Phospholipase C-related catalytically inactive protein participates in the autophagic elimination of Staphylococcus aureus infecting mouse embryonic fibroblasts.

Kae Harada-Hada,Motoyuki Sugai,Takashi Kanematsu,Kana Harada,Junzo Hisatsune,Satoshi Asano,Michinaga Ogawa,Isei Tanida,Fuminori Kato,Masato Hirata,Maasaki Komatsu

doi:10.1371/journal.pone.0098285

Abstract

Autophagy is an intrinsic host defense system that recognizes and eliminates invading bacterial pathogens. We have identified microtubule-associated protein 1 light chain 3 (LC3), a hallmark of autophagy, as a binding partner of phospholipase C-related catalytically inactive protein (PRIP) that was originally identified as an inositol trisphosphate-binding protein. Here, we investigated the involvement of PRIP in the autophagic elimination of Staphylococcus aureus in infected mouse embryonic fibroblasts (MEFs). We observed significantly more LC3-positive autophagosome-like vacuoles enclosing an increased number of S. aureus cells in PRIP-deficient MEFs than control MEFs, 3 h and 4.5 h post infection, suggesting that S. aureus proliferates in LC3-positive autophagosome-like vacuoles in PRIP-deficient MEFs. We performed autophagic flux analysis using an mRFP-GFP-tagged LC3 plasmid and found that autophagosome maturation is significantly inhibited in PRIP-deficient MEFs. Furthermore, acidification of autophagosomes was significantly inhibited in PRIP-deficient MEFs compared to the wild-type MEFs, as determined by LysoTracker staining and time-lapse image analysis performed using mRFP-GFP-tagged LC3. Taken together, our data show that PRIP is required for the fusion of S. aureus-containing autophagosome-like vacuoles with lysosomes, indicating that PRIP is a novel modulator in the regulation of the innate immune system in non-professional phagocytic host cells.

Highlights

Autophagy, an evolutionarily conserved intracellular catabolic pathway in eukaryotic cells, delivers intracellular materials, such as damaged cytosolic components, into the lysosomes for degradation
To investigate whether phospholipase C-related catalytically inactive protein (PRIP) affects bacterial infectioninduced autophagosome-like vacuole formation, mRFP-light chain 3 (LC3) transfected mouse embryonic fibroblasts (MEFs) were prepared from WT and PRIP-DKO mice and were infected with S. aureus ATCC 29213
In the WT MEFs, a peak in the number of S. aureus entrapped in the LC3-positive vacuoles was at 3 h post-infection (Figure 1C), consistent with the results shown by Schnaith et al using HeLa cells [8]

Summary

Introduction

An evolutionarily conserved intracellular catabolic pathway in eukaryotic cells, delivers intracellular materials, such as damaged cytosolic components, into the lysosomes for degradation. We recently reported that phospholipase C (PLC)-related catalytically inactive protein (PRIP) is a modulator for canonical autophagy [2]. It is unknown whether PRIP is involved in the autophagy-mediated clearance of intracellular pathogens. The multiple steps of autophagy generally consist of the formation of a phagophore, which is the membrane precursor of the autophagosome; the elongation and closure of the membrane; and the maturation of autophagosomes by fusion with lysosomes, resulting in the formation of autolysosomes, acquiring an acidic compartment for degradation [5]

Methods

Results

Conclusion