Molecular Microbiology (2014) ■

doi:10.1111/mmi.12830

Protease IV, a quorum sensing-dependent protease of Pseudomonas aeruginosa modulates insect innate immunity Su-Jin Park,† Soo-Kyoung Kim,† Yong-In So, Ha-Young Park, Xi-Hui Li, Doo Hwan Yeom, Mi-Nan Lee, Bok-Luel Lee and Joon-Hee Lee* College of Pharmacy, Pusan National University, Pusan, 609-735, Korea.

Summary In Pseudomonas aeruginosa, quorum sensing (QS) plays an essential role in pathogenesis and the QS response controls many virulence factors. Using a mealworm, Tenebrio molitor as a host model, we found that Protease IV, a QS-regulated exoprotease of P. aeruginosa functions as a key virulence effector causing the melanization and death of T. molitor larvae. Protease IV was able to degrade zymogens of spätzle processing enzyme (SPE) and SPE-activating enzyme (SAE) without the activation of the antimicrobial peptide (AMP) production. Since SPE and SAE function to activate spätzle, a ligand of Toll receptor in the innate immune system of T. molitor, we suggest that Protease IV may interfere with the activation of the Toll signaling. Independently of the Toll pathway, the melanization response, another innate immunity was still generated, since Protease IV directly converted Tenebrio prophenoloxidase into active phenoloxidase. Protease IV also worked as an important factor in the virulence to brine shrimp and nematode. These results suggest that Protease IV provides P. aeruginosa with a sophisticated way to escape the immune attack of host by interfering with the production of AMPs.

Introduction Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen that is widely found in diverse environments (Driscoll et al., 2007; Coggan and Wolfgang, 2012). It causes a variety of infections in patients with burn wounds, cystic fibrosis, pneumonia, cancer, and acquired immunodeficiency syndrome (AIDS) (Engel et al., 1998a; Hancock and Speert, 2000; Willcox et al., 2008). P. aerAccepted 11 October, 2014. *For correspondence. E-mail joonhee@ pusan.ac.kr; Tel. (+82) (0)51 510 2821; Fax (+82) (0)51 513 6754. † These authors equally contributed to this work.

© 2014 John Wiley & Sons Ltd

uginosa also infects higher plants like Arabidopsis, sweet basil, and lettuce (Rahme et al., 1995; 1997; Walker et al., 2004; Starkey and Rahme, 2009), insects like the moth and fruit fly (Jander et al., 2000; Miyata et al., 2003; Lutter et al., 2012), and a nematode, Caenorhabditis elegans (Mahajan-Miklos et al., 1999). Many virulence factors of P. aeruginosa, such as alginate, proteases, toxins, phenazines, pyocyanin, and lipopolysaccharide play important roles in the infection (Coggan and Wolfgang, 2012; Rada and Leto, 2013). While the virulence factors required for the infection of insects, plants, and animals have been suggested to be similar (Rahme et al., 1995; 2000; Jander et al., 2000), the host-related association or specificity of the virulence factors are not fully understood. Many hosts have been studied to better understand the relation between the P. aeruginosa virulence factors and host immunity (Tan et al., 1999; Jander et al., 2000). Insects have been considered as a favorable model system due to their fast growth and convenience for biological analyses. For defense against pathogenic bacterial invasion, invertebrates mainly depend on innate immune reactions like coagulation, melanization, the activation of Toll receptor, and complement-like reactions. These are generally initiated from the recognition of bacteriaspecific materials, so-called PAMPs (pathogen-associated molecular patterns) such as peptidoglycan (PG), lipopolysaccharide, lipoteichoic acid, and glucan by the specific recognition proteins (pattern recognition receptors; PRRs) (Iwanaga and Lee, 2005; Cerenius et al., 2010; Tsakas and Marmaras, 2010). This recognition signal of PAMPs is amplified via proteolytic cascade similar to the vertebrate complement system. Well-known proteolytic cascades are the Toll pathway to produce antibacterial peptides (AMPs), and the prophenoloxidase (proPO) system to synthesize melanin pigments (Cerenius et al., 2008; 2010; Tsakas and Marmaras, 2010). In insects, the host defense by the innate immunity is mainly exerted through the AMP production and the PO activation (Iwanaga and Lee, 2005; Cerenius et al., 2010; Tsakas and Marmaras, 2010). A mealworm beetle, Tenebrio molitor was recently used for studies of the identification of host components involved in the activation mechanism of the Toll signaling and melanization cascades (Kan et al., 2008; Kim et al., 2008). In T. molitor, these cascades are triggered by the recognition of bacterial Lys-type PG by the PGRP (PG

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recognition protein) and the recognition signals are amplified by a series of serine proteases, such as modular serine protease (MSP), SPE-activating enzyme (SAE), and spätzle processing enzyme (SPE), which converts pro-spätzle to spätzle that is a ligand of the Toll receptor (Kan et al., 2008). The active SPE also cleaves zymogen forms of serine protease homologue 1 (SPH1) and proPO into their active forms of melanin complexes, which lead the melanin synthesis (Kan et al., 2008; Kim et al., 2008). Although the recognition signals of these pathways are specifically amplified by host protease system, pathogenic bacteria also produce many proteases as virulence factors during infection (Ingmer and Brondsted, 2009). So, it could be hypothesized that bacterial proteases modulate the melanization and AMP production by cleaving or degrading the host protease components. Some studies reported several examples about this postulation. A major cysteine protease of Streptococcus pyogenes, an important human pathogen degraded multiple complement factors of human, affecting the survival of bacteria in serum (Honda-Ogawa et al., 2013). Protease-activated receptors (PARs) that belong to a G protein-coupled receptor (GPCR) family are activated by host serine proteases via specific cleavage, but during inflammation, PARs can be activated by various proteases released from pathogens, such as a fungus, Alternaria (Shpacovitch et al., 2007; Matsuwaki et al., 2009; Yike, 2011). These interventions by pathogen-produced proteases can modulate host immunity between the initial PAMP recognition and final immune effectors. Pseudomonas aeruginosa also produces many extracellular proteases that are possibly involved in pathogenicity, for examples, elastase (LasB, pseudolysin), alkaline protease (AprA, aeruginolysin), LasA (staphylolysin), LasD (staphylolysin), small protease (PasP), large extracellular protease (LepA), and Protease IV (lysyl endopeptidase) (Blackwood et al., 1983; Traidej et al., 2003; Kida et al., 2011; Lee et al., 2011). Among them, Protease IV has been considered an important virulence factor and suggested to be involved in some Pseudomonas infections such as corneal infection (Engel et al., 1998b; Caballero et al., 2004). Mature Protease IV (encoded by piv, PA4175) is a 26 kDa exoprotease but its gene-encoded full length is 48 kDa, which is initially expressed in cytoplasm (called as a pre-proenzyme), then cleaved to 45 kDa (called as a proenzyme), and further processed to 26 kDa mature protease with secretion (Traidej et al., 2003). A transcriptome analysis showed that piv is strongly induced by quorum sensing (QS), a cell density-dependent cell-to-cell communication mechanism (Schuster et al., 2003). Like many other pathogenic bacteria, P. aeruginosa expresses a large number of genes related to the production of virulence factors, motility, and biofilm formation through QS regulation (Davies et al., 1998; Bassler, 2002). Most Gram-

negative bacteria including P. aeruginosa use acyl homoserine lactones (acyl-HSLs) as signal molecules for the QS regulation and have signal-receptor systems. P. aeruginosa has the acyl-HSL-based QS systems consisting of LasI-R, RhlI-R and QscR. LasI and RhlI synthesize the N-3-oxododecanoyl-L-HSL (3OC12-HSL) and N-butyryl-L-HSL (C4-HSL), which specifically bind to their cognate receptors, LasR and QscR (for 3OC12-HSL), and RhlR (for C4-HSL) respectively (Fuqua et al., 2001; Schuster et al., 2004; Lee et al., 2006; Schuster and Greenberg, 2006). The signal-receptor complexes regulate the transcription of their target genes; LasR and RhlR control the genes related to the production of extracellular proteases, rhamnolipid, and some toxic secondary metabolites, and QscR regulates PA1897 and its own gene, qscR (Davies et al., 1998; Schuster et al., 2003; Lee et al., 2006; Lequette et al., 2006; Ha et al., 2012). QscR can respond to non-P. aeruginosa signals through its relaxed signal specificity (Lee et al., 2006; Ha et al., 2012). Since many virulence factors are regulated by these QS systems, researchers have tried to control the virulence of P. aeruginosa by modulating the QS signaling (Rasmussen and Givskov, 2006; Kim et al., 2009; Kohler et al., 2010). In this study, we intended to unravel the link between the QS regulation of P. aeruginosa and its effect on virulence in T. molitor as a host model. We first found that a QS-dependent secreted protein factor of P. aeruginosa could induce the melanization and killing of T. molitor, and further explored which factor was responsible for the virulence. Here, we provide the evidence that Protease IV of P. aeruginosa is a key virulence factor that may modulate the host innate immune system.

Results A QS-dependently secreted protein factor of P. aeruginosa is crucially involved in the melanization and killing of T. molitor larvae To investigate the virulence factor of P. aeruginosa responsible for the melanization and killing of T. molitor larvae, the cell-free culture supernatant (CCS) of wild type (PAO1) was prepared and injected into T. molitor larvae, which strongly induced the melanin synthesis (Fig. 1A). Since the control fraction that passed through the 10 kDa filter failed to induce the melanization, it was apparent that extracellular macromolecules whose molecular weights are higher than 10 kDa are responsible for the melanization. When we treated the CCS with heat at 95°C for 10 min before injection, it failed to induce the melanin synthesis, indicating that the responsible factor is most likely protein (Fig. 1B). As well as the melanization, the CCS showed remarkable killing effect on T. molitor larvae, compared with the control fraction (Fig. 1C). Consistently, the killing © 2014 John Wiley & Sons Ltd, Molecular Microbiology

Role of Protease IV in the Pseudomonas virulence 3

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Fig. 1. Secreted protein factor from P. aeruginosa induced the melanization and killing of T. molitor larvae. A and B. (A) The cell-free culture supernatant (CCS) prepared from P. aeruginosa was concentrated 10 times by ultra-filtration (cut-off size; 10 kDa) and injected into T. molitor larvae, and melanin synthesis was investigated. The pass-through fraction in the ultra-filtration was used as a control (Con). (B) The CCS was heat-treated at 95°C for 10 min and injected into T. molitor larvae to see the melanization at 40 h. C. Survival of the larvae was investigated in the same condition.

effect of CCS disappeared with heat treatment, confirming that the factor is a secreted protein(s) (Fig. 1C). Since it has been well documented that QS is very important in the virulence of P. aeruginosa, we investigated whether the production of the factor is QS-dependent, or not. When the CCS was prepared from QS mutant strain, MW1 (lasI− rhlI−) that is incapable of producing the QS signals, and injected into T. molitor larvae, it failed to induce the melanin synthesis (Fig. 2A). The killing effect of the CCS of MW1 was also much alleviated compared with that of wild type (Fig. 2B). In addition, these phenotypes were well complemented by the exogenous supplement of the synthetic QS signal molecules, 3OC12-HSL and C4-HSL © 2014 John Wiley & Sons Ltd, Molecular Microbiology

during cultivation (Fig. 2A and B). As expected, all heatinactivated CCSs and control fractions failed to induce the melanization and killing effects (Fig. 2A and B). These results consistently demonstrated that the factor responsible for the melanin synthesis and killing of larvae is a secreted protein that is expressed in a QS-dependent manner. Protease IV, a lysyl endopeptidase is responsible for the virulence in T. molitor larvae Since the early part of the insect innate immunity is amplified by a proteolytic cascade, we suspected that one of the

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Fig. 2. The expression of the factor was QS-dependent. A. The CCSs were prepared from wild type (PAO1, left column) and a QS mutant (lasI−, rhlI−) (MW1, middle column), concentrated 10 times by ultra-filtration, and injected into T. molitor larvae. In right column, synthetic QS signals, C4-HSL and 3OC12-HSL were supplemented to medium at 5 μM during the cultivation of MW1, and the CCS was prepared to be injected to T. molitor larvae. In top row, the pass-through fraction in the ultra-filtration was used as control (Con). In bottom row, the CCSs were heat inactivated at 95°C for 10 min before injection. B. Survival of T. molitor larvae was counted in the same condition.

exoproteases produced by P. aeruginosa intervened in the host immunity to exert virulence. Based on genome database and QS-transcriptome analysis of P. aeruginosa (Schuster et al., 2003), we screened the proteases that are QS-dependently induced and extracellularly secreted. Eight proteases (LasA, LasB, Protease IV, PA2939, PA1249, PA0355, PA4171, and PA3535) were found in this

category (Table 1). They were cloned into pJN105 and expressed in P. aeruginosa under the control of arabinose. When the CCSs prepared from the proteaseoverexpressing P. aeruginosa cells were injected into T. molitor larvae, the CCS of the cells expressing Protease IV strongly induced the melanin synthesis even without the concentration of the CCS (Fig. 3A). It also showed strong © 2014 John Wiley & Sons Ltd, Molecular Microbiology

Role of Protease IV in the Pseudomonas virulence 5

Table 1. Extracellular proteases that are induced by QS system. Gene number

Gene name and product description

PA0355 PA1249 PA1871

pfpI, protease PfpI aprA, alkaline metalloproteinase precursor lasA, LasA protease precursor, staphylolytic protease preproenzyme pepB, probable aminopeptidase eprS, probable serine protease lasB, elastase, neutral metalloproteinase Probable protease Piv (prpL), Protease IV, probable endoproteinase Arg-C precursor, Pvds-regulated endoprotease, lysyl class

PA2939 PA3535 PA3724 PA4171 PA4175

killing effect (Fig. 3B), indicating that Protease IV is the factor responsible for the melanization and killing of T. molitor larvae. To confirm this result, the Protease IV-deficient mutant (piv−) was tested for the melanin synthesis and killing activity. The CCS prepared from piv− mutant failed to melanize and kill T. molitor larvae (Fig. 4A and B). This attenuation of the piv− mutant was fully complemented with the episomal expression of Proteases IV (Fig. 4A and B). Finally, when we parenterally infected the live cells to T. molitor larvae by the microsyringe injection, the piv− mutant showed the significantly attenuated virulence (Fig. 4C), like in the CCS experiments. This result was confirmed by complementation test in that only PIV-expressing plasmid was able to restore the virulence of the piv− mutant (Fig. 4D). We note that there is no significant difference in growth between wild type and the piv− mutant. A previous study reported that Protease IV of P. aeruginosa can be inhibited by N-p-tosyl-L-chloromethyl ketone (TLCK), a serine protease inhibitor (Engel et al., 1998a). When we treated the CCS prepared from the Protease IV-overexpressing PAO1 with TLCK and inject it to T. molitor larvae, the melanization and killing of the larvae was significantly reduced (Fig. 5A and B). This result also demonstrates that Protease IV is mainly responsible for the melanization and killing of T. molitor larvae. Similarly, when the live cells of Protease IV-overexpressing P. aeruginosa was treated with TLCK, virulence was much attenuated (Fig. 5C), confirming the important role of Protease IV in virulence. We note that heat treatment inhibited the CCSmediated virulence more dramatically than the TLCK treatment. This may be due to minor contribution of other proteases such as PA3535 or PA4171 that contributed to virulence marginally when overexpressed (Fig. 3B). We think that while TLCK specifically inhibits a certain set of proteases including Protease IV, but heat inactivates all proteases. We also note that the total protease activity was not significantly altered by the piv− mutation, while the over© 2014 John Wiley & Sons Ltd, Molecular Microbiology

expression of Protease IV augmented the total protease activity (Supplementary Fig. S1). This is likely due to the redundancy of exoproteases, since P. aeruginosa produce many proteases besides Protease IV. No reduction in total protease activity but significant alleviation of virulence demonstrates that Protease IV specifically functions as a virulence factor on T. molitor. For more direct confirmation, we prepared recombinant Protease IV to directly inject into T. molitor larvae. We fused the histidine tag to C-terminus of Protease IV for easy purification, and overexpressed the His-tagged Protease IV (PIVh) in P. aeruginosa, since Protease IV has signal peptide at N-terminus and undergoes specific multiple processing in P. aeruginosa during secretion (Traidej et al., 2003). We purified the secreted PIVh from the culture supernatant and confirmed its purity and protease activity (Fig. 6A and B). The molecular mass of Protease IV is 26 kDa but the purified PIVh was slightly bigger than 26 kDa due to His-tag. When the PIVh was injected into T. molitor larvae, it induced the melanin synthesis in dosedependent manner (Fig. 6C and D). PIVh also killed the T. molitor larvae in a similar pattern (Fig. 6E), but the heatinactivated PIVh failed to induce the melanization and killing effect, indicating that the melanin synthesis and killing effect was apparently caused by the protease activity of Protease IV. Taken together, these results suggested that Protease IV is a crucial virulence factor of P. aeruginosa for infection to T. molitor larvae. Protease IV blocks the production of antimicrobial peptides (AMPs) by interrupting Toll signaling pathway of T. molitor larvae Protease IV is known to have a lysine-specific endopeptidase activity (Traidej et al., 2003). We investigated whether this activity could modulate the Toll proteolytic cascade of host innate immune system (see Fig. 11). Using in vitro reconstitution and Western analysis, we tested the reaction between the purified PIVh and the protease components of the T. molitor innate immune system, such as SAE, SPE, SPH1, or PO which function as triggers of AMP production and melanin synthesis. When SAE or SPE were mixed with the purified PIVh, they were degraded to a fragment other than the active form or fully degraded (Fig. 7A and B), suggesting that Protease IV of P. aeruginosa can interfere with the proteolytic cascade of T. molitor. Consistent with this, when we injected the purified PIVh to T. molitor larvae, the AMPs were not produced (Fig. 7C). An independent study about the P. aeruginosa virulence with another insect, Galleria mellonella showed that a sublethal concentration of elastase B (LasB) elicited the humoral immune response of the host (Andrejko and Mizerska-Dudka, 2011). We also purified (Fig. 6A) and injected LasB into

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Fig. 3. Protease IV was responsible for the melanin synthesis and killing. A. The CCSs were prepared from various exoprotease-overexpressing P. aeruginosa cells and directly injected into T. molitor larvae without concentration to see the melanization. Same volume of insect saline (IS) and the CCS prepared from empty vector-carrying cells (vector control; VC) were injected for controls. PIV, Protease IV. B. Survival of the larvae was investigated in the same condition. VC, vector control; PIV, Protease IV.

© 2014 John Wiley & Sons Ltd, Molecular Microbiology

Role of Protease IV in the Pseudomonas virulence 7

Fig. 4. The piv− mutant was much less virulent. A and B. The CCSs were prepared from wild-type PAO1 (WT), Protease IV-deficient mutant strain (piv−), and their complemented strains with episomal expression of Protease IV (WT/PIV and piv−/PIV respectively). WT and piv− strains also carried the empty plasmid (pJN105) as vector control. The CCSs were concentrated five times and injected to T. molitor larvae to measure the melanin synthesis (A) and survival (B) of the larvae. IS, insect saline. C. Live cells of wild-type or the piv− mutant were diluted in IS and injected to T. molitor larvae by microsyringe. IS was injected for control. Survival was measured at fourth day. *P-value < 0.05. D. piv− mutant was complemented by various plasmid-encoded exoproteases. The live cells harboring the complementing plasmids were diluted in IS and injected to T. molitor larvae. Survival was measured at second day. VC indicates pJN105, the parental plasmid for a vector control. PIV, Protease IV. ***P-value < 0.0005 (between PIV and VC); **P-value < 0.01 (between PIV and other proteases).

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Fig. 5. The Protease IV-mediated virulence was inhibited by a protease inhibitor. A and B. The CCS prepared from Protease IV-overexpressing PAO1 (WT/PIV) was treated with 1 mM TLCK, a serine protease inhibitor, and directly injected to T. molitor larvae without concentration. Heat-treated CCS was injected for comparison. The melanization (A) and survival (B) were measured. ***P-value < 0.0005. B. The live cells of Protease IV-overexpressing PAO1 (WT/PIV) were treated with 1 mM TLCK. After treatment, the cells were diluted in insect saline and injected into T. molitor larvae. For comparison, the same amount of live PAO1 cells harboring pJN105 was diluted and injected into T. molitor larvae (WT). Survival of larvae was measured at second day. IS, insect saline. *P-value < 0.05.

T. molitor larvae. High dosage of LasB induced the AMP production to some extent (Fig. 7C). However, in the case of Protease IV, AMPs were not produced over wide range of dosages from very small amount of PIVh (4 ng) that is a sublethal dose to large amount of PIVh (400 ng) that is

a fully lethal dose (Supplementary Fig. S2). This indicates that PIVh does not activate the host Toll signaling cascade. In order to confirm this result, we directly investigated the induction of AMPs by measuring the transcription level of AMPs. In T. molitor, four Tenecins that are © 2014 John Wiley & Sons Ltd, Molecular Microbiology

Role of Protease IV in the Pseudomonas virulence 9

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Fig. 6. Purified Protease IV induced the melanization and killing. A. The purified PIVh and LasB were electrophoresed in 12% SDS-PAGE gel and stained by Coomassie Brilliant Blue R-250. M, molecular size marker. B. The activity of Histidine-tagged Protease IV (PIVh) was confirmed using the chromogenic substrate, N-p-Tosyl-Gly-Pro-Lys-4-nitroanilide that gives off color when it is specifically cleaved. Twenty nanograms of PIVh and heat-treated PIVh as a control were incubated in 50 mM Tris-HCl (pH 8.0) containing 200 mM N-p-Tosyl-Gly-Pro-Lys-4-nitroanilide and the absorbance (A410) was measured. C and D. Various amounts of the purified PIVh was injected to T. molitor larvae and the melanin synthesis was observed (C) and plotted (D). One hundred nanograms of heat-treated PIVh was also injected for comparison. Injection volume was same and the protein storage buffer was injected for 0 ng sample. IS was injected as a control. E. Survival of the larvae was measured after the injection of PIVh. IS and heat-treated PIVh were injected as a control.

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Fig. 7. Protease IV degraded the innate immune components for the AMP production. A and B. Zymogen forms of SAE and SPE were incubated with the purified PIVh and the cleavage was investigated by Western analysis. Antibodies against SAE (A) and SPE (B) were used to detect the cleaved product. Z and A indicate zymogen and active forms. zSAE, and zSPE are zymogen forms, and aSAE and aSPE are active forms of SAE and SPE. A, Lane 1, zymogen form of SAE; lane 2, active form of SAE; lane 3–5, zymogen form of SAE plus different amount of purified PIVh (1, 10, 100 ng respectively). B, Lane 1, zymogen form of SPE; lane 2, active form of SPE; lane 3–5, zymogen form of SPE plus different amount of purified PIVh (1, 10, 100 ng respectively). C. The AMP production in hemolymph was measured. After injection of purified PIVh and LasB into T. molitor larvae, hemolymph was taken from the larvae and loaded on the bacterial lawn of E. coli. Insect saline was injected for a negative control (IS) and 5 × 105 cfu of S. aureus was injected for a positive control (PC) as an inducer of Toll pathway. Hemolymph with no treatment was also injected (NT).

AMPs belonging to the family of defensins have been reported (Lee et al., 1996; Roh et al., 2009; Chae et al., 2012). As shown in Fig. 8, none of 4 Tenecins were induced by the injection of purified PIVh. We note that

Tenecin 3 was not induced even by bacterial injection (Fig. 8C). Tenecin 3 has been suggested as an antifungal protein that is active against Candida albicans (Kim et al., 2001). Since the proteolytic cascade was not activated by PIVh, it was questionable how the melanin synthesis could occur. When we mixed the CCS prepared from the Protease IV-overexpressing P. aeruginosa with the hemolymph of T. molitor, the PO activity was significantly augmented (Fig. 9A). More directly, when we mixed the purified PIVh with proPO and SPH1, the proPO was activated proportionally to the dose of PIVh (Fig. 9B). When we added dopamine, the substrates for melanine synthesis to the mixture of PIVh, proPO, and SPH1, the melanin synthesis increased (Fig. 9C). We also investigated the cleavage of proPO by PIVh in hemolymph using Western blot analysis. When PIVh was added to hemolymph, specifically cleaved bands were detected by anti-proPO antibody (Fig. 9D). This cleavage pattern was similar to the in vitro cleavage pattern by active SPE that was previously reported (Kan et al., 2008). The hemolymph treated by β-1.3-glucan, a well-known inducer of melanization also included the similar sized bands (Fig. 9D). All these results consistently demonstrated that Protease IV of P. aeruginosa can activate the T. molitor proPO system and melanin synthesis independently of the upstream components. Protease IV also works as a virulence factor in other hosts In order to know whether Protease IV is also working on other invertebrate systems, we investigated the virulence of a wild type and a piv− mutant to brine shrimp (Artemia), an aquatic crustacean and C. elegans, a nematode. Interestingly, when we fed brine shrimps with wild type and a piv− mutant, the piv− mutant cells showed dramatic attenuation (Fig. 10). This attenuation was complemented by the episomal expression of Protease IV (Fig. 10). When this Protease IV-overexpressing P. aeruginosa was treated with TLCK, virulence was much attenuated again (Fig. 10), confirming the important role of Protease IV in virulence. Similarly, when C. elegans was fed with P. aeruginosa cells, the virulence of piv− mutant was slightly, but significantly alleviated by the deficiency of Protease IV (Supplementary Fig. S3). These results suggest that Protease IV may work as an important virulence factor of P. aeruginosa in other invertebrate hosts.

Discussion Like the complement system of mammalians, insects have a similar protease cascade system that mediates the signal amplification from the initial recognition of pathogens to the © 2014 John Wiley & Sons Ltd, Molecular Microbiology

Role of Protease IV in the Pseudomonas virulence 11

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Fig. 8. AMPs were not induced by Protease IV. Fifty nanograms of the purified PIVh and LasB proteins were injected to T. molitor larvae and the expression of Tenecins, the AMPs of T. molitor was measured by quantitative real-time PCR analysis (A, Tenecin 1; B, Tenecin 2; C, Tenecin 3; D, Tenecin 4). A total of 5 × 105 cfu of S. aureus was injected for the positive control (PC) and insect saline (IS) was injected for buffer control. *P-value < 0.05; **P-value < 0.01; ***P-value < 0.0005.

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activation of Toll pathway and melanin synthesis. Since proteases produced by pathogens are also important virulence factors, it implied possible interaction between the proteases from pathogens and from hosts. In this study we addressed this idea using the P. aeruginosa–T. molitor model system and showed that a QS-dependently produced bacterial protease, Protease IV modulates the innate immune system of T. molitor, which plays a significant role in the infection process. While this is an example for the interruption of host immune system by the pathogenic factor, probably this is a common mode of the host–pathogen interaction. So far, both melanization and AMP production of T. molitor were known to be triggered by the recognition of PAMPs, such as Lys-PG or β-1,3-glucan as shown in Fig. 11. Our study shows that P. aeruginosa has its own active way to oppose the host immune system in which it interrupts the Toll proteolytic cascade of T. molitor toward the AMP production using Protease IV. Since the AMPs are critical weapons to defeat pathogens, the blocking AMP production is obviously advantageous for P. aeruginosa, and provides a clever strategy for successful infection. We © 2014 John Wiley & Sons Ltd, Molecular Microbiology

PC

PIVh LasB

note that Protease IV can provoke the melanization of T. molitor larvae independently of the AMP production (Fig. 11). It is not clear why Protease IV induces the melanization even though it inactivates the upstream protease cascade. While the melanization is generally considered to be important in invertebrate innate immunity, some insects do not seem to be dependent on PO activity and melanin synthesis for the defense against pathogens (Cerenius et al., 2008). Probably, the melanization may not be effective for the defense without the AMP production in the interaction between P. aeruginosa and T. molitor. A transcriptome study showed that piv encoding Protease IV is strongly induced in P. aeruginosa by 3OC12HSL, a major QS signal (Schuster et al., 2003). This result and our study explain why QS mechanism is important for the virulence of P. aeruginosa to insects. While the crucial role of QS in the P. aeruginosa infection to insects is often documented (Chugani et al., 2001; D’Argenio et al., 2001), the responsible virulence effectors regulated by QS were not fully clarified. Some studies have suggested that the factors related with twitching motility that is under the control of QS are important in the Pseudomonas viru-

12 S.-J. Park et al. ■

A

B 2.4

PO activity (A520)

PO activity (A520)

3

2

1

0 hemolymph

1.6

0.8

0 +

+

+ 1

+

+

+

2

+

+

+

+ proPO + SPH1

2.5 13 100 (ng)

3 (hours)

CCS

C

D

proPO antibody 1 2 3 4 5

Melanin synthesis (A400)

1.2 140 97 0.8 48 0.4

0 proPO + SPH1 +

35 30

+

+

lence in Drosophila model system (Glessner et al., 1999; D’Argenio et al., 2001), and elastase B (LasB), another QS-regulated exoprotease of P. aeruginosa can stimulate the humoral immune response at early stage of infection, and then degrade some immune components at later stage in G. mellonella model system (Andrejko and Mizerska-Dudka, 2011; 2012). However, it was reported that the twitching motility is not directly related to the fly killing (D’Argenio et al., 2001), and our result showed that LasB is not crucially involved in the virulence to T. molitor (Fig. 3). Therefore, in the P. aeruginosa infection to T. molitor, Protease IV is a direct effector exerting the virulence derived from the QS regulation. As mentioned above, although both T. molitor and G. mellonella belong to holometabolous insect, elastase B and Protease IV differently work in each host for the

Fig. 9. Protease IV induced the melanin synthesis independently of the Toll signaling cascade. A. The activation of phenoloxidase (PO) by the CCS prepared from the Protease IV-overexpressing P. aeruginosa was measured in hemolymph. The PO activity was assayed by adding 4-methylcatechol (4-MC) and 4-hydroxyproline ethyl ester HCl (4-HP), the PO substrates. After mixing the CCS with T. molitor hemolymph, the PO substrates were added and the PO activity was hourly measured by absorbance (A520). All reactions contained hemolymph. From the left, LB, the blank medium for a negative control (−), β-1,3-glucan, an inducer of the melanin synthesis for a positive control, and the CCS were added and incubated for 1, 2, and 3 h respectively. B. The PO activation was reconstituted by mixing the purified PIVh with purified proPO and SPH1, instead of hemolymph. All reactions contained proPO and SPH1, and either of protein storage buffer (−), 150 ng aSPE (active form of SPE), or different amount of PIVh (2.5, 13, and 100 ng) were added from the left. After mixing the purified proteins, the PO substrates were added and the PO activity was measured by absorbance (A520) at 10 min. The protein storage buffer and aSPE were used for the negative and positive controls respectively. C. The melanin synthesis was measured in the reconstitution. After mixing the purified PIVh with the purified proPO and SPH1, 1 mM dopamine, a substrate of melanine synthesis was added and absorbance was measured at A400. All reactions contained proPO and SPH1. From the left, protein storage buffer (−), 150 ng aSPE, and purified PIVh were added respectively. D. PIVh was added to hemolymph and Western analysis was carried out to see the cleavage of proPO with antibody against proPO. Lane 1, only hemolymph; lane 2, hemolymph plus β-1,3-glucan; lane 3–5, hemolymph plus purified PIVh (1, 10, and 100 ng respectively).

Pseudomonas virulence. Some studies also demonstrated that elastase B is crucial for the cytotoxicity to human cells (Blackwood et al., 1983; Lee et al., 2011). These results suggest the specific association between the virulence factor of pathogen and host immune components, which may be a determinant of the host– pathogen relationship. SPE of T. molitor has a similar structure with PPAF-I, II, III (Phenoloxidase activating factors) of Holotrichia diomphalia, a colepteran insect, in that they have similar protrusions of an activation loop (Piao et al., 2007). A recent study showed that the activating loop of PPAF-I of H. diomphalia was cleaved by a lysyl endopeptidase of Achromobacter lyticus, a bacterium belong to β-proteobacteria (Piao et al., 2007). Since Protease IV is also a lysyl endopeptidase, it might be able to cleave the activating loop of © 2014 John Wiley & Sons Ltd, Molecular Microbiology

Role of Protease IV in the Pseudomonas virulence 13

***

***

***

Survival (%)

100 75 50 25 0

Fig. 10. Protease IV works as a virulence factor in the brine shrimp infection. Live cells of wild type (WT), MW1, piv− mutant, and the complemented strain with episomal expression of Protease IV (piv−/PIV) were fed to brine shrimps nauplii in seawater and the survival of the nauplii was daily counted after challenge. For the control, same amount of the killed cells were applied in the same manner. The live cells of the piv−/PIV strain were treated with 1 mM TLCK at 25°C for 30 min to inhibit Protease IV before feeding to nauplii (piv−/PIV+TLCK). Only TLCK was added to nauplii to exclude the chemical effect of TLCK. ***P-value < 0.0005.

SPE like the A. lyticus lysyl endopeptidase. Our results showed that Protease IV of P. aeruginosa can cleave SPE of T. molitor, but to inactive form. The determination of the three-dimensional structures of SPE and Protease IV is needed to better understand the exact mechanism of the cleavage.

merase chain reaction) using the primers that have suitable restriction sites for the cloning into an expression plasmid, pJN105 (Supplementary Tables S1 and S2). This plasmid is designed to express a gene under the control of an arabinoseinducible promoter, pBAD, and replicable in both P. aeruginosa and Escherichia coli. Amplified DNA fragments were digested by EcoRI (for PA4175, PA1871 and PA3724) or NheI/XbaI (for PA4171), or EcoRI/XbaI (for PA2939, PA0355, PA1249, and PA3535), and ligated into the EcoRI- or NheI/XbaI-, or EcoRI/ XbaI-digested pJN105. To construct pQF21c-PIV, the plasmid overexpressing the C-terminal His-tagged Protease IV (PIVh) in P. aeruginosa, the DNA fragment including the ORF of piv gene was amplified from genomic DNA by PCR with primers in Supplementary Table S2, digested with NdeI and XhoI, and cloned into the NdeI/XhoI-digested pQF21c. pQF21c that is replicable in P. aeruginosa was constructed by inserting a Pseudomonas replication origin (ori1600) into pET21c, a commercial E. coli overexpression plasmid. The correct construction was confirmed by restriction enzyme-digestion pattern and sequencing.

Lys-PG or β-1,3-glucan

PGRP

PAMP/PGRP/GNBP1 complex

GNBP1

proMSP

MSP

SAE

proSAE

proSPE

SPE

Experimental procedures Bacterial strains, plasmids, and culture condition The bacterial strains and plasmids used in this study are listed in Supplementary Table S1. In general, P. aeruginosa was cultured in Luria–Bertani medium (LB; 5 g l−1 yeast extract, 10 g l−1 bacto-tryptone, 5 g l−1 NaCl) at 37°C with vigorous shaking. Cell growth was measured by optical density at 600 nm (OD600). For the induction of protein expression, arabinose (for the pJN105-based plasmids, Table S1) and IPTG (Isopropyl-1-thio-β-D-galactopranoside, for pQF21c-PIV, Table S1) were added at 0.2% and 1 mM respectively. Antibiotics were used as following concentrations: carbenicillin, 150 μg ml−1; gentamicin, 50 μg ml−1; tetracycline, 60 μg ml−1; HgCl, 7.5 μg ml−1 respectively. For the complementation of MW1 with synthetic QS signals, 5 μM of synthetic 3OC12-HSL and C4-HSL was supplemented to culture media.

Construction of recombinant plasmids For the construction of plasmids to overexpress the candidate proteases (Table 1), each gene of the proteases were amplified from genomic DNA of P. aeruginosa PAO1 by PCR (poly© 2014 John Wiley & Sons Ltd, Molecular Microbiology

Protease IV

proSpz proSPH1 proPO Spz SPH1 PO Toll receptor

Melanine production

AMP production

Fig. 11. Proposed role of Protease IV in Pseudomonas infection. The Toll signaling pathway of T. molitor innate immune system is schematically illustrated with slight modification from previous publications (Kan et al., 2008; Roh et al., 2009), which shows current understanding about how the melanin synthesis and AMP production are induced by infection. The proposed role of Protease IV in the P. aeruginosa infection is indicated with grey arrows, in which Protease IV degraded the key components (SAE and SPE in dotted line) of T. molitor innate immune system to block the AMP production, but still provokes the melanization of the larvae by activating the PO/SPH1 system independently of the Toll pathway.

14 S.-J. Park et al. ■

Virulence assay with T. molitor Tenebrio molitor were maintained in a terrarium with wheat bran and virulence assay using T. molitor larvae was performed as described elsewhere (Yeom et al., 2013). The melanization was measured after parenteral injection of live cells, or cell-free culture supernatants (CCSs) of P. aeruginosa or a purified protein (PIVh) into T. molitor larvae. PAO1 or its isogenic mutants, or the strains harboring the pJN105-based plasmids expressing the candidate proteases were grown in LB medium up to OD600 = 3.0. For the live cell injection, these cells were diluted by 50-fold with insect saline (130 mM NaCl, 5 mM KCl, and 1 mM CaCl2) and 5 μl was injected into T. molitor larvae by microsyringe. To prepare the CCSs, cells were removed from the cultures by centrifugation at 6000 r.p.m. for 20 min at 4°C and filtration 0.2 μm filter (Satorious). Five microlitres of the CCSs was directly injected into T. molitor larvae by microsyringe, or otherwise, the CCSs were further concentrated to 5–20 times by ultra-filtration with a 10 kDa cut-off membrane (Satorious, Vivaspin 500). All CCSs were freshly prepared before each injection and maintained on ice. As a control, the same volume of insect saline or the passthrough fraction from the 10 kDa cut-off ultra-filtration were used for injection. The purified protein was injected into T. molitor larvae in the same way and the protein storage buffer was injected as a control. After injection, the larvae were incubated in Petri dishes at 30°C for 2–4 days, and the number of melanin-induced or live/dead larvae were counted. In order to inhibit the Protease IV activity, the CCS or live cells were treated with a serine protease inhibitor, N-p-tosyl-Lchloromethyl ketone (TLCK, Sigma, USA). The TLCK treatment was carried out at 25°C for 30 min at 1 mM concentration before injection to T. molitor. The heat treatment of CCSs or purified PIVh was performed at 95°C for 10 min.

1 × 105 cfu (colony-forming units) of bacterial cells were added to the seawater to infect brine shrimps, and incubated at 28°C for 2 days. As a control, the same number of killed bacterial cells were added to shrimps and incubated in the same manner. The survival of shrimps was scored daily after the addition of bacteria. This experiment was independently repeated three times. To inhibit the Protease IV activity, live bacterial cells were treated with TLCK at 25°C for 30 min at 1 mM concentration before feeding to brine shrimps.

Purification of His-tagged Protease IV (PIVh) and LasB

Caenorhabditis elegans, a nematode was maintained by feeding E. coli OP50 on nematode growth medium (NGM; 3 g l−1 NaCl, 17 g l−1 agar, 2.5 g l−1 peptone, 3 g l−1 KH2PO4, 0.5 g l−1 K2HPO4, 1 mM CaCl2, 5 mg l−1 cholesterol, and 1 mM MgSO4). For virulence assay, cell lawns of P. aeruginosa strains were prepared by dropping 50 μl of overnight-cultured cells on to the NGM plates and incubating 18 h. The equal amount of E. coli OP50 cells was dropped on the NGM plates in the same manner as a control. Twenty of C. elegans worms at L4 stage were placed on the lawns and incubated at 25°C for 8 days with daily transfer of live worms to fresh cell lawns on new plates. Live or dead worms were counted every day and statistically analyzed in software supplied by Pohang University of Science and Technology (Yang et al., 2011). This experiment was independently repeated three times.

Pseudomonas aeruginosa PAO-T7 was transformed with pQF21c-PIV that has T7-RNA polymerase gene on chromosome under the control of IPTG-inducible promoter. The transformant was grown in 1 l of LB broth up to OD600 = 0.5 and IPTG was added to 0.1 mM. After 16 h cultivation, cells were removed by centrifugation at 12 000 r.p.m. for 30 min at 4°C and the supernatant was passed through 0.2 μm filter. The resulting supernatant was applied to the binding buffer (20 mM Tris-HCl, 500 mM NaCl, pH 7.9)-equilibrated Ni-NTA agarose resin (Novagen) in column chromatography. The bound proteins were washed with the binding buffer and eluted by the stepwise addition of the imidazole-containing elution buffer (20 mM Tris-HCl, 500 mM NaCl, 20–500 mM imidazole, pH 7.9). The fractions containing PIVh without impurity were pooled and dialyzed with storage buffer (50 mM Tris-HCl, 50 mM NaCl, 50 mM KCl, 40% glycerol, pH 7.5). The resulting purified PIVh was aliquoted and stored at −80°C. Protease IV activity was confirmed by using the chromogenic substrate, N-p-Tosyl-Gly-Pro-Lys-4-nitroanilide (acetate salt, Sigma). This substrate gives off color when it is specifically cleaved. Purified PIVh (1–10 ng) was mixed with 2 μl of commercial chromogenic substrate (final concentration = 200 μM) in 100 μl of reaction buffer (50 mM Tris-HCl, pH 8.0). The reaction was incubated at 37°C for 30 min and the absorbance was measured at 410 nm (A410). For the purification of LasB, pSP201, the LasB-overexpressing plasmid was introduced into the PAO1 and the transformed strain was cultivated in LB medium at 37°C with vigorous shaking. Arabinose (0.8%) was added to induce LasB and further cultivated for 16 h. Cells were then removed by centrifugation and the culture supernatant was taken. LasB was precipitated from the culture supernatant by slowly adding ammonium sulfate up to 60% saturation (39 g per 100 ml). After centrifugation, the pellet was dissolved in 50 mM TrisHCl (pH 8.0) and the remained salt was completely removed by dialysis. This LasB-containing solution was applied to DEAE sepharose column chromatography and eluted by NaCl gradient. The fractions containing pure LasB were collected and dialyzed in the storage buffer. The purified LasB was aliquoted and stored at −80°C.

Virulence assay with brine shrimp

Disruption of piv encoding Protease IV

Virulence tests with brine shrimp (Artemia salina) were performed as described previously (Brackman et al., 2008). Briefly, after hatching from eggs, 20 brine shrimp nauplii were transferred into Petri dish (35 × 10 mm) containing 20 ml of autoclaved artificial seawater (Sigma, S9883). A total of

A piv− mutant (PW8077) was obtained from University of Washington genome center in Seattle (Jacobs et al., 2003). To equalize the genetic background, we moved the mutant allele to our wild-type PAO1 by transformation as follows. Genomic DNA was isolated from PW8077 and 7 μg of the

Virulence assay with C. elegans

© 2014 John Wiley & Sons Ltd, Molecular Microbiology

Role of Protease IV in the Pseudomonas virulence 15

DNA was very carefully mixed with the competent cells of PAO1 strain that were prepared by MgCl2 method. The mixture was kept on ice for 30 min and quickly moved to 37°C for heat shock. After 3 min incubation at 37°C, it is quickly moved on ice again. LB broth was added for regeneration and the mixture was incubated at 37°C for 1 h with shaking. The mixture was spread on LB plate containing 60 μg ml−1 tetracycline and incubated at 37°C for 12–24 h for selection. False positive colonies were excluded by successive selection on the tetracycline plates, and then the correct transfer of mutant allele was finally confirmed by PCR with specific primers (Supplementary Table S2). There was no significant difference in growth between wild-type PAO1 and this piv− mutant in all conditions we used in this study.

Total protease activity assay The total protease activity was measured by skim milk plate method. Five microliters of CCFs from P. aeruginosa overnight cultures (OD600 = 3) were spotted on the discs on the skim milk plates (0.5% skim milk, 0.5% peptone, 0.1% glucose, and 1.5% agar), and further incubated at 37°C for 14 h. As the degradation of the proteins in skim milk by proteases makes a clear zone, the protease activity was estimated by the diameter of the clear zones.

Western analysis The zymogens and active forms of SPE, SAE, and PO, and their antibodies were obtained as previously described (Kan et al., 2008; Kim et al., 2008; Roh et al., 2009). Each immune components or hemolymph were mixed with the indicated amount of PIVh and incubated for 1 h at 30°C. Whole proteins in the reaction were electrophoresed in SDS-PAGE and transferred to PVDF membrane in Western transfer buffer (192 mM glycine, 25 mM Tris, 0.02%, 20% methanol) at 4°C. Blocking of the PVDF membrane and hybridization with antibody were done in 5% skim milk. After several washing in TBST (20 mM Tris, 150 mM NaCl, 0.1% Tween 20, pH 7.4), HRP (horseradish peroxidase)-conjugated secondary antibody was added and incubated for 1 h at room temperature. After several washing in TBST, the bands were visualized on X-ray film by chemiluminescence.

Measurement of phenoloxidase (PO) activity and melanine synthesis The PO activity was assayed with purified proPO or CCS in hemolymph and reconstitution system by adding 4-methylcatechol (4-MC) and 4-hydroxyproline ethyl ester HCl (4-HP) as PO substrates. The CCS prepared from the Protease IV-overexpressing P. aeruginosa or purified PIVh were mixed with 300 μg of T. molitor hemolymph (for hemolymph analysis) or 1 μg proPO and 300 ng SPH1 (for reconstitution analysis) in 30 μl of 20 mM Tris-HCl (pH 8.0) solution. The mixture was pre-incubated at 30°C for 1 h. In the hemolymph analysis, 3 μg of β-1,3-glucan was used for a positive control and in the reconstitution analysis, active form of SPE (aSPE) was used for a positive control. After pre-incubation, the PO © 2014 John Wiley & Sons Ltd, Molecular Microbiology

reaction was carried out by adding substrate solution containing 10 mM CaCl2, 2 mM 4-MC, and 2 mM 4-HP in 500 μl reaction solution. The PO activity was measured by absorbance at 520 nm (A520). The PO-induced melanin synthesis was estimated as previously described (Kan et al., 2008). Briefly, proPO (3 μg) and SPH1 (1 μg) were mixed with PIVh in 50 μl reaction buffer (10 mM CaCl2, 20 mM Tris-Cl, pH 8.0) and pre-incubated at 30°C for 5 min. One hundred and fifty microliters of substrate solution (1 mM dopamine, 10 mM CaCl2, 20 mM Tris-Cl, pH 8.0) was added and incubated at 30°C for 30 min. The absorbance was measured at 400 nm (A400), which increases proportionally with the melanin synthesis. aSPE (150 ng) was used for a positive control.

Assay of antimicrobial activity in hemolymph The antimicrobial activity in hemolymph was measured against Staphylococcus aureus and E. coli. The hemolymph was taken from T. molitor larvae at 18 h after the injection of the purified PIVh or Lys-PG as a positive control, and collected in decoagluation buffer (15 mM NaCl, 30 mM trisodium citrate, 26 mM citric acid, and 20 mM EDTA). After centrifugation at 15 000 r.p.m. at 4°C for 15 min, the supernatant was taken and heated at 95°C to inactivate the high-molecularweight proteins. After another centrifugation at 15 000 r.p.m. at 4°C for 15 min, the supernatant was desalted in sep-pak C18 column and eluted with 80% acetonitrile. The acetonitrile was completely evaporated by speed vacuum and resolved in 1 M Tris-HCl (pH 7.5). This solution was dropped on the discs on the bacterial lawn of S. aureus and E. coli.

RNA preparation, cDNA synthesis, and quantitative real-time PCR analysis Tenebrio molitor larvae were injected with 50 ng of purified LasB and Protease IV. After 17 h, fat body of the larvae was taken and disrupted by sonication. Total RNA was purified by MINI-total RNA purification Kit (Qiagen) according to the manufacturer’s protocol and quantified by spectrophotometer. cDNA was synthesized from 5 μg of total RNA by using HelixCripTM Thermo Reverse Transcriptase Kit (NanoHelix, Korea) as the supplied protocol. For the quantitative real-time PCR, 5 ng of cDNA and the specific primers were mixed in 10 μl of SYBR Premix Ex Taq (TaKaRa, Japan) and analyzed in Thermal CyclerTM Real-time PCR system TP8000 (TaKaRa, Japan). The standards for the quantification were obtained from various multiples of cDNA synthesized from non-treated RNA. For the positive control, 5 × 105 cfu of S. aureus cells was injected and insect saline was injected for buffer control.

Statistical analysis In order to ensure the significance of the results in the virulence analyses, the data were statistically analyzed using t-test (two-sample assuming equal variances) in MS office Excel (Microsoft, USA). If the P-value was lower than 0.05, it was considered significant.

16 S.-J. Park et al. ■

Acknowledgments This work was supported by National Research Foundation of Korea Grant funded by the Korean Government (20100015901). This research was also supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2013R1A1A2012220).

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