephen A Renshaw,Annemarie H Meijer

doi:10.1080/15548627.2020.1739443

Tomasz K Prajsnar, Justyna J Serba + Show 7 more

Open Access

https://doi.org/10.1080/15548627.2020.1739443

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Abstract

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.

Highlights

Staphylococcus aureus is a highly successful human pathogen causing a wide range of diseases [1]
In agreement with previous work, we observed both neutrophils and macrophages to take up S. aureus, with most bacteria detected within tyramide signal amplification (TSA)-negative infected macrophages [25]
Lc3bacteria associations were lost upon genetic (Figure 4A–C) and diphenyleneiodonium (DPI)-mediated chemical inhibition of NADPH oxidase (Figure 4D,F) in macrophagedepleted, neutrophil-enriched larvae. recruitment of Lc3 to S. aureus in both macrophages and these results suggest that Cyba and the neutrophils, and we propose that this response NADPH oxidase complex plays an important role in the represents Lc3-associated phagocytosis (LAP)

Summary

Introduction

Staphylococcus aureus is a highly successful human pathogen causing a wide range of diseases [1]. This microorganism is a leading cause of fatal bacteremia, with mortality rates reaching 30% [2] making multidrug resistance a particular concern [3]. Neutrophils, shown to play a role in immunity against S. aureus [7], can provide an intracellular niche for staphylococcal dissemination or persistence [8,9,10,11]. There is a need for a better understanding of the interactions of intracellular S. aureus with phagocytes to develop therapies based on immunomodulatory approaches [12]

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