EprS, an autotransporter protein of Pseudomonas aeruginosa, possessing serine protease activity induces inflammatory responses through protease-activated receptors

Authors


For correspondence. E-mail kuwano@med.kurume-u.ac.jp; Tel. (+81) 942 31 7548; Fax (+81) 942 31 0343.

Summary

PA3535 (EprS), an autotransporter (AT) protein of Pseudomonas aeruginosa, is predicted to contain a serine protease motif. The eprS encodes a 104.5 kDa protein with a 30-amino-acid-long signal peptide, a 51.2 kDa amino-terminal secreted passenger domain and a 50.1 kDa carboxyl-terminal outer membrane channel formed translocator. Although the majority of AT proteins have been reported to be virulence factors, little is known about the functions of EprS in the pathogenicity of P. aeruginosa. In this study, we performed functional analyses of recombinant EprS secreted by Escherichia coli. The proteolytic activity of EprS was markedly decreased by changing Ser to Ala at position 308 or by serine protease inhibitors. EprS preferred to cleave substrates that terminated with arginine or lysine residues. Thus, these results indicate that EprS, a serine protease, displays the substrate specificity, cleaving after basic residues. We demonstrated that EprS activates NF-κB-driven promoters through protease-activated receptor (PAR)-1, -2 or -4 and induces IL-8 production through PAR-2 in a human bronchiole epithelial cell line. Moreover, EprS cleaved the peptides corresponding to the tethered ligand region of PAR-1, -2 and -4 at a specific site with exposure oftheir tethered ligands. Collectively, these results suggest that EprS activates host inflammatory responses through PARs.

Introduction

The Gram-negative bacterium Pseudomonas aeruginosa is responsible for a variety of diseases, including lung infection with cystic fibrosis, burn wound infections, hospital-acquired pneumonia and keratitis (Lyczak et al., 2000). Major virulence factors produced by this pathogen include secreted proteases that damage host tissues. Several P. aeruginosa proteases have been isolated and shown to be involved in pathogenesis. Of the proteases analysed, alkaline protease (Kharazmi, 1991), elastase (Suter, 1994; Preston et al., 1997), protease IV (O'Callaghan et al., 1996; Engel et al., 1998), small protease (Marquart et al., 2005; Tang et al., 2009), immunomodulating metalloprotease (Bardoel et al., 2012) and large extracellular protease (LepA) (Kida et al., 2008; 2011) have been characterized extensively.

Proteins secreted by Gram-negative bacteria are transported across the cell envelope, which is composed of the inner membrane (IM), the peptidoglycan-containing periplasm and the outer membrane (OM). Six different classes of secretion systems have been described, which are identified as type I secretion system (T1SS) up to type VI secretion system (T6SS) (Economou et al., 2006; Filloux et al., 2008). Except for T4SS, P. aeruginosa possesses all the other secretion machines described in Gram-negative bacteria (Bleves et al., 2010; Filloux, 2011). Among them, the type V secretion system (T5SS) comprises diverse branches, the autotransporter (AT or type Va and type Vc) pathway and two-partner secretion (TPS or type Vb) pathway (Henderson et al., 2004; Jacob-Dubuisson et al., 2004; Benz and Schmidt, 2011). T5SS composes a two-step process (Mazar and Cotter, 2007; Leo et al., 2012; Leyton et al., 2012). First, proteins cross the IM via the Sec export machinery; second, they are transported through an OM channel formed by a β barrel protein/module. Finally, exoproteins either remain associated with the outer face of the OM or are released into the extracellular milieu after cleavage by autoproteolysis or by a protease. AT proteins contain their own transporter domain, covalently attached to the carboxyl-terminal of the secreted passenger domain, while the TPS system is composed of two separate proteins, with TpsA being the secreted protein and TpsB its specific transporter. The transporter domains of T5SS are highly homologous, while the passenger domains are very diverse. The majority of characterized passenger protein domains have been implicated in virulence by displaying enzymatic activity (protease, peptidase, lipase and esterase); mediating host invasion; acting as adhesins, immunomodulatory proteins and cytotoxins (Henderson and Nataro, 2001; Jacob-Dubuisson et al., 2001; Wells et al., 2007; 2010).

Three typical AT proteins have been predicted from the genome sequence of P. aeruginosa strain PAO1 (Stover et al., 2000). PA5112 (also termed EstA) is the most characterized AT protein in P. aeruginosa (Wilhelm et al., 1999). EstA has been shown to hydrolyse glycerol esters with short- or long-chain fatty acids, which are involved in the production of rhamnolipids. An estA defective mutant has been demonstrated to be deficient in twitching, swarming, swimming motilities and biofilm formation (Wilhelm et al., 2007). Among two other AT proteins, PA0328 has more recently been reported to be an arginine-specific aminopeptidase and an important virulence factor playing a significant role in the successful establishment of P. aeruginosa infections (Luckett et al., 2012). Although PA3535 (also termed EprS) is predicted to encode a subtilisin-like serine protease (Ma et al., 2003), there is currently no published experimental evidence to confirm this. On the other hand, mining the PAO1 genome identified at least six putative TpsA–TpsB proteins, Tps1: PA0041–PA0040; Tps2: PA0690–PA0692; Tps3: PA2462–PA2463; Tps4: PA2542–PA2543; Tps5: PA4541 (also termed LepA)–PA4540 (also termed LepB); Tps6: PA4625 (also termed CdrA)–PA4624 (also termed CdrB) (Yen et al., 2002; Ma et al., 2003). Only two, Tps5 and Tps6, have been characterized. In the case of Tps5 (LepA–LepB), we previously reported that a protease, LepA, activates the critical transcription factor nuclear factor-κB (NF-κB) for host inflammatory and immune responses through protease-activated receptors (PARs) (Kida et al., 2008) and contributes to the in vivo virulence and growth of P. aeruginosa (Kida et al., 2011). As for Tps6 (CdrA–CdrB), a cyclic-di-GMP-regulated adhesin, CdrA, has been shown to promote P. aeruginosa biofilm formation and bacterial auto-aggregation in liquid culture (Borlee et al., 2010).

Intriguingly, the serine protease ATs of the Enterobacteriaceae (SPATEs) have been reported to be virulence factors implicated in bacterial pathogenesis (Dutta et al., 2002; Yen et al., 2008; Wells et al., 2010). Thus, we hypothesized that P. aeruginosa-derived EprS of the non-SPATEs would play a role in the pathogenicity of P. aeruginosa. In this study, we performed functional analyses of P. aeruginosa EprS produced by Escherichia coli. Our results indicate that EprS require PARs to activate NF-κB, thereby modulating host inflammatory and immune responses such as cytokine production. Herein, we for the first time describe the biological function of EprS.

Results

Structure of EprS

The eprS gene of P. aeruginosa strain PAO1 is annotated as an autotransporter protein with a subtilisin-like protease motif (http://www.pseudomonas.com). The 2988-bp-long gene encodes a hypothetical 995-amino acids (aa)-long protein with a theoretical molecular size of 104.5 kDa. The molecular features of EprS are shown as a schematic representation in Fig. 1A. SignalP4.0 (http://www.cbs.dtu.dk/services/SignalP) demonstrated that the signal sequence is 30-aa-long with the potential cleavage site between residues 30 and 31. An analysis of the amino acid sequence of EprS using the NCBI protein database (http://www.ncbi.nlm.nih.gov/protein) revealed the presence of a subtilisin-like protease domain between residues 40 and 374. MEROPS protease database (http://merops.sanger.ac.uk) indicated that the protein belongs to the family of serine proteases with Asp79, His122 and Ser308 forming the putative catalytic triad of serine protease. InterProScan (http://www.ebi.ac.uk/Tools/pfa/iprscan) demonstrated that EprS contains an outer membrane AT barrel domain between residues 521 and 995.

Figure 1.

Schematic representation of the structure of P. aeruginosa PAO1-derived EprS.

A. Molecular features of EprS. The amino acids Asp79, His122 and Ser308 that form the putative catalytic triad of serine protease.

B. Expression constructs of recombinant EprS (rEprS). Each expression plasmid was generated by inserting a six histidine tag between residues 32 and 33 downstream of the putative amino-terminal amino acid. Each arrow indicates the putative signal sequence cleavage site. rEprS WT, six histidine-tagged EprS; rEprS S308A; six histidine-tagged EprS harbouring a putative active-site serine mutant.

Purification and proteolytic activity of recombinant EprS

To purify and characterize EprS, expression plasmids of six histidine-tagged recombinant EprS (rEprS WT) and its putative active-site serine mutant rEprS S308A were constructed (Fig. 1B). rEprS WT or rEprS S308A was overexpressed in E. coli TOP10, each protein was purified from the culture supernatant as described in Experimental procedures. As shown in Fig. 2A, each protein was purified to a nearly single band at ∼52 kDa. The results correlated well with the predicted molecular weight (51.2 kDa) for the putative secreted passenger domain of EprS (31–520 aa).

Figure 2.

Purification and proteolytic activity of rEprS.

A. SDS-PAGE of rEprS. The gel was stained with Coomassie blue G-250. Lane M, molecular weight markers; lane 1, rEprS WT contained in flow-through fraction of Q-Sepharose; lane 2, rEprS WT contained in flow-through fraction of Ni-Sepharose; lane 3, purified rEprS WT; lane 4, rEprS S308A contained in flow-through fraction of Q-Sepharose; lane 5, rEprS S308A contained in flow-through fraction of Ni-Sepharose; lane 6, purified rEprS S308A. Arrow indicates the size of rEprS.

B. Proteolytic activity of rEprS. Five micrograms of rEprS was added to 100 μM fluorescein thiocarbamoyl (FTC)-casein solutions of substrates. Reactions were carried out as described in Experimental procedures. Enzyme activity was presented as the absorbance reading at 492 nm. Values represent the mean ± standard deviation (SD) from triplicate determinations of a representative experiment. Similar results were obtained in two independent experiments. WT, rEprS WT; S308A, rEprS S308A. **P < 0.01 (compared with WT).

To test whether purified rEprS WT has proteolytic activity, we assessed for hydrolysis of FTC-casein. Figure 2 shows that rEprS WT is able to hydrolyse casein as a substrate. The proteolytic activity was greatly diminished by changing Ser to Ala at position 308, suggesting the involvement of Ser308 in a catalytic residue. Furthermore, the activity of rEprS WT was markedly decreased in the presence of serine protease inhibitors such as PMSF (Table 1). Pepstatin, E-64 and metallo-protease inhibitors were not inhibitory. Together, these results suggest that P. aeruginosa EprS is a serine protease.

Table 1. Effect of selected inhibitors on the proteolytic activity of rEprS WT
InhibitorInhibitor classActivity (%)
  1. Five micrograms of rEprS WT was pre-incubated with the inhibitor (1 mM) for 1 h at 4°C, and the residual activity was measured as described in Experimental procedures. All absorbance measurements were normalized to those obtained for untreated controls. Values represent the mean ± SD from triplicate determinations of a representative experiment. Similar results were obtained in two independent experiments. PMSF, phenylmethyl-sulfonyl fluoride; EDTA, ethylenediamine tetraacetate; EGTA, ethyleneglycol bis(2-aminoethylether) tetraacetate; DTT, dithiothreitol; E-64, N-[N-(l-3-Trans-carboxirane-2-carbonyl)-l-leucyl]-agmatine.
None 100
Benzamidine 14.6 ± 0.8
PMSFSerine2.4 ± 0.5
LeupeptinSerine/Cysteine8.2 ± 0.8
AprotininSerine10.2 ± 0.8
PepstatinAspartic90.5 ± 1.9
EDTAMetallo94.6 ± 2.0
EGTAMetallo96.1 ± 1.3
1, 10-PhenanthrolineMetallo98.3 ± 1.5
E-64Cysteine97.7 ± 1.6

Oligopeptide cleavage profiles of EprS

To characterize the preference of cleavage of EprS, the relative rates of hydrolysis of various synthetic peptides conjugated at the carboxyl-terminus with 4-methylcoumaryl-7-amide (MCA) were compared. EprS was reacted with a variety of MCA-conjugated peptides and fluorescence of 7-amino-4-methylcoumarin (AMC) liberated by hydrolysis was measured. As shown in Table 2, EprS preferred to cleave substrates that terminated with arginine or lysine residues linked to MCA or substrates with a single arginine or lysine linked to MCA. In contrast, EprS could not efficiently hydrolyse substrates with small or hydrophobic residues, such as Suc-AAA-MCA, Suc-AAPF-MCA, Suc(OMe)-AAPV-MCA, L-MCA, etc. Thus, these results indicate that EprS exhibits the substrate specificity, cleaving at the carboxyl side of basic residues.

Table 2. Substrate specificity of EprS
SubstrateEnzyme typeActivity (%)
  1. Five micrograms of rEprS WT was added separately to 100 μM solutions of MCA-conjugated peptides, and the activity was determined as described in Experimental procedures. The maximum fluorescence value obtained was set to 100%, and other readings were normalized accordingly. Values represent the mean ± SD from triplicate determinations of a representative experiment. Similar results were obtained in two independent experiments. –, non-detectable activity (i.e. < 0.5%); MCA, 4-methylcoumaryl-7-amide; Z, benzyloxycarbonyl; Boc, butyloxycarbonyl; Bz, benzoyl; Suc(OMe), methoxysuccinyl; Pyr, pyroglutamyl; Suc, succinyl; Glt, glutaryl.
R-MCACathepsin H100
Z-FR-MCACathepsin B/L67.2 ± 0.9
Z-RR-MCACathepsin B58.6 ± 4.2
Z-LR-MCACathepsin K/S/V54.3 ± 2.1
Boc-FSR-MCATrypsin45.6 ± 1.4
Boc-LGR-MCATrypsin-like peptidase42.0 ± 1.0
K-MCAAminopeptidase38.4 ± 0.9
PFR-MCAKallikrein38.2 ± 2.1
Bz-R-MCATrypsin29.9 ± 1.0
Boc-VLK-MCAPlasmin23.2 ± 1.0
Z-GPR-MCACathepsin K20.5 ± 1.5
Boc-VPR-MCAThrombin10.4 ± 0.9
Boc-IEGR-MCAFactor Xa7.0 ± 0.9
Z-Pyr-GR-MCAFactor Xa2.1 ± 0.1
Suc(OMe)-AAPV-MCAElastase1.3 ± 0.2
Suc-GPLGP-MCACollagenase-like peptidase0.8 ± 0.2
L-MCAAminopeptidase
M-MCAAminopeptidase
F-MCAAminopeptidase
A-MCAAminopeptidase
Suc-AAPF-MCAChymotrypsin
Glt-AAF-MCAChymotrypsin
Suc-LLVY-MCAChymotrypsin
Suc-AAA-MCAElastase
Suc-APA-MCAElastase
Suc-GP-MCAProlyl endopeptidase
Pyr-MCAPyroglutamyl peptidase
GP-MCAX-prolyl dipeptidyl-aminopeptidase

PARs activation by EprS

Some bacterial proteases have been shown to modulate host inflammatory and immune responses through PAR-1, -2 and -4 (Lourbakos et al., 2001a; 2001b; Chung et al., 2004; Dommisch et al., 2007). We previously reported that P. aeruginosa LepA induces NF-κB activation through PAR-1, -2 and -4 (Kida et al., 2008). We then tested whether rEprS WT activates NF-κB-driven promoter through PAR-1, -2 and -4. To this end, COS-7 cells without functional expression of endogenous PARs were used for transfection experiments. As shown in Fig. 3A, co-transfection of COS-7 cells with human PAR-1, -2 and -4 expression plasmids activated NF-κB-driven promoter in response to stimulation with rEprS WT in a dose-dependent manner with a plateau at 128–512 nM. In contrast, rEprS WT failed to activate NF-κB-driven promoter in COS-7 cells co-transfected with a mock plasmid. Furthermore, proteolytic activity-deficient rEprS S308A abrogated the activation of NF-κB-driven promoter through PAR-1, -2 and -4 (Fig. 3B). The results indicate that the proteolytic activity of EprS is required for activation of PAR-1, -2 and -4. On the other hand, thrombin, used as a positive control for activation of PAR-1 and -4, could induce NF-κB-driven promoter activity through PAR-1 or -4 (Fig. 3B). In addition, trypsin, used as a positive control for activation of PAR-1, -2 and -4, was able to augment transactivation of NF-κB-driven promoter through PAR-1, -2 or -4 (Fig. 3B). Thus, these results suggest that like thrombin and trypsin, EprS can also activate NF-κB-driven promoter through human PAR-1, -2 or -4.

Figure 3.

EprS-induced transactivation of NF-κB-driven promoter in COS-7 cells transfected with human PAR-1, -2 or -4. COS-7 cells were transfected with 100 ng of the appropriate plasmid (phPAR-1, phPAR-2 or phPAR-4 as human PARs expression plasmid), 95 ng of pNF-κB-Luc as a reporter plasmid and 5 ng of phRG-TK as an internal control plasmid. The pcDNA3.1(+) was used to ensure that all transfection mixtures had a total of 200 ng of DNA. After 48 h, transfected cells were stimulated with or without the indicated concentrations of rEprS, thrombin or trypsin. After a further 6 h of incubation, the cells were lysed and assayed for luciferase activity. The data are presented as the relative luciferase activity. The results for each set of transfections were normalized for Renilla luciferase activity and to the unstimulated samples. Values represent the mean ± SD from triplicate determinations of a representative experiment. Similar results were obtained in three independent experiments.

A. Transactivation of NF-κB-driven promoter in response to stimulation with increasing amounts of rEprS WT in human PAR-1, -2 or -4-transfected COS-7 cells.

B. Induction of NF-κB-driven promoter activity stimulated with rEprS WT (250 nM), rEprS S308A (250 nM), thrombin (25 nM) and trypsin (100 nM) in human PARs-transfected COS-7 cells. WT, rEprS WT; S308A, rEprS S308A; Thr, Thorombin; Try, Trypsin. *P < 0.05; **P < 0.01 (compared with unstimulated cells).

EprS induces IL-8 production in a human bronchiole epithelial cell line, EBC-1 cells

We previously reported that Serratia marcescens serralysin and P. aeruginosa LepA induce inflammatory responses through PAR-2 in a human bronchiole epithelial cell line, EBC-1 cells (Kida et al., 2007; 2008). We then determined by ELISA whether rEprS WT can induce IL-8 production in EBC-1 cells. As shown in Fig. 4A, rEprS as well as thrombin and also trypsin induced IL-8 production in EBC-1 cells; however, proteolytic activity-deficient rEprS S308A failed to stimulate IL-8 production. Thus, the results indicate that the proteolytic activity of EprS is required for induction of IL-8 production. Next, we examined whether PAR-2 participates in rEprS WT-stimulated IL-8 production in EBC-1 cells. As shown by the results in Fig. 4B, pre-treatment with PAR-2 antagonist peptides, FSLLRY-NH2 and LSIGRL-NH2, significantly reduced IL-8 production in response to stimulation with rEprS WT. We observed that neither FSLLRY-NH2 nor LSIGRL-NH2 inhibits the proteolytic action of rEprS WT in the protease activity assay (data not shown). In addition, rEprS WT-induced IL-8 production was significantly inhibited by pre-treatment with anti-PAR-2 mAb (Fig. 4C). Therefore, these results suggest that EprS has the ability to induce IL-8 production through PAR-2 in EBC-1 cells.

Figure 4.

EprS induces IL-8 secretion in EBC-1 cells. EBC-1 cells were stimulated with or without rEprS (250 nM), thrombin (25 nM) or trypsin (100 nM) for 16 h and then IL-8 levels in the culture supernatants were determined by ELISA. To inhibit PAR-2 activation, EBC-1 cells were incubated with human PAR-2 antagonist peptides (200 μM) or PAR-2 mAb (10 μg ml−1) for 1 h prior to stimulation.

A. rEprS WT as well as thrombin and also trypsin induce IL-8 production.

B. Human PAR-2 antagonist peptides reduce IL-8 production in response to stimulation with rEprS WT.

C. Anti-PAR-2 mAb blocks rEprS WT-stimulated IL-8 production.

Values represent the mean ± SD from triplicate determinations of a representative experiment. Similar results were obtained in three independent experiments. WT, rEprS WT; S308A, rEprS S308A; Thr, Thorombin; Try, Trypsin. **P < 0.01 (compared with unstimulated cells); ##P < 0.01 (compared with WT-stimulated cells in the absence of the inhibitors for PAR-2 activation); NS, not significant.

Cleavage of PARs tethered ligand region by EprS

The above results indicate that EprS would have the proteolytic activity required to cleave human PAR-1, -2 and -4 at a specific site with exposure of their tethered ligands, as in the cases of thrombin and trypsin. To test this possibility, the peptides corresponding to the region surrounding the tethered ligand of human PAR-1, -2 and -4 were incubated with rEprS WT and proteolytic fragments were analysed by reversed-phase HPLC and MALDI-TOF MS. Thrombin, an agonist for PAR-1 and -4, and trypsin, an agonist for PAR-1, -2 and -4 were used as positive controls. As shown in Fig. 5, the PAR-1 peptide was cleaved at the R41-S42 site by rEprS WT, thrombin or trypsin. The two peptide fragments were identified as NATLDPR and SFLLR, and the measured molecular masses (786.309 and 635.334 for rEprS WT; 786.373 and 635.323 for thrombin; 786.368 and 635.335 for trypsin) were in good agreement with the calculated values of 785.89 and 634.79. The analysis also demonstrated that rEprS WT as well as trypsin can cleave the PAR-2 peptide at the R36-S37 site, generating the two major peptide fragments of SSKGR and SLIGKVDGT (Fig. 5B). The measured molecular mass (534.180 and 889.366 for rEprS WT; 534.142 and 889.350 for trypsin) was compatible with the calculated values of 533.59 and 889.03. In addition, rEprS WT cleaved the PAR-4 peptide at the R47-G48 site, as in the cases of thrombin and trypsin (Fig. 5C). The two major peptide fragments were identified as SILPAPR and GYPGQV, and the molecular masses (753.011 and 620.066 for rEprS WT; 753.111 and 620.003 for thrombin; 753.282 and 620.137 for trypsin) were in good agreement with the calculated values of 752.94 and 619.70. In contrast to rEprS WT, proteolytic activity-deficient rEprS S308A could not cleave the tethered ligand regions of PAR-1, -2 and -4 (Fig. 5A–C). Therefore, these results suggest that EprS has the protease activity required to activate human PAR-1, -2 and -4.

Figure 5.

Analysis of the EprS-cleaved human PARs tethered ligand region peptides. The peptide (50 μM) was incubated for 30 min 37°C with 125 nM rEprS, 25 nM thrombin or 50 nM trypsin and the products were separated by reversed-phase HPLC, then each peak was analysed by MALDI-TOF MS.

A. Treatment of hP1-35/46 with rEprS WT. P1, uncleaved hP1-35/46; P2, NATLDPR; P3; SFLLR.

B. Treatment of hP2-32/45 with rEprS WT. P1, uncleaved hP2-32/45; P2, SSKGR; P3; SLIGKVDGT.

C. Treatment of hP4-41/53 with rEprS WT. P1, uncleaved hP4-41/53; P2, GYPGQV; P3; SILPAPR.

WT, rEprS WT; S308A, rEprS S308A; Thr, Thorombin; Try, Trypsin. AUFS, absorbance units at full scale. The results shown are representative of those for two different experiments.

Secretion of the EprS passenger domain from P. aeruginosa

To elucidate whether P. aeruginosa secretes the endogenous EprS passenger domain, we examined cell-free culture supernatants from P. aeruginosa strains by Western blot analysis using anti-EprS pAb raised to a part of the EprS passenger domain. As shown in Fig. 6, anti-EprS pAb was able to recognize the purified rEprS WT and rEprS S308A proteins (∼52 kDa) used as controls (lanes 1 and 2). As expected, a band with a molecular mass of ∼52 kDa was detected in the supernatant of P. aeruginosa wild-type KU2 strain (lane 3). This size is largely consistent with the predicted molecular weight (51.2 kDa) for the putative secreted passenger domain of EprS (31–520 aa). Conversely, KU2ΔeprS strain, isogenic eprS mutant of P. aeruginosa KU2, failed to produce a band corresponding to that seen with wild-type KU2 (lane 4). In addition, the mutant strain bearing the wild-type eprS gene cloned into an arabinose-inducible expression vector, pCF430, secreted a ∼52 kDa protein identical in size to that secreted by wild-type KU2 strain (lane 6). However, this phenotype was not restored in the mutant strain harbouring mock vector (lane 5). Thus, these results indicate that P. aeruginosa freely secretes the EprS passenger domain into the extracellular milieu.

Figure 6.

Detection of the EprS passenger domain secreted into the culture supernatants of P. aeruginosa strains by Western blot analysis. Culture supernatants of P. aeruginosa strains were collected followed by centrifugal ultrafiltration. The retentates were analysed by Western blot. Purified rEprS WT and rEprS S308A were used as positive controls. Lane 1, rEprS WT (5 ng) purified from E. coli TOP10 harbouring pBAD-eprS/6H WT; lane 2, rEprS S308A (5 ng) purified from E. coli TOP10 harbouring pBAD-eprS/6H S308A; lane 3, proteins (25 μg) secreted from P. aeruginosa KU2; lane 4, proteins (25 μg) secreted from P. aeruginosa KU2ΔeprS; lane 5, proteins (25 μg) secreted from P. aeruginosa KU2ΔeprS harbouring pCF430; lane 6, proteins (25 μg) secreted from P. aeruginosa KU2ΔeprS harbouring pCF-eprS WT. The data shown are representative of those for two different experiments.

Discussion

Of the AT proteases analysed, the SPATEs have been characterized extensively (Henderson et al., 2004; Wells et al., 2010). The SPATEs represent a group of large-sized, multi-domain exoproteins found only in pathogenic enteric bacteria. These proteins include EspC from enteropathogenic E. coli (EPEC), EspP and EpeA from enterohaemorrhagic E. coli (EHEC), EatA from enterotoxigenic E. coli (ETEC), plasmid-encoded toxin (Pet) from enteroaggregative E. coli (EAEC), Sat and PicU from uropathogenic E. coli (UPEC), Tsh from avian pathogenic E. coli, Pic from Shigella flexneri and EAEC, and SepA also from S. flexneri (Yen et al., 2008). Interestingly, although the SPATEs show high homologies among their passenger domains, their substrate specificities do not reveal a correlation with the pathogenetic functions (Dutta et al., 2002). In this article, we describe that P. aeruginosa EprS of the non-SPATEs is a serine protease (Fig. 2 and Table 1) and prefers to hydrolyse substrates that terminated with arginine or lysine residues (Table 2). Using blast analysis, EprS, despite no significant homology with many of the SPATEs, is 35% identical (56% similar) to the sequence of Proteus toxic agglutinin (Pta), a serine protease AT from Proteus mirabilis. The amino acid sequences of the putative catalytic domains forming the catalytic triad of serine protease are highly conserved between EprS and Pta. The homology between them in the putative catalytic domains is as follows: the catalytic Asp-containing domain, 60% identical (80% similar); the catalytic His-containing domain, 80% identical (100% similar); and the catalytic Ser-containing domain, 91% identical (100% similar). However, Pta that functions both as a cytotoxin and as an agglutinin has been demonstrated to exhibit the disparate substrate specificity, cleaving at the carboxyl side of hydrophobic residues such as leucine and phenylalanine (Alamuri and Mobley, 2008). Thus, it is feasible that, despite the homology observed between EprS and Pta, these proteins may have different pathogenetic functions only partly dependent on their substrate specificities.

As shown by the results in Table 2, EprS was able to hydrolyse synthetic aminopeptidase substrates such as R-MCA and K-MCA as well as the synthetic peptide substrates of endopeptidase like Boc-FSR-MCA, Boc-LGR-MCA, Bz-R-MCA, Boc-VPR-MCA, etc. Accordingly, EprS is assumed to possess both exopeptidase and endopeptidase activities in at least our experiments using synthetic substrates. Since cathepsin H that preferentially cleaves the substrates at the carboxyl side of basic residues has been demonstrated to be an aminopeptidase and also display endopeptidase activity (Barrett and Kirschke, 1981; Rothe and Dodt, 1992; Vasiljeva et al., 2003), it is likely that the enzymatic properties of EprS are similar to those of cathepsin H.

Some members of the AT protease family, such as Hap and immunoglobulin A1 protease from Haemophilus influenzae, have been reported to rely on autoproteolysis involving the serine protease active site for processing and release of the passenger domain from the outer membrane (Hendrixson et al., 1997; Henderson et al., 1998; Fink et al., 2001). On the other hand, the passenger domain of the SPATEs bearing mutations within the catalytic triad of serine protease is normally released from the bacterial surface via a process that does not involve this particular motif (Henderson et al., 1999; Navarro-Garcia et al., 1999; Maroncle et al., 2006). Using blast analysis, the serine protease AT from S. marcescens (Ssp) as well as Pta has homology to EprS (41% identical and 55% similar). The mutant protein of Ssp with no proteolytic activity because of the change of the catalytic residue Ser to Thr was still secreted into extracellular milieu (Shikata et al., 1993). Indeed, we demonstrated that proteolytic activity-deficient rEprS can be purified from culture supernatants of E. coli TOP10 expressing rEprS S308A (Fig. 2). Therefore, it is possible that EprS serine protease activity would not affect the translocation of the mature passenger domain and the release of EprS into the culture supernatant, suggesting other endogenous proteases are involved in the release of EprS from the bacterial surface.

PARs are expressed in various tissues and cells, including circulatory, gastrointestinal, respiratory, immune, haematopoietic and central nervous systems (Macfarlane et al., 2001; Hollenberg and Compton, 2002; Ossovskaya and Bunnett, 2004). Therefore, PARs activation contributes to a variety of physiological and pathophysiological roles, including development, growth, immunity, inflammation, tissue repair and pain (Ramachandran and Hollenberg, 2008; Ramachandran et al., 2012). To date, four PARs, named in the chronological order of their discovery as PAR-1, -2, -3 and -4, have been identified. Three of them (PAR-1, -3 and -4) are thrombin receptors, whereas PAR-1 and -4 can also be activated by trypsin. In contrast, the fourth (PAR-2) is not activated by thrombin but by a large number of other proteases, including trypsin, tryptase, factor Xa, neutrophil protease 3, membrane-tethered serine protease 1 and house dust mite proteases (Ossovskaya and Bunnett, 2004). In particular, PAR-3 does not signal but rather can act as an accessory receptor for either PAR-1 or PAR-4 (Nakanishi-Matsui et al., 2000; McLaughlin et al., 2007), while thrombin-mediated PAR-3 activation has been shown to be able to trigger signals independent of other thrombin receptors (Ostrowska and Reiser, 2008). The activation of PAR-1, -2 and -4 has been reported to modulate host inflammatory and immune responses (Shpacovitch et al., 2007; Potempa and Pike, 2009; Rothmeier and Ruf, 2012). However, our understanding of PAR-3 signalling and its functional role, in contrast to the other members of the PAR family, is still unclear and confined to relatively few published findings. In this report, we demonstrated that EprS activates host inflammatory responses through PAR-1, -2 and -4 (Figs 3 and 4) and cleaves the peptides corresponding to the tethered ligand region of human PAR-1, -2 and -4 at a specific site with exposure of their tethered ligands (Fig. 5), whereas whether EprS can activate PAR-3 remains to be elucidated.

In this study, we showed that P. aeruginosa EprS has the substrate specificity, cleaving at the carboxyl side of arginine or lysine residues (Table 2). Moreover, EprS cleaved the peptides corresponding to the tethered ligand region of human PAR-1, -2 and -4 at a specific site with exposure of their tethered ligands, as in the cases of thrombin and trypsin (Fig. 5). The activation of PARs has been reported to be initiated by proteolytic cleavage at a specific basic amino acid residue in the amino-terminal domain of the receptor, resulting in the generation of a new tethered ligand that interacts with the receptor within the extracellular loop 2 (Macfarlane et al., 2001; Hollenberg and Compton, 2002). Also, PARs have been shown to be activated by proteases such as thrombin and trypsin that cleave peptide bonds after arginine or lysine (Ossovskaya and Bunnett, 2004; Shpacovitch et al., 2007). Thus, these findings support our data that like thrombin and trypsin, EprS can also activate host inflammatory responses through PAR-1, -2 or -4.

Although little is known about the expression levels of EprS protein in P. aeruginosa, we showed that P. aeruginosa secretes the EprS passenger domain into culture supernatant (Fig. 6). Recently, the transcriptional level of eprS gene has been demonstrated to be upregulated by quorum-sensing (QS) response in P. aeruginosa (Gilbert et al., 2009). In this organism, QS response plays an important role in the pathogenicity of P. aeruginosa (Lyczak et al., 2000; Deng and Barbieri, 2008) and controls the expression of numerous virulence factors including extracellular proteases (alkaline protease, elastase, protease IV, etc.) and toxins (exotoxin A, exoenzyme S, haemolysin, etc.) (Rumbaugh et al., 2000; Smith and Iglewski, 2003; Girard and Bloemberg, 2008; Jimenez et al., 2012). Hence, EprS, the expression of which is regulated by QS response may also be involved in the virulence of P. aeruginosa.

In summary, to investigate the biological function of P. aeruginosa EprS, we purified and characterized rEprS secreted by E. coli. Our results suggest that EprS, a serine protease, exhibits the substrate specificity, cleaving at the carboxyl side of basic residues. Furthermore, we demonstrated that EprS has the proteolytic activity required to activate human PAR-1, -2 and -4. Thus, these results suggest that bacterial proteases such as an EprS would require PARs or other unknown mechanisms to modulate various host responses against bacterial infection. In addition, we demonstrated that P. aeruginosa is able to secrete the endogenous EprS passenger domain into the extracellular environment. To further investigate whether EprS participates in the pathogenicity of P. aeruginosa, the evaluation of eprS-disrupted strain with respect to the virulence is now in progress in our laboratory.

Experimental procedures

Bacterial strains and plasmids

The bacterial strains and plasmids used in this study are listed in Table S1. All bacterial strains were grown in Luria–Bertani (LB) medium (LB-Miller; Nacalai Tesque, Kyoto, Japan) unless otherwise noted. Vogel–Bonner minimal medium (VBMM) was used for selection of transconjugants from conjugation mixtures (Schweizer, 1992). VBMM is VB medium (Vogel and Bonner, 1956) containing 0.3% trisodium citrate and is selective for P. aeruginosa since E. coli cannot utilize citrate. Antibiotics were used in selection media at the following concentrations: ampicillin, 100 μg ml−1 (E. coli); carbenicillin, 500 μg ml−1 (P. aeruginosa); gentamicin, 100 μg ml−1 (P. aeruginosa); and tetracycline (10 μg ml−1 for E. coli and 100 μg ml−1 for P. aeruginosa).

Reagents

All protease inhibitors were obtained from Nacalai Tesque. Bovine pancreas-derived endotoxin-free trypsin (endotoxin contents, < 0.2 ng mg-protein−1) and human plasma-derived thrombin (endotoxin contents, < 0.1 ng mg-protein−1) were purchased from Calbiochem (San Diego, CA). Normal mouse immunoglobulin G2a (IgG2a) and mouse anti-PAR-2 monoclonal antibody (mAb) (clone, SAM11; isotype, IgG2a) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). The peptides of human PARs tethered ligand region were synthesized by solid-phase methods at Invitrogen (Carlsbad, CA): hP1-35/46 (corresponding to human PAR-1 residues 35–46, N35ATLDPRSFLLR46), hP2-32/45 (corresponding to human PAR-2 residues 32–45, S32SKGRSLIGKVDGT45) and hP4-41/53 (corresponding to human PAR-4 residues 41–53, S41ILPAPRGYPGQV53) (tethered ligand sequences are underlined). The peptides of human PAR-2 antagonist (FSLLRY-NH2 and LSIGRL-NH2) were synthesized with amidated carboxyl-terminus by solid-phase methods at Sigma-Aldrich (St. Louis, MO). These antagonist peptides have been shown not to inhibit the proteolytic activity of trypsin but to block trypsin-induced activation of PAR-2 by a mechanism through which they possibly interact with a tethered ligand receptor-docking site (Al-Ani et al., 2002). All peptides were > 95% purity by HPLC and mass spectrometry analysis. Stock solutions were prepared in 50% DMSO and stored at −20°C.

Cells

A human lung squamous cell carcinoma, EBC-l cells (Cell Resource Center for Biomedical Research Institute of Development, Aging and Cancer, Tohoku University, Sendai, Japan), was maintained in RPMI 1640 medium (Nissui Pharmaceutical, Tokyo, Japan) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Gibco, Rockville, MD), 2 mM l-glutamine, 0.2% sodium bicarbonate, 100 U ml−1 penicillin and 100 μg ml−1 streptomycin (Kida et al., 2006). An African green monkey kidney fibroblast-like cell line, COS-7 cells (Health Science Research Resources Bank, Osaka, Japan), which has no functional expression of endogenous PARs, was maintained in Dulbecco's modified Eagle's medium (DMEM; Nissui Pharmaceutical) supplemented with 10% heat-inactivated FBS, 4 mM l-glutamine, 0.35% d-glucose, 0.37% sodium bicarbonate, 100 U ml−1 penicillin and 100 μg ml−1 streptomycin (Molino et al., 1997). Cells were passaged without the use of trypsin by non-enzymatic cell dissociation solution (Gibco) to minimize the proteolytic activation of the PARs.

Construction of plasmids

The 3.0 kb gene eprS was amplified from P. aeruginosa PAO1 genomic DNA by PCR. The following eprS-specific primers were used: sense, 5′-CAGGAGGAATTAACCATGACCGACGACCACTCCTTCCGCCCTCGCCCC-3′ (underline indicates 15 bp region required for cloning); and antisense, 5′-GGAGACCGTTTAAACTCAGAAACGCCAGTCGACGGCCAGTCCGACGCC-3′ (underline indicates 15 bp region required for cloning). To prepare the vector for cloning by an In-Fusion cloning kit (Takara), pBAD/myc-His A (Invitrogen) was amplified by PCR using a sense primer, 5′-GTTTAAACGGTCTCCAGCTTGGCTGTTTTGGCGG-3′ and an antisense primer, 5′-GGTTAATTCCTCCTGTTAGCCCAAAAAACGGGTATGG-3′. PCR was performed with a PrimeSTAR GXL DNA polymerase (Takara, Ohtsu, Japan) according to the manufacturer's protocol. The PCR-amplified eprS was cloned into the PCR-amplified pBAD/myc-His A by an In-Fusion cloning kit according to the manufacturer's protocol. The resultant plasmid was named pBAD-eprS wild-type (WT).

To construct the expression plasmid for six histidine-tagged EprS protein, pBAD-eprS WT was mutated by PCR-based mutagenesis (Imai et al., 1991). The following primers were used: sense, 5′-CATCATCACGTCGAAGCCGGTCGGCCCGGC-3′ (underline indicates mutated sequences encoding three histidine residues); antisense, 5′-GTGGTGATGATAGGCGGCCGTGGCCGGCTG-3′ (underline indicates mutated sequences encoding three histidine residues). The mutated construct was designated pBAD-eprS/6H WT. To generate Ser308 to Ala308 point mutation of EprS protein, pBAD-eprS/6H WT was mutated by PCR using a sense primer, 5′-CAAGTCCGGTACGGCCATGGCGGCCCCCCACGCCACC-3′ (underline indicates mutated sequences), and an antisense primer, 5′-GGTGGCGTGGGGGGCCGCCATGGCCGTACCGGACTTG-3′ (underline indicates mutated sequences). The resultant plasmid was designated pBAD-eprS/6H S308A. Each expression plasmid was generated by inserting a six histidine tag between residues 32 and 33 of downstream of the putative amino-terminal amino acid. The presence of the correct sequences was confirmed by sequencing these plasmids.

Purification of recombinant EprS

Escherichia coli TOP10 harbouring pBAD-eprS/6H WT or pBAD-eprS/6H S308A was grown in LB broth containing ampicillin (200 μg ml−1) at 21°C with rotary shaking at 150 r.p.m. (AT-12R shaker; Thomas, Tokyo, Japan) to an optical density at 600 nm of 0.5. Recombinant EprS (rEprS) expression was induced by the addition of l-arabinose at a final concentration of 0.002%, and the incubation was continued for an additional 20 h. Bacterial cells were removed from the medium by centrifugation (8000 g, 30 min, 4°C) and rEprS was purified from the supernatant. Briefly, proteins in the supernatant were precipitated with ammonium sulfate (80% saturation). The precipitate was dissolved in 20 mM Tris-HCl (pH 8.0), dialysed, and loaded onto a Q-Sepharose anion-exchange column (GE Healthcare, Uppsala, Sweden) equilibrated with the same buffer. Then, the flow-through fractions containing rEprS were collected and adjusted to a buffer composition of 20 mM Tris-HCl (pH 8.0) containing 500 mM NaCl, 20 mM imidazole and 1% CHAPS (equilibration buffer), followed by application to a Ni-Sepharose immobilized metal ion affinity column (GE Healthcare) equilibrated with equilibration buffer. The column was washed with equilibration buffer until non-specific material was removed. Elution of rEprS was carried out using 100 mM imidazole in 20 mM Tris-HCl (pH 8.0) containing 500 mM NaCl, followed by dialysis against phosphate-buffered saline (PBS). The purity of rEprS was examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The endotoxin level of purified rEprS was estimated with a Limulus Amebocyte Lysate QCL-1000 (Cambrex, Walkersville, MD) and was revealed to be < 1 pg ml−1 when suspended in PBS at a protein concentration of 100 nM. Protein concentration was determined with a Coomassie Protein Assay Reagent (Pierce, Rockford, IL) using bovine serum albumin as a standard.

Proteolytic activity assay

The enzyme activities of the purified proteins were determined with a protease assay kit (Calbiochem). In brief, 5 μg of the purified proteins was added to 100 μM fluorescein thiocarbamoyl (FTC)-casein solutions of substrates in a buffer containing 50 mM Tris-HCl (pH 8.0) and 5 mM CaCl2. Reactions were performed in a total volume of 100 μl at 37°C for 20 h. Then, the reactions were stopped by adding 250 μl of 5% trichloroacetic acid (TCA), followed by centrifugation and measurement of absorbance at 492 nm of the TCA-soluble fractions using a DU730 spectrophotometer (Beckman Coulter, Brea, CA). Enzyme activity was expressed as the absorbance reading at 492 nm. To determine the effect of inhibitors, the enzyme (5 μg) was pre-incubated with the inhibitor (1 mM) for 1 h at 4°C in the above-mentioned buffer and the residual activity was measured as described above. The absorbance value obtained for untreated controls was set to 100%, and other readings were normalized accordingly. All reactions were performed in triplicate, and the mean ± standard deviation (SD) was calculated.

Oligopeptide cleavage profile

Peptidyl-4-methylcoumaryl-7-amide (peptidyl-MCA) substrates (Peptide Institute, Osaka, Japan) were prepared as 10 mM stock solutions in DMSO. Five micrograms of rEprS WT was added separately to 100 μM solutions of MCA-conjugated peptides in a buffer containing 50 mM Tris-HCl (pH 8.0) and 5 mM CaCl2. Reactions were performed in a total volume of 100 μl in 96-well microtitre plates (Nalge Nunc, Rochester, NY). After 6 h of incubation at 37°C, end-point fluorescence of released 7-amino-4-methylcoumarin (AMC) was measured using a Twinkle LB970 microplate fluorometer (Berthold, Wildbad, Germany) with excitation at 355 nm and emission at 460 nm. Background values derived from the enzyme-free control samples were subtracted from the fluorescence measurements. The maximum fluorescence value obtained was set to 100%, and other readings were normalized accordingly. All reactions were performed in triplicate, and the mean ± SD was calculated.

Transient transfection of COS-7 cells and luciferase assay

COS-7 cells maintained as described above were detached from the culture flask with non-enzymatic cell dissociation solution and washed three times in serum-free DMEM/F-12 (Gibco). Then, the cells were seeded into 24-well plates (Costar, Cambridge, MA) at a density of 5 × 104 cells per well in serum-free DMEM/F-12 and incubated for 24 h. One hour before transfection, the growth medium was replaced by fresh serum-free DMEM/F-12. Transient transfections were performed with 100 ng of the appropriate plasmids (phPAR-1, phPAR-2 and phPAR-4 as human PARs expression plasmids), 95 ng of pNF-κB-Luc as a reporter plasmid and 5 ng of phRG-TK (Promega, Madison, WI) as an internal control plasmid using the FuGENE HD transfection reagent (Promega), according to the manufacturer's protocol. After 48 h, transfected cells were stimulated with or without the indicated concentrations of rEprS, thrombin or trypsin. After a further 6 h of incubation, cells were lysed and assayed for luciferase activity using a Dual-Glo Luciferase Assay System (Promega). Both firefly and Renilla luciferase activities were monitored with a GloMax 96 microplate luminometer (Promega). Normalized reporter activity was expressed as the firefly luciferase value divided by the Renilla luciferase value. Relative fold induction was calculated as the normalized reporter activity of the stimulated samples divided by the unstimulated samples.

Cleavage of human PARs tethered ligand region by rEprS WT

The peptides of human PARs tethered ligand region (50 μM): hP1-35/46, hP2-32/45 and hP4-41/53 were incubated for 30 min at 37°C with 125 nM rEprS WT in 20 mM Tris-HCl (pH 8.0) containing 1 mM CaCl2, then the digested materials were separated by reversed-phase HPLC on a Cosmosil 5C18-AR-300 column (4.6 × 250 mm; Nacalai Tesque) using a linear gradient of acetonitrile (5–45% for hP1-35/46, 0–40% for hP2-32/45 and 5–45% for hP4-41/53) in 0.1% TFA for 40 min at a flow rate of 1 ml min−1 and each peak was analysed by a matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) (Autoflex; Bruker Daltonics, Bremen, Germany). As controls, the peptides were incubated under the same experimental conditions with 25 nM thrombin, which cleaves human PAR-1 and -4 tethered ligand region, or with 50 nM trypsin, which cleaves human PAR-1, -2 and -4 tethered ligand region.

Measurement of IL-8 secretion

EBC-1 cells prepared as described above were washed three times in serum-free DMEM/F-12 (Gibco). The cells were then seeded into 24-well plates (Costar) at a density of 3 × 105 cells per well in serum-free DMEM/F-12 and incubated for 16 h. One hour before stimulation, the growth medium was replaced by fresh serum-free DMEM/F-12. Cells were stimulated with or without rEprS (250 nM), thrombin (25 nM) or trypsin (100 nM) for 16 h and the culture supernatants were collected and stored at −80°C until assayed. To block PAR-2 activation, EBC-1 cells were incubated with human PAR-2 antagonist peptides (200 μM) or PAR-2 mAb (10 μg ml−1) for 1 h prior to stimulation. The levels of IL-8 in the culture supernatants were measured using enzyme-linked immunosorbent assay (ELISA) kit (R&D systems, Minneapolis, MN) according to the manufacturer's protocol.

Replacement of the eprS region of P. aeruginosa KU2

Allele replacement of the eprS was performed by a modified method of Schweizer (Schweizer, 1992). In brief, a 4.0 kb PCR fragment containing eprS was amplified from P. aeruginosa PAO1 genomic DNA by using primers U/epr (5′-GGAAACTCTAGACAACAGCGCCGCATCCTGGCCGGGACGAATG-3′, underline indicates XbaI restriction site) and D/epr (5′-GGAAACAAGCTTTCAGAAACGCCAGTCGACGGCCAGTCCGACGCC-3′, underline indicates HindIII restriction site). After digestion with XbaI and HindIII, the resulting fragments were cloned into pUC18Not, producing plasmid pYK6. A 0.8 kb PCR fragment containing a Genr cassette was amplified from pJN105 by using primers Gen-F (5′-GGAAACAGTACTGCGGCGTTGTGACAATTTACCGAACAACTCC-3′, underline indicates ScaI restriction site) and Gen-R (5′-GGAAACCTGCAGCCCAGTTGACATAAGCCTGTTCGGTTCGTAAAC-3′, underline indicates PstI restriction site). After digested with ScaI and PstI, the resulting fragments were cloned into pYK6, which was digested with EcoRV and PstI to yield plasmid pYK6-Gm. The ΔeprS::Genr fragment was then subcloned into the NotI site of the suicide vector pYK1-T, which has the oriT for conjugative transfer and the counter selectable-marker sacB, producing plasmid pYK7. This plasmid was used for allelic exchange and conjugated from E. coli S17-1λpir into P. aeruginosa KU2 on LB agar using filters.

Merodiploid single-cross-over mutants were selected from the conjugation mixture by plating on VBMM agar containing 100 μg ml−1 gentamicin. Purified single-cross-over mutants were cultured overnight in LB broth without antibiotics. This culture was then serially diluted in saline and plated on LB agar containing 100 μg ml−1 gentamicin and 7% sucrose to select against the sacB marker present on the pYK7 vector, and hence select for strains that had undergone a second homologous recombination event resulting in loss of the pYK7 vector. This was confirmed by the loss of the vector-encoded carbenicillin resistance. In addition, the double-cross-over mutants were confirmed by PCR using the primers U/epr and D/epr (data not shown).

For complementation, wild-type copy of eprS was cloned into an arabinose-inducible broad-host-range expression vector, pCF430. Briefly, the eprS was amplified from P. aeruginosa PAO1 genomic DNA by PCR using a sense primer, 5′-GGAAACGCTAGCATGACCGACGACCACTCCTTCCGCCCTCGCCCC-3′ (underline indicates NheI restriction site) and an antisense primer, 5′-GGAAACAAGCTTTCAGAAACGCCAGTCGACGGCCAGTCCGACGCC-3′ (underline indicates HindIII restriction site). The PCR products were digested with NheI and HindIII and cloned into a pCF430. The resultant plasmid was named pCF-eprS WT. This plasmid was used for complementation and conjugated from E. coli S17-1λpir into P. aeruginosa KU2ΔeprS on LB agar using filters. Cells were suspended in saline and plated on VBMM agar containing 100 μg ml−1 tetracycline. After overnight incubation at 37°C, tetracycline-resistant colonies were obtained. To identify the clones of P. aeruginosa KU2ΔeprS containing the pCF-eprS WT plasmid, the plasmid DNA was isolated from tetracycline-resistant strains, followed by application to agarose gel electrophoresis (data not shown).

Preparation of P. aeruginosa-secreted proteins

Pseudomonas aeruginosa KU2 or KU2ΔeprS was grown in LB broth for 24 h at 37°C with rotary shaking at 150 r.p.m. P. aeruginosa KU2ΔeprS harbouring pCF430 or pCF-eprS WT was grown in LB broth containing tetracycline (25 μg ml−1) and l-arabinose (5%) as described above. Culture supernatants were harvested by centrifugation (13 000 g, 10 min, 4°C) and passed through a 0.2-μm-pore-size filter to remove residual bacteria. Cell-free culture supernatants were concentrated with 10 kDa cut-off spin filter devices (Millipore, Billerica, MA) and the concentration of protein in the retentates was determined as described above.

Western blot analysis

Rabbit anti-EprS polyclonal antibody (pAb) was raised against keyhole limpet haemocyanin-conjugated synthetic peptide corresponding to EprS residues 466–479, L466SGDSTYRGPTLVD479. The antiserum was purified by peptide affinity chromatography. Proteins were separated by SDS-PAGE on a 10% gel and transferred onto nitrocellulose membrane. Non-specific binding was blocked by incubation of the membrane in 5% skimmed milk (Difco, Detroit, MI) in Tris-buffered saline (TBS) buffer [20 mM Tris-HCl (pH 8.0) and 0.15 M NaCl] containing 0.1% Tween-20 (TBS-T) for 1 h at room temperature. The membrane was rinsed in TBS-T and incubated with the rabbit anti-EprS pAb (1:1000 dilution) in 0.5% skimmed milk in TBS-T. After 1 h incubation at room temperature, the membrane was incubated for 1 h with peroxidase-conjugated goat anti-rabbit immunoglobulin (Cappel Research, Durham, NC) in 0.5% skimmed milk in TBS-T at room temperature. Bands were visualized by an ImmunoStar LD (Wako, Osaka, Japan) and analysed with LAS-4000 (GE Healthcare).

Statistical evaluation

Data were analysed by Student's paired t-test. Data with a P-value of < 0.05 were considered significant.

Acknowledgements

This work was supported in part by a Grant-in-Aid for Young Scientists (B) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan and by the research funds from the Kurume University Millennium Box Foundation for the Promotion of Science and the Takeda Science Foundation.

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