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Phospholipase C-Related Catalytically Inactive Protein Participates in the Autophagic Elimination of Staphylococcus aureus Infecting Mouse Embryonic Fibroblasts

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.


Introduction
Autophagy, an evolutionarily conserved intracellular catabolic pathway in eukaryotic cells, delivers intracellular materials, such as damaged cytosolic components, into the lysosomes for degradation. Autophagy also plays an important role in eliminating invading pathogens by targeting them to the lysosome [1]. We recently reported that phospholipase C (PLC)-related catalytically inactive protein (PRIP) is a modulator for canonical autophagy [2]. However, it is unknown whether PRIP is involved in the autophagy-mediated clearance of intracellular pathogens.
In the autophagy pathway, a part of the cytoplasm is sequestered by autophagosomes, which in mammals are doublemembrane vacuoles characterized by the presence of specific structures containing microtubule-associated protein 1 light chain 3 (LC3), a homologue of yeast autophagy-related protein 8 (Atg8) [3,4]. 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, thus acquiring an acidic compartment for degradation [5].
Xenophagy, an autophagic pathway triggered by microbial infection to combat intracellular pathogens, is a host defense mechanism that serves to restrict bacterial growth and thus the infection of neighboring cells. For some pathogens, however, autophagosomes may be beneficial to the invading microbe in terms of supporting replication and pathogenesis, and thus promoting the pathogen life cycle [6]. Coxiella burnetti, Brucella abortus, and Porphyromonas gingivalis, for example, can reside in the autophagosome and utilize the nutrients sequestered by the vesicle for their growth and proliferation [7].
Staphylococcus aureus is a pathogen that causes serious diseases including pneumonia, endocarditis, and osteomyelitis, in addition to wound infection. The proliferation of accessory gene regulator (agr)-positive S. aureus strains has been reported to be markedly impaired in mouse embryonic fibroblasts (MEFs) deficient of the autophagy protein Atg5, indicating an essential role for the autophagic pathway in S. aureus replication [8]. On the other hand, Mauthe and his colleagues recently reported that intracellular agrpositive S. aureus is sequestered by autophagosome-like vacuoles decorated with WD repeat domain phosphoinositide-interacting protein 1 (WIPI-1), and that, like in the canonical autophagic pathway, these vacuoles become more abundant upon lysosomal inhibition. From these findings, the authors concluded that S. aureus is degraded by xenophagy [9]. To date, it remains unclear whether S. aureus is eliminated by the autophagic pathway or whether it is sequestered by autophagosomes, where it proliferates and then escapes into the cytoplasm.
We have recently demonstrated that PRIP regulates amino acid starvation-induced autophagic flux by binding to LC3 [2]. Therefore, we used MEFs prepared from PRIP-DKO mice and wild-type (WT) mice to examine whether PRIP is involved in the elimination of S. aureus by the autophagic pathway, and, thus, determined that PRIP is a novel modulator for maintenance of the innate immune system in non-professional phagocytic host cells.

Bacterial strains and growth conditions
S. aureus strains MW2 and ATCC 29213 were grown on tryptic soy agar (Becton Dickinson, Franklin Lakes, NJ). A colony from the agar plates was cultured in tryptic soy broth (Becton) at 37uC until the mid-logarithmic phase of growth before being used in the infection assays. S. aureus ATCC 29213 was transformed with a modified pS1-GFP plasmid [8] using the Gene Pulser II electroporation system (Bio-Rad, Hercules, CA). A positive clone on a chloramphenicol-containing tryptic soy agar plate was used in the subsequent experiments.

S. aureus infection of MEFs
In vitro MEF infection was performed as previously described [30], with minor modifications. Briefly, plasmid-transfected MEFs adhered to glass coverslips were supplied with fresh culture medium and incubated for 1 h. S. aureus (3610 5 cfu) was added to each dish containing MEFs. After 1.5 h incubation at 37uC, the cells were washed three times with culture medium, after which 100 mg/mL gentamicin was added to the culture medium to kill any extracellular S. aureus. Infected cells were incubated in culture medium containing 100 mg/mL gentamicin for the duration of the assay.

Immunostaining and confocal microscopy
To distinguish between intracellular and extracellular S. aureus, protein A, a cell wall protein of S. aureus, was immunostained under non-permeabilizing conditions, after which 49,6-diamidino-2-phenylindole (DAPI, Kirkegaard & Perry Laboratories, Inc., Gaithersburg, MD) was used to stain cell nuclei under permeabilizing conditions. S. aureus cells stained only with DAPI were identified as intracellularly localized bacteria. Briefly, infected cells were fixed with 4% paraformaldehyde in phosphate buffered saline (PBS) for 10 min, then treated with 50 mM NH 4 Cl/PBS for 10 min, and blocked with 2% bovine serum albumin/PBS for 30 min at room temperature. The cells were then incubated with mouse anti-protein A antibodies (1:1000) (P2921, Sigma-Aldrich) for 1 h at room temperature. After washing with PBS, the cells were incubated with Alexa 488-conjugated anti-mouse IgG antibodies (1:1000) for 30 min at room temperature. Cells were permeabilized with 0.2% Triton X-100/PBS for 10 min at room temperature and incubated with 1 mg/mL DAPI/PBS for 1 h at 37uC. To stain autolysosomes, red fluorescent protein (RFP)-LC3transfected MEFs were infected with green fluorescent protein (GFP)-expressing S. aureus (ATCC 29213) for 1.5 h. After washing with culture medium containing 100 mg/mL recombinant lysostaphin (Wako, Osaka, Japan), the cells were further incubated until 3 or 4.5 h post-infection. LysoTracker Blue DND-22 (100 nM, Life Technologies) was added to the medium 15 min before the end of the incubation period. After fixation with 4% paraformaldehyde for 10 min at room temperature, the samples were mounted with PermaFluor aqueous mounting medium (Thermo Fisher Scientific, Runcorn, UK). Images were acquired using a laser scanning confocal microscope (FV10i, Olympus, Tokyo, Japan). For analysis, at least 25 cells were randomly selected from three independent experiments.

Live-cell imaging
MEFs were grown on glass-bottom dishes (Matsunami Glass, Osaka, Japan) in culture medium without antibiotics. Following bacterial infection, dishes were mounted onto the microscope stage of a fluorescent microscope (BZ-9000, Keyence, Osaka, Japan) equipped with a humidified environment chamber (with 5% CO 2 at 37uC). Images were acquired every 90 sec using the BZ-9000 microscope equipped with a CFI Plan Apo 106 oil immersion objective (Nikon, Tokyo, Japan), and were processed using the Keyence Bz-II application (Keyence). For the visualization of autophagosomes and autolysosomes, we used the mRFP-GFP-LC3 plasmid, which labels autophagosomes in yellow due to the dual luminescence of RFP and GFP, and autolysosomes in red due to the quenching of green fluorescence in the autolysosomes [31].

Colony formation assay
Colony formation assays were performed as previously described [8], with some modifications. Briefly, MEFs were cultured to subconfluence in 35-mm diameter dishes, at which point they were supplied with fresh culture medium. S. aureus was then added to the culture medium at a multiplicity of infection (MOI) of 100:1. After 1.5 h, the cells were washed and treated with 100 mg/mL recombinant lysostaphin for 15 min to remove extracellular bacteria, after which the cells were washed three times to remove traces of lysostaphin and dead bacteria. A lysis buffer (PBS containing 0.1% Triton X-100) was added to some culture dishes (1.5 h control samples), while the remaining dishes were incubated until 3 h post-infection before washing and lysis. Lysates were diluted with PBS and spread onto tryptic soy agar plates for colony formation.

Statistical analysis
Statistical analyses were performed using unpaired two-tailed t tests with Welch's correction, or Kruskal-Wallis tests followed by Dunn's multiple comparison tests. A p-value of ,0.05 was considered statistically significant. Graphs show mean 6 standard error of the mean (SEM).

Results
S. aureus numbers in LC3-positive S. aureus-containing autophagosome-like vesicles (SAcVs) are higher in PRIP-DKO MEFs PRIP is a newly identified LC3-binding protein that regulates canonical autophagy [2]. Autophagy has recently been highlighted as an important component of the immediate autonomous cell defense mechanism by degrading intracellular pathogens. Therefore, to investigate whether PRIP affects bacterial infectioninduced autophagosome-like vacuole formation, mRFP-LC3 transfected MEFs were prepared from WT and PRIP-DKO mice and were infected with S. aureus ATCC 29213. Cells were then immunostained, followed by confocal microscopic observation.  [8]. On the other hand, in PRIP-DKO MEFs, the number of S. aureus was robustly increased during the 1.5-4.5 h period postinfection ( Figure 1C). The number of S. aureus was 5-and 15-fold higher than the control at 3 and 4.5 h post-infection, respectively. Thereby, more cytosol-located bacteria were consistently observed in PRIP-DKO MEFs, suggesting that S. aureus accumulates (and probably proliferates) in the LC3-positive autophagosome-like vacuoles in PRIP-DKO MEFs before escaping into the cytosol.
As observed in canonical autophagy [2], PRIP was co-localized to LC3-positive vacuolar membranes entrapping S. aureus, as determined using S. aureus-infected WT MEFs transiently transfected with GFP-PRIP and mRFP-LC3 ( Figure S1A). Rab7 is a member of the small GTPase Rab family, participating in the formation of pathogen-containing large vacuoles [32] and the fusion step of autophagosomes with lysosomes in canonical autophagy [33,34]. We therefore investigated the subcellular distribution of S. aureus using MEFs transiently transfected with mRFP-LC3 and GFP-Rab7. S. aureus cells were more frequently seen to have accumulated in Rab7

Autophagosomal maturation is suppressed in PRIP-DKO MEFs
The tandem fluorescent-tagged LC3 (mRFP-GFP-LC3) is a convenient tool to monitor autophagic flux based on the different pH stabilities of the EGFP and mRFP fluorescent proteins. To elucidate the role of PRIP in the autophagy maturation process, MEFs transiently expressing mRFP-GFP-LC3 were infected with S. aureus. As shown in Figure Figure 2B).
To investigate whether the fusion of autophagosomes with lysosomes in canonical autophagy is also disturbed in PRIP-DKO MEFs, an autophagy flux assay was performed using mRFP-GFP-LC3. The number of GFP and RFP-double positive LC3 dots was increased in PRIP-DKO MEFs before and after 1 h of nutrient starvation, compared with WT MEFs (Figure S2A), consistent with our previously reported observations [2]. However, the starvation triggered a significant decrease in the number of RFP-single positive LC3 dots in PRIP-DKO MEFs than that in WT MEFs, indicating that PRIP positively regulates the fusion of autophagosomes with lysosomes.
Acidification of autophagosomes is prevented in S.

aureus-infected PRIP-DKO MEFs
To confirm the existence of autolysosome-like acidic vacuoles, we analyzed the acidification of SAcVs in MEFs using Lyso-Tracker blue DND-22, a probe for acidic compartments. LC3positive SAcVs stained with LysoTracker were observed 3 h post-infection in WT MEFs, but there were fewer apparent in PRIP-DKO MEFs ( Figure 3A). The acidic compartments were then counted; in WT MEFs, approximately 10% and 30% of LC3-positive SAcVs were stained with LysoTracker at 3 h and 4.5 h post-infection, respectively. However, in PRIP-DKO MEFs, there were only about 2% and 10% at 3 h and 4.5 h, respectively ( Figure 3B). The acidification process of SAcVs is, therefore, likely attenuated in PRIP-DKO MEFs, since there is no quantitative difference in lysosomes between WT and PRIP-DKO MEFs before the infection ( Figure S2B).
To evaluate the elimination of S. aureus in PRIP-DKO MEFs at a lower magnitude than that in the control, a colony count assay was performed using cell homogenates of S. aureus-infected MEFs collected 1.5 h and 3 h after the infection. No significant difference between the mean numbers of S. aureus colonies between WT and PRIP-DKO MEFs was observed at 1.5 h postinfection, but the ratio of the colony number at 3 h post-infection relative to that at 1.5 h post-infection was significantly higher in PRIP-DKO cells than in WT cells ( Figure 3C), suggesting that S. aureus infected into PRIP-DKO MEFs proliferated in the autophagosomes.
The lifetime of autophagosome-like vacuoles is prolonged in PRIP-DKO MEFs PRIP appears to positively regulate the fusion process of autophagosomes with lysosomes, and thus, the acidification process would be markedly inhibited in the PRIP-DKO MEFs. To monitor the acidification process of SAcVs, we finally performed live-cell imaging using double-tagged LC3 to monitor the fusion of autophagosomes with lysosomes. MEFs were transiently transfected with the mRFP-GFP-LC3 plasmid, followed by infection with S. aureus (agr-positive strain MW2). As shown in Figure 4A, mRFP-GFP-LC3 appeared as ring-shaped, yellowish structures from about 3 h post-infection in both WT and PRIP-DKO genotypes. From 3 h to 6.5 h after the infection in WT cells, newly formed RFP(+)GFP(+) autophagosome-like structures were frequently converted into RFP(+)GFP(2) structures (Movie S1). In PRIP-DKO cells, however, rapidly formed yellow-colored autophagosomes rarely changed to red (Movie S2). Using the time lapse movies, we measured how long the yellowcolored period of vesicles lasted, i.e., the time required for change from yellow to red. The mean ''yellow periods'' were less than 120 min in 90% of WT cells. In PRIP-DKO cells, ''yellow periods'' were significantly longer: in ,40% and 50% of cells, the periods were .180 min and between 120-180 min, respectively ( Figure 4B, C).

Discussion
Autophagy is not only a cytosolic catabolic process, but also an innate defense mechanism against invading pathogenic bacteria in eukaryotic cells. We have previously reported that PRIP binds LC3, a pivotal component of the autophagic machinery, and is implicated in starvation-induced canonical autophagy [2]. In this study, we demonstrate that PRIP participates in the elimination of S. aureus in non-professional phagocytic cells by promoting the acidification of autophagic vacuoles; i.e., PRIP promotes the process of the fusion between autophagosomes and lysosomes. To our knowledge, this is the first report on the important role of LC3-mediated autophagic flux via PRIP in host innate immunity against bacterial infection.
In yeast, Atg8 is crucial to the regulation of the autophagic process, specifically in the elongation of the phagophore membrane by mediation of hemifusion events [35,36]. At least eight mammalian Atg8 orthologs, LC3A, LC3B, LC3B2, LC3C, GABARAP, GABARAPL1 (GEC1), GABARAPL2 (GATE-16), and GABARAPL3 have been identified [37]. The various roles of LC3B have been extensively studied in the process of mammalian autophagy. However, the functions of the other mammalian Agt8 orthologs in autophagy are not fully understood, despite a recent report of the involvement of GABARAP/GATE-16 in autophagosome biogenesis [38]. GABARAP was identified as a PRIPbinding protein by yeast two-hybrid analysis [19], and then we examined that LC3 [2] and GATE-16 (unpublished data) were also able to bind PRIP. In this study, we demonstrated the colocalization of PRIP with LC3 on vacuolar membranes containing S. aureus and used LC3 as a tracer protein to visualize autophagosomes and to monitor the autophagy flux in S. aureusinfected cells.
Compared with WT MEFs, the number of S. aureus cells entrapped in autophagosome-like vesicles was significantly increased in PRIP-DKO MEFs, and the S. aureus proliferation efficiency was upregulated in these cells. Yamaguchi et al. reported that Rab7 localizes group A streptococcus-containing autophagosome-like vacuoles (GcAV) and mediates the early phase of GcAV formation in NIH3T3 cells [32]. We investigated the colocalization of SAcV with the small GTPase Rab7. Our findings showed that approximately 25% of S. aureus entrapped in LC3and/or Rab7-positive vacuoles was observed in LC3-single positive vacuoles (results not shown), indicating that, unlike GcAV formation, Rab7 may not be essential for the early phase of SAcV formation in MEFs. Therefore, a role for Rab7 in the early phase of SAcV formation is currently unknown. Rab7 is also reported to be directly involved in the fusion process of late endocytic structures with lysosomes [39], and is required for fusion of autophagosomes with lysosomes in the canonical autophagy pathway [33,34,40]. Our results showing that more S. aureus were entrapped in autophagosome-like vacuoles in PRIP-DKO MEFs indicate that PRIP may be participating in the proper formation of autophagosomes in collaboration with Rab7.
To determine whether PRIP may possibly participate in the later steps of autophagy, including fusion between SAcV and lysosome, we performed autophagy flux assays using an mRFP-GFP-tagged LC3 plasmid. The acidification process in LC3positive SAcVs in the PRIP-DKO MEFs was significantly inhibited. Similar effects were observed in autophagy flux assays using another agr-positive S. aureus strain, MW2. We also elucidated the involvement of PRIP in the starvation-induced autophagosome/lysosome fusion process. Based on these results, we concluded that PRIP participates in the fusion of autophagosomes with lysosomes, and that insufficient autophagosome/ lysosome fusion in PRIP-DKO MEFs promotes agr-positive S. aureus replication in autophagosomes, leading to propagation of the bacteria.
The ability of autophagy to either eliminate pathogenic organisms or to provide a niche for their replication depends on the nature of the pathogen [41]. It has been reported that intracellular agr-positive, but not agr-negative S. aureus, becomes sequestered by and replicates in autophagosome-like vesicles following autophagosome/lysosome fusion blockage in HeLa cells [8]. However, it was recently reported that intracellular agrpositive S. aureus was efficiently entrapped in WIPI-1 positive autophagosome-like vesicles and targeted for lysosomal degradation in non-professional phagocytic cells (human osteosarcoma U2OS cells, ATCC) [9], indicating that agr-positive S. aureus is eliminated by the autophagy system followed by degradation in the lysosome. The fate of S. aureus in a host cell may partly depend on a balance between the ability of the pathogen to escape from autophagosomes and the ability of host cells to eliminate the pathogen by autophagy. In our experiments, approximately 30% of SAcVs in WT MEFs were fused with lysosomes, which is consistent with the previously reported findings of Schnaith et al.
WIPI-1-decorated autophagosome-like vacuoles entrapping S. aureus induce the degradation of bacteria by lysosomes [9]. WIPI-1 and WIPI-2 are the mammalian orthologs of yeast Atg18, and contain a specific phospholipid-binding region [42]. Moreover, tectonin domain-containing protein 1 (TECPR1) regulates the selective autophagy pathway against Shigella in combination with WIPI-2 [43]. TECPR1 has also been shown to be involved in the fusion process of autophagosomes with lysosomes in the canonical autophagy pathway, rigorously mediating the process by associating with both the Atg12-Atg5 conjugate and phosphatidylinositol 3-phosphate [PtdIns(3)P] [44]. Indirect downregulation of PtdIns(3)P levels by Listeria phospholipases, phospholipases C A and B, protects Listeria from autophagy-mediated clearance [45]. We have shown that PRIP has a pleckstrin homology domain that binds phosphoinositides, and an X-Y phospholipase C catalyticlike domain with no catalytic activity [11,12]. A recombinant fulllength PRIP1 and a recombinant X-Y phospholipase C catalyticlike domain of PRIP were found to be able to bind phosphoino- sitides including PtdIns(3)P (unreported observation). It is therefore also tempting to speculate that PRIP may regulate autophagy maturation by affecting the functions of TECPR1 through phosphoinositide metabolism in the process of bacterial elimination. Further experiments are needed to fully elucidate the PRIPregulated autophagosome/lysosome fusion mechanism.
In this study, we show that PRIP is involved in the entrapment of pathogens by LC3-positive autophagosome-like vacuoles and contributes to the autophagic clearance of bacterial pathogens as part of an innate defense system in eukaryotic non-professional phagocytic cells. In PRIP-DKO cells, S. aureus can escape the host defense system via autophagy due to the autophagosome/lysosome fusion process being disabled. Revealing the functional mechanisms of PRIP-mediated elimination of S. aureus from infected host cells gives us new insight into potentially effective treatments for infectious diseases.