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

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.

Figure 1. PRIP deficiency in mouse embryonic fibroblasts (MEFs) induces S. aureus accumulation in autophagosome-like vacuoles.

(A–C) Comparison between S. aureus-containing autophagosome-like vacuoles in wild-type (WT) and PRIP double-knockout (DKO) MEFs. MEFs transiently expressing RFP-LC3 were incubated with S. aureus (ATCC 29213). After 1.5 h incubation, cells were washed with 100 µg/mL of gentamicin-containing medium and further incubated with gentamicin-containing medium until 3 or 4.5 h post-infection. Extracellular (ext.) S. aureus cells were immunostained with anti-protein A antibody (green), and intracellular S. aureus cells were defined as DAPI single-positive signals (A). Images at 3 h post-infection obtained by confocal laser microscopy are shown. Enlarged images of the boxed areas of the left image (scale bar: 10 µm) are shown in the middle and right images (scale bar: 2 µm). Representative images from three independent experiments are shown. The graph in (B and C) shows the numbers of intracellular S. aureus cells and of S. aureus cells localized to LC3-positive autophagosome-like vacuoles per cytosol area (0.01 mm²), respectively. Values represent means ±SEM (n = 20 cells analyzed at each time-point). Reproducible results were obtained from three independent experiments. *p<0.05 and ***p<0.001 relative to corresponding WT values.

Figure 2. Impairment of autophagosomal maturation in PRIP-DKO MEFs.

(A, B) MEFs transiently expressing mRFP-GFP-LC3 were incubated with S. aureus (ATCC 29213) for 3 h (A) and 4.5 h (B). Images were obtained by confocal laser microscopy. A set of representative images from three independent experiments using wild-type (WT) and PRIP-DKO (DKO) MEFs are shown. Bacteria were stained with DAPI (blue). LC3-positive autophagosome-like vacuoles with RFP(+)GFP(−) signal indicate the formation of autolysosomes (arrowheads). Scale bars represent 20 µm in the left panel and 5 µm in the three right panels. (C) The graph shows the ratio of the number of S. aureus cells entrapped in RFP(+)GFP(−) vacuoles vs. the number of S. aureus cells entrapped in RFP(+) vacuoles. Values are expressed as means ±SEM (n = 60 cells for each genotype and each infection time from three independent experiments). WT, 5.4±1.3% (3 h) and 25.7±3.8% (4.5 h); DKO, 0.9±0.3% (3 h) and 3.7±1.3% (4.5 h). *p<0.05, ***p<0.001.

Figure 3. Decreased LysoTracker-positive vacuoles containing S. aureus in PRIP-DKO MEFs.

(A) Co-localization of LC3-positive S. aureus-containing autophagosome-like vacuoles (SAcVs) and lysosomal marker (LysoTracker). PRIP-DKO (DKO) and wild-type (WT) MEFs transiently expressing mRFP-LC3 were infected with GFP-expressing S. aureus (ATCC 29213), after which extracellular bacteria were killed by lysostaphin treatment 1.5 h post-infection. The images were acquired using a fluorescent microscope. Scale bars: 20 µm (left) and 5 µm (right). Asterisks and arrowheads show LC3-positive SAcVs staining with and without LysoTracker, respectively. (B) Comparison of the number of S. aureus in LysoTracker-positive vacuoles vs. the number of S. aureus in mRFP-LC3-positive signals from three independent experiments. WT, 9.6±4.5% (3 h, n = 25) and 28.6±4.7% (4.5 h, n = 28); DKO, 1.4±0.6% (3 h, n = 26) and 11.9±4.3% (4.5 h, n = 25). (C) Colony count assay. Homogenates of S. aureus-infected MEFs prepared 1.5 or 3 h post-infection were plated on tryptic soy agar and colony numbers were counted after 16 h incubation at 37°C. Graph represents relative number of colonies (the number of colonies at 3 h vs. the number of colonies at 1.5 h). Data represent the means ±SEM (n = 3; WT, 4.3±1.3%; DKO, 9.0±3.3%). *p<0.05, **p<0.01.

Figure 4. Impairment of autophagic flux in PRIP-DKO MEFs.

(A–C) Time-lapse analysis of S. aureus-infected MEFs. MEFs transiently transfected with the mRFP-GFP-LC3 plasmid were cultured with S. aureus (MW2) for 1.5 h, after which extracellular bacteria were killed by lysostaphin treatment. In wild-type (WT) cells (upper panels), a RFP(+)GFP(+)-LC3-labeled autophagosome (arrowhead) appeared at 3 h 30 min post-infection and changed to a RFP(+)GFP(−)-LC3-labeled autolysosome at 4 h 21 min post-infection (asterisk). In PRIP-DKO images (DKO, lower panels), an RFP(+)GFP(+)-LC3-labeled autophagosome (arrowhead) appeared at 4 h 30 min post-infection, and the RFP(+)GFP(+) signal (yellow) was not altered during the experiment (terminated at 6 h 24 min post-infection). The closed bars in graphs (B) represent the elapsed time for RFP(+)GFP(+) autophagosome formation in WT (upper) and PRIP-DKO (lower) MEFs. Asterisks in (B) indicate the experiment replicates corresponding to images in (A). The graph in (C) shows the proportion of the elapsed times (<120 min: dark shading, 120–180 min: hatched shading, and >180 min: no shading) in WT and PRIP-DKO cells.