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(Investigative Ophthalmology and Visual Science. 2003;44:190-196.)
© 2003 by The Association for Research in Vision and Ophthalmology, Inc.
DOI:  10.1167/iovs.02-0458
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Molecular Analysis of Pseudomonas aeruginosa Protease IV Expressed in Pseudomonas putida

Mullika Traidej,1 Armando R. Caballero,1 Mary E. Marquart,1 Brett A. Thibodeaux,1 and Richard J. O’Callaghan1,2

1From the Department of Microbiology, Immunology, and Parasitology, Louisiana State University Health Sciences Center, New Orleans, Louisiana; and the 2Louisiana State University Eye Center, New Orleans, Louisiana.


   Abstract
Top
 Abstract
Methods
 Results
 Discussion
 References
 
PURPOSE. In this study, the protease IV gene of Pseudomonas aeruginosa was expressed in the nonocular pathogenic host, Pseudomonas putida, to elucidate the molecular properties and virulence contribution of the enzyme. Recent determination of the protease IV gene sequence suggests that the protein of 463 amino acids contains a signal sequence, a propeptide domain, and a mature protease. The only form of this protein that has been detected previously is the extracellular mature protease.

METHODS. The protease IV gene was cloned and expressed in a protease IV-negative Pseudomonas species, P. putida. The cloned protease IV gene product was analyzed to identify biochemical, enzymatic, and immunologic properties and its contribution to corneal virulence.

RESULTS. P. putida expressing the cloned protease IV gene had significantly greater extracellular enzyme activity than P. aeruginosa. These P. putida cell extracts produced a protein with the same molecular mass as mature protease IV and two other polypeptides representing larger precursors, all of which were recognized by protease IV–specific antibodies. P. putida producing protease IV, relative to P. putida with the vector alone, caused a threefold increase in ocular inflammation and tissue damage when intrastromally injected into rabbit corneas.

CONCLUSIONS. The present study demonstrates for the first time that protease IV is synthesized as a large precursor that is processed intracellularly through an intermediate form and secreted into the extracellular milieu as a mature protease. The results also confirm a significant correlation between production of protease IV and corneal virulence.

The Gram-negative bacterium Pseudomonas aeruginosa is a major opportunistic human pathogen. Individuals with cystic fibrosis, AIDS, burn wounds, or certain cancers are highly predisposed to P. aeruginosa infection.1 2 3 4 P. aeruginosa is also well recognized as an ocular pathogen that causes severe keratitis, especially in contact lens wearers.5 6 7 P. aeruginosa produces several extracellular enzymes that are important in its pathogenicity. Among these, protease IV, a lysine-specific endoprotease, has been demonstrated to correlate with corneal virulence.8 9 10 11 Several lines of evidence support this correlation: The production of protease IV correlates with corneal virulence in rabbit and mouse models of keratitis; P. aeruginosa deficient in protease IV produces significantly reduced corneal damage; exogenous protease IV can restore corneal virulence to a protease IV–deficient mutant; and protease IV is capable of degrading a variety of host defense proteins including the complement protein C3 and immunoglobulin G.9 10 11

Little is known about the processing and transport of protease IV. Our laboratory has recently sequenced the protease IV gene from P. aeruginosa strain PA103-29, which is deficient in exotoxin A, elastase, and alkaline protease activity. The protease IV gene is located on a 1388-bp open reading frame, encoding a protein of 463 amino acids. Computer analysis of the amino acid sequence predicts that a 48.2 kDa full-length protease IV consists of three domains: signal sequence, propeptide domain, and mature protease (Fig. 1) .12 13 However, there has been no report describing the presence of the predicted protease IV precursors. The only molecular form of protease IV that has been identified is the mature protease.



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  		FIGURE 1. Primary translation product of the protease IV gene. Computer analysis of the amino acid sequence predicts that a 48.2-kDa, full-length protease IV consists of three domains: signal sequence (SS), propeptide domain (P) and mature protease (M). Arrows: predicted cleavage sites after processing. The first site is located between amino acids 24 (Alanine) and 25 (Alanine), separating the signal sequence from the propeptide and mature protease. ASA/AP represents the amino acid sequence at the first cleavage site. The second site is located at Lys-211 (K) and releases the mature protease from the propeptide domain.

 
The 26-kDa mature protease is an extracellular enzyme that specifically cleaves substrates on the carboxyl side of lysine residues.14 Purified mature protease IV could undergo autodigestion before or during secretion, because a lysine residue is present between the putative propeptide domain and the mature protease.8

The study of protease IV has been hindered because of the low production of the enzyme under the control of its native promoter on the P. aeruginosa chromosome. In the present study, this limitation was overcome by the construction of a plasmid with the protease IV gene under the control of a lac promoter. This construct expressed functional protease IV in a new host species, Pseudomonas putida. P. putida was selected as a heterologous host for expression of the protease IV gene, because it is a nonocular pathogen in humans, is devoid of protease IV production, is closely related to P. aeruginosa, and is useful as an expression host for other P. aeruginosa extracellular proteins (e.g., elastase B).15 In addition, P. putida has been determined to be nonvirulent in rabbit corneas (Traide JM, unpublished findings, 2002). Our results demonstrate, for the first time, that protease IV is synthesized as a precursor protein that is processed and then secreted extracellularly as a 26-kDa mature protease. The full-length 48-kDa precursor and the 45-kDa intermediate that contained the propeptide and mature protease domains of protease IV were detected only when the protease IV gene was present in P. putida. In addition, expression of protease IV in a heterologous host has shown protease IV to be a corneal virulence factor in the rabbit cornea.


   Methods
Top
 Abstract
 Methods
Results
 Discussion
 References
 
Bacterial Strains, Plasmids, and Growth Conditions
P. aeruginosa strain PA103-29 and P. putida KT2440 (American Type Culture Collection no.47054; ATTC, Manassas, VA) were cultivated in tryptic soy broth (TSB; Difco, Detroit, MI) or M9 minimal medium containing 50 mM monosodium glutamate, 1 mM MgSO4, and 1% glycerol. Growth of bacterial cultures was performed at 37°C, as described previously.8 When appropriate, carbenicillin (100 µg/mL) was incorporated into the medium for plasmid selection. P. aeruginosa strain PA103-29 was kindly provided by Paul V. Phibbs (East Carolina University, Greenville, NC) and was originally described by Ohman et al.16 P. putida was purchased from ATTC. The plasmid pUCP20 was obtained from Jeffery A. Hobden (Wayne State University, Detroit, MI) and was originally described by West et al.17

Construction of Plasmids Expressing Protease IV
The protease IV gene was amplified from P. aeruginosa strain PA103-29, using the GC-rich PCR system (Roche, Indianapolis, IN) under the following conditions: 3 minutes at 95°C followed by 30 cycles of 30 seconds at 95°C for denaturing, 30 seconds at 60°C for annealing, and 2 minutes at 72°C for extension and ending with an incubation at 72°C for 7 minutes. Oligonucleotide primers were designed to amplify the promoterless protease IV gene with recognition sites for the restriction enzyme EcoRI at the locus coding for the N-terminal amino acids (5'-cggaattccatgcataagagaacgtacctgaat-3') and the restriction enzyme BamHI site at the locus coding for the C-terminal amino acids (5'-ggatcctcagggcgcgaagtagcgggagat-3'). Oligonucleotide primers were synthesized by Core Laboratories, Louisiana State University Health Sciences Center. The PCR products were ligated into a PCR cloning vector (TOPO TA cloning; Invitrogen, Carlsbad, CA) and transformed into chemically competent TOP10 Escherichia coli, as described by the manufacturer. Plasmid DNA was purified from transformants, and the protease IV gene was excised from the cloning vector by EcoRI-BamHI restriction digestion. The EcoRI-BamHI DNA fragment containing the protease IV gene was subcloned into the E. coli- Pseudomonas shuttle vector, pUCP20, and transformed into P. putida (as described later). The resultant plasmid with the protease IV gene was designated pPIV.

Purification of plasmid DNA was performed by the alkali lysis method using a kit (QIAprep Spin Miniprep; Qiagen Inc., Valencia, CA). Large-scale plasmid preparations were also performed by a kit (Plasmid Midi; Qiagen). Restriction enzymes and T4 DNA ligase were purchased from New England Biolabs, Inc. (Beverly, MA).

Agarose Gel Electrophoresis and DNA Fragment Isolation
Electrophoresis was performed as described by Sambrook et al.18 with 1% agarose (Sigma, St. Louis, MO) in Tris-acetate-EDTA (TAE) buffer. The presence of DNA was visualized by addition of ethidium bromide (0.5 µg/ml). For DNA fragment isolation, samples were electrophoresed in a 1% agarose gel (SeaPlaque GTG; BioWhittaker Molecular Applications, Rockland, ME) in TAE buffer, and fragments were purified with a gel extraction kit (QIAquick; Qiagen).

Transformation
P. putida was transformed by chemical treatment with MgCl2 with some modifications.19 Bacteria were grown in TSB to optical density at 650 nm (OD650) = 0.2 and centrifuged at 8000g for 15 minutes to pellet the cells. Bacteria were resuspended in 0.15 M MgCl2 and incubated on ice for 5 minutes. This step was repeated by resuspending the cells in 0.15 M MgCl2 and incubating on ice for 20 minutes. Bacteria were centrifuged, the supernatant was decanted, and the cells were resuspended in 0.15 M MgCl2. The plasmid DNA (1–5 µg) was added to 200-µL aliquots of bacteria (103 CFU) and incubated on ice for 1 hour. The bacteria were then heat pulsed at 37°C for 3 minutes and incubated on ice for 5 minutes, after which 500 µL TSB was added to the suspension. The cells were then incubated at 37°C for 1 to 2 hours. Transformants were selected by plating on tryptic soy agar (TSA, Difco) containing 100 µg/mL carbenicillin.

Purification of Protease IV
The purification of protease IV was performed as described by Engel et al.8 with some modifications. Briefly, P. aeruginosa strain PA103-29 or P. putida carrying a plasmid with the protease IV gene was grown overnight at 37°C in M9 minimal medium and then subcultured in the same medium (1 L) to log phase. The log phase culture was added to 15 L M9 medium at 37°C and incubated with vigorous stirring and aeration. The culture was centrifuged (8000g for 20 minutes) to pellet the cells. The supernatant was filtered through a capsule filter (0.45 µm; Versapor Membrane; Pall Life Sciences, Ann Arbor, MI). The filtrate was concentrated to approximately 100 mL by using an ultrafiltration device with a 10-kDa cutoff spiral cartridge filter membrane, and sodium azide was added to a concentration of 0.02% (wt/vol). The concentrated supernatant was dialyzed overnight against 10 mM ammonium acetate buffer (pH 6.4). The dialyzed supernatant was applied to a cation exchange matrix (CM; Bio-Rad Laboratories, Hercules, CA) and was washed with 10 mM ammonium acetate buffer (pH 6.4). A pH gradient, generated by mixing 10 mM ammonium acetate buffer at pH 9.0 with 10 mM ammonium acetate buffer at pH 6.4, was used to elute the protein. The eluted fractions containing protease IV were assayed for their reactivity with a chromogen substrate (Chromozym PL; [tosyl-gly-pro-lys-p-nitroanilide]; Sigma), and active fractions were pooled. The pooled fractions were then concentrated to 1.0 mL by using a stirred ultrafiltration cell with a 10-kDa cutoff filter membrane (YM10; Amicon Inc., Beverly, MA) and loaded onto a molecular sieve column (Sephacryl S-300; Pharmacia Biotech, Uppsala, Sweden). The fractions were eluted with Tris-HCl buffer (10 mM, pH 7.0). Fractions were assayed for protease activity using the chromogen. Active fractions were pooled and concentrated to 1.0 mL. Total protein was determined with the bicinchoninic acid assay (Sigma). The purity of the sample was determined by 12% SDS-PAGE and visualized by silver staining (Bio-Rad).

Colorimetric Substrate Assay for Protease IV Activity
Protease IV activity was determined by the hydrolysis of the chromogen substrate (Chromozym PL; Sigma) as described by O’Callaghan et al.11 Briefly, 10 µL of concentrated supernatants or cell lysates was mixed with 20 µL of chromogen (2 mg/mL) in a reaction buffer containing 50 mM Tris (pH 8.0) and 150 mM NaCl. The reactions were incubated at 37°C for 30 minutes or as stated in each experiment, and the optical density (A410) was measured with a plate reader (model MR5000; Dynatech Laboratories, Chantilly, VA). In the inhibitor reactivity assays, 100 mM EDTA or 1 mM tosyl-L-lysine chloromethyl ketone (TLCK; Sigma) was added to the reaction mixtures.

Kinetic analysis was performed by reading the optical density every 2 minutes for 30 minutes to measure the increase in optical density (absorbance) per minute ({Delta}A per minute). One unit of activity was defined as the amount of enzyme that caused an optical density increase at 410 nm of 1 A/min under the assay conditions. The unit activity was calculated by

The total assay volume was 120 µL, sample volume was 10 µL, the extinction coefficient of the product at 410 nm (E410) was 9.75, and the light path was 0.53 cm.

SDS-PAGE and Immunoblotting
SDS-PAGE was performed on 4% stacking gels and 12% separating gels. Samples were electrophoresed under reducing conditions (50 mM Tris [pH 6.8], 2% SDS, 1% ß-mercaptoethanol, 10% glycerol, 0.1% bromophenol blue, with heating at 100°C for 5 minutes). Electrophoretic transfer of proteins from gels to polyvinylidene difluoride (PVDF) membrane (Immun-Blot; Bio-Rad) was performed in prechilled methanol-Tris-glycine buffer (20 mM Tris base, 150 mM glycine, and 20% methanol), and the membrane was blocked with 5% skim milk (Difco). The membrane was washed three times with Tris-buffered saline (pH 7.6, 20 mM Tris base and 137 mM sodium chloride). The primary antibody was rabbit anti-protease IV prepared against a recombinant form of mature protease IV and used at a 1:2000 dilution. After incubation at room temperature for 2 hours with the primary antibody, the membrane was washed three times with Tris-buffered saline (pH 7.6) and incubated with horseradish peroxidase–conjugated donkey anti-rabbit IgG at a 1:10,000 dilution (Amersham Pharmacia Biotech, Inc., Piscataway, NJ). Proteins were detected by enhanced chemiluminescence Western blot detection reagents (ECL; Amersham Pharmacia Biotech) and exposed to film (Biomax MR; Eastman Kodak Co., Rochester, NY).

Amino-Terminal Sequence Analysis
Samples were subjected to SDS-PAGE and electrotransferred to PVDF membrane (Sequi-blot; Bio-Rad) by using carbonate transfer buffer (10 mM NaCHO3, 3 mM Na2CO3, 20% methanol [pH 9.9]). The membrane was then immersed in 0.5% Coomassie blue R-250 prepared in 50% methanol for 1 to 5 minutes. The membrane was destained in 10% acetic acid prepared in 50% methanol until protein bands became clearly visible. The membrane was washed in deionized water, and bands were cut out and allowed to air dry at room temperature. Amino-terminal amino acids were determined by Core Laboratories, Louisiana State University Health Sciences Center.

Rabbit Intrastromal Inoculation Model of Keratitis
New Zealand White rabbits were treated and maintained in strict accordance with the tenets of the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. All rabbits were anesthetized by subcutaneous injection of a 1:5 mixture of xylazine (100 mg/mL; Rompum; Miles Laboratories, Shawnee, KS) and ketamine hydrochloride (100 mg/mL; Ketaset; Bristol Laboratories, Syracuse, NY). Eyes were topically anesthetized with proparacaine hydrochloride (0.5% Alcaine; Alcon Laboratories, Fort Worth, TX) before intrastromal injection. The corneal inoculum for each strain was grown to log phase in TSB, then diluted to approximately 105 colony forming units (CFU)/mL, based on spectrophotometry. Each cornea (n ≥ 6 per strain) was intrastromally injected with 10 µL containing approximately 1000 CFU per cornea. This inoculum was also plated onto TSA to confirm the number of CFU injected into the corneas.

Evaluation of Ocular Pathogenesis in Rabbits
Inflammation of rabbit eyes was observed by two masked observers by biomicroscope (Topcon; Koaku Kikai KK, Tokyo, Japan). Slit lamp examination (SLE) scoring was graded on seven ocular parameters (conjunctival injection, conjunctival chemosis, iritis, stromal infiltrate, stromal edema, fibrin in the anterior chamber, and formation of hypopyon) on a scale of 0 (none) to 4 (severe). The parameter grades were totaled to produce a single SLE score ranging from 0 (normal eye) to a theoretical maximum of 28.

Bacterial Quantification
After SLE, rabbits were killed with an overdose of pentobarbital sodium (Sigma). Corneas were removed aseptically, dissected, and homogenized in sterile phosphate buffered saline (PBS) using a tissue homogenizer (Ultra-Turrex; Tekmar, Cincinnati, OH). Aliquots of each corneal homogenate were serially diluted 1:10 in PBS and inoculated in triplicate onto TSA plates (Difco) and incubated for 24 hours at 37°C. Colonies were counted and the number of viable bacteria per cornea was expressed as base 10 logarithms.

Statistical Analysis
Data were analyzed on computer (Statistical Analysis System; SAS, Cary, NC), as described previously.11 For determination of CFUs, analysis of variance and protected Student’s t-tests between least-square means from each group were performed. For SLE scores, nonparametric, one-way analysis of variance (Kruskal-Wallis test) and the Wilcoxon test were used for comparison among groups. P ≤ 0.05 was considered significant.

Nucleotide Sequence Accession Number
The nucleotide sequence data for protease IV had been deposited in the GenBank database under accession number AY062882 (GI: 17978564; http://www.ncbi.nlm.nih.gov/Genbank; provided in the public domain by the National Center for Biotechnology Information, Bethesda, MD).

Analysis of the Primary Translation Product of Protease IV
The amino acid sequence data of protease IV was analyzed with the Expert Protein Analysis System (ExPaSy) proteomic server of the Swiss Institute of Bioinformatics (www.expasy.ch, Geneva, Switzerland).


   Results
Top
 Abstract
 Methods
 Results
Discussion
 References
 
Expression of Protease IV Gene from P. aeruginosa
The protease IV gene from P. aeruginosa strain PA103-29 was cloned and transformed into a protease IV-negative Pseudomonas species, P. putida. The presence of the protease IV gene in the new host strain was verified by restriction analysis, sequencing of the plasmid DNA, and a positive PCR reaction for the protease IV gene (data not shown).

To determine the expression of plasmid encoded protease IV in the new host strain, P. putida bearing the protease IV encoding plasmid (pPIV) was cultivated in M9 medium, and culture supernatants were tested for protease activity. Three assay methods were used to analyze protease activity: casein digestion, hydrolysis of chromogenic substrate, and inhibitor reactivity. The concentrated supernatant of P. putida carrying pPIV or P. aeruginosa PA103-29 produced a zone of proteolysis around the well in a skim milk agar plate (data not shown). No protease activity could be detected from concentrated supernatants of P. putida harboring the vector without the protease IV gene insert, even when reactions were incubated for 3 days. The concentrated supernatants were further examined for the ability to hydrolyze the chromogen substrate (Chromozym PL; Sigma). Kinetic analysis showed that protease activity was present in the culture supernatant of P. putida harboring pPIV and was significantly higher (approximately fivefold) than that of PA103-29 (Fig. 2A) . There was insignificant activity in the control, P. putida harboring the vector alone. These results indicated that the cloned protease IV gene was actively expressed in P. putida and that the secretion system for protease IV was operative in both P. aeruginosa and P. putida.



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  		FIGURE 2. Colorimetric substrate and inhibitor assays for protease IV activity. (A) Chromogen activity. Aliquots (10 µL) of concentrated supernatants (2 mg/mL total protein) of P. putida/pUCP20, P. putida/pPIV, and P. aeruginosa PA103-29 were reacted with a chromogenic substrate over numerous time points. Bars represent the mean of protease activity units of three replicates and error bars represent SEM. *Significantly different from P. putida/pUCP20 (P < 0.01). (B) Inhibitor reactivity. Aliquots (10 µL) of concentrated supernatants of each strain were incubated with the chromogen substrate for 30 minutes in the presence or absence of EDTA (100 mM) or TLCK (1 mM). Bars: the mean of the optical density at 410 nm of three replicates; error bars, SEM.

 
Protease IV activity has been shown to be inhibited by TLCK, a serine protease inhibitor, whereas EDTA, a metalloprotease inhibitor, has no inhibitory effect.14 End point analysis showed that the chromogen cleavage activities of concentrated supernatants of P. putida harboring pPIV and PA103-29 were completely inhibited by TLCK (1 mM), but not by EDTA (100 mM; Fig. 2B ). Taken together, these results suggest that P. putida harboring pPIV secretes approximately five times more protease activity than that produced by the prototype P. aeruginosa strain PA103-29 and that protease IV from P. putida carrying pPIV has the same sensitivity to inhibitors as the wild-type protease IV.

The protease IV protein in the culture supernatant of P. putida harboring pPIV was sequentially purified by ultrafiltration, ion exchange, and gel filtration chromatography. All fractions collected from gel filtration chromatography were tested for protease IV activity by reactivity with the chromogen. Fractions with activity formed a single broad peak on gel filtration chromatography with a molecular weight range of 17 to 44 kDa (Fig. 3A) . The proteins in the gel filtration peak produced two bands on SDS-PAGE. One band had a molecular mass of 26 kDa, and the other band had a molecular mass of 17 kDa. The SDS-PAGE profile was identical with that of protease IV purified from P. aeruginosa strain PA103-29 (Fig. 3B) . The band at approximately 26 kDa has been shown to be the mature protease and the band at 17 kDa has been previously reported to be an enzymatically active breakdown product of protease IV formed by autodigestion.8 The N-terminal amino acid sequence of the 26-kDa protein encoded by the plasmid pPIV was analyzed and found to be AGYRDGFGAS, which was identical with that of the mature protease IV secreted from P. aeruginosa PA103-29. These results suggest that protease IV is efficiently produced and secreted by P. putida and is active both in processing and proteolytic activity.



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  		FIGURE 3. Purification of protease IV from culture supernatants of P. putida harboring pPIV. (A) Fractionation of protease IV activity by gel filtration chromatography. Fractions from gel filtration chromatography of concentrated proteins derived from ion exchange chromatography were tested for reactivity with chromogen substrate during the purification process. (B) SDS-PAGE analysis of the purified protease IV. Protease IV purified by ion exchange and gel filtration chromatography was electrophoresed on SDS-PAGE. Protein bands were visualized by silver staining. Lane 1: molecular weight marker; lane 2: purified protease IV from P. putida/pPIV; and lane 3: purified protease IV from P. aeruginosa PA103-29.

 
Maturation of Protease IV
Computer analysis of the primary translation product of protease IV predicts that the protease IV gene encodes a protein of 48.2 kDa that undergoes cleavage to release the mature protease IV molecule (see Fig. 1 ). To determine the nature of precursors to the mature protease IV protein, high molecular weight forms of protein reacting with antibody specific for protease IV were sought in both cell extracts and culture supernatants of P. putida carrying pPIV. Analysis of the cell-free supernatant by Western blot analysis with rabbit polyclonal antibody reactive with the mature protease demonstrated a 26-kDa protein in the supernatants of P. putida carrying pPIV and PA103-29 (Fig. 4A) . This protein was not detected in the concentrated supernatant of P. putida carrying the vector alone. Western blot analysis of the cell extracts of P. putida carrying pPIV revealed proteins equivalent to the mature protease (26 kDa) and two other polypeptides representing precursor forms at 48 and 45 kDa (Fig. 4B) . Neither of these forms was detected in P. putida cell extracts carrying the vector alone. The mature protease was barely detectable in PA103-29 cell extracts and no proteins of 45 or 48 kDa appeared in the Western blot analysis of the PA103-29 cell extract.



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  		FIGURE 4. Proteolytic processing of P. aeruginosa protease IV expressed in P. putida. Concentrated supernatants (A) and whole-cell extracts (B) were subjected to SDS-PAGE under reducing conditions and immunoblotted with rabbit polyclonal antisera directed against mature protease IV. Lane 1: P. aeruginosa PA103-29; lane 2: P. putida/pUCP20; lane 3: P. putida/pPIV; and lane 4: purified protease IV from PA103-29. P1, a full-length protease IV gene product; P2, an intermediate precursor with no signal sequence; M, mature protease IV.

 
We further investigated whether the intracellular protease IV was enzymatically active. Cell lysates of P. putida carrying pPIV were subjected to the chromogen-reactivity test for ability to hydrolyze a lysine-containing peptide. The cell lysates of P. putida carrying pPIV, but not P. putida with the vector alone or PA103-29, showed proteolytic activity, although the activity detected was approximately 10-fold lower than that of the culture supernatants (data not shown).

In Vivo Activity of P. putida Expressing Protease IV
To examine the importance of the protease IV gene to corneal virulence, rabbit eyes were injected intrastromally with P. putida expressing protease IV, PA103-29, or P. putida carrying the vector alone, pUCP20. Evaluation of ocular virulence was determined by SLE scoring, as described in the Methods section. The SLE scores of eyes infected with P. putida expressing protease IV were significantly greater at 32 hours after infection than eyes infected with P. putida carrying the vector without the protease IV gene (P = 0.004; Table 1 ). PA103-29 had significantly more corneal virulence than the P. putida strains with or without the protease IV gene (P ≤ 0.0001). Eyes infected with any of the three strains tested contained similar numbers of CFU per cornea at 32 hours after infection (P ≥ 0.1671).


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  		TABLE 1. Virulence of P. putida Expressing Protease IV 32 Hours after Infection

 
Eyes infected with P. putida carrying the vector alone had mildly reddened conjunctivae, and there was no infiltrate in the corneal stroma (Fig. 5A) . Eyes infected with P. putida producing protease IV showed redness and swelling of conjunctivae, moderately inflamed irides, and stromal infiltrate covering approximately half the corneal surface (Fig. 5B) . No epithelial erosion was observed. Eyes infected with PA103-29 had fibrin in the anterior chamber and stromal infiltrate covered almost 100% of the cornea (Fig. 5C) . The irides were barely visible, because the corneas were nearly opaque.



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  		FIGURE 5. In vivo action of P. putida expressing protease IV. Rabbit eyes were intrastromally injected with 1000 CFU of (A) P. putida/pUCP20, (B) P. putida/pPIV, or (C) PA103-29 and photographed 32 hours after injection.

 

   Discussion
Top
 Abstract
 Methods
 Results
 Discussion
References
 
Production of protease IV was achieved in P. putida, a species essentially avirulent for the rabbit cornea and devoid of protease IV activity. Production of protease IV significantly increased the corneal virulence of P. putida, as evidenced by a threefold increase in the SLE score of eyes infected with protease IV-producing versus nonproducing strain of P. putida. The cloned protease IV gene was efficiently expressed and the enzyme was secreted in vitro by P. putida, as evidenced by an approximate fivefold increase in enzyme activity in supernatants of P. putida carrying pPIV relative to that of P. aeruginosa PA103-29. Because of the increased enzyme expression by the cloned gene, intracellular precursor forms of protease IV could be identified for the first time. These precursors were detected only in P. putida harboring the protease IV gene and not in the same strain carrying the vector without a protease IV gene insert. These findings support computer analysis of the protease IV primary translation product as consisting of three domains: a signal sequence, a propeptide, and a mature domain. The inability to detect significant amounts of intracellular protease IV in P. aeruginosa PA103-29 has been described.8 The absence of detectable protease IV in P. aeruginosa cell extracts could be due to rapid secretion from the cell on translation, the relatively low level of protease IV protein produced, or the degradation of protease IV inside the cell.

Because the cloned protease IV is processed and secreted into the extracellular milieu by P. putida, it is possible that the mature protease IV can use the type II secretory pathway, which has been identified in P. putida.20 21 The processing and subsequent secretion of protease IV could be mediated in part by an autodigestive event. Based on our observations, we propose the following model of maturation for protease IV (Fig. 6) . Protease IV could be synthesized as a large precursor of 48 kDa with an N-terminal signal sequence. Within the cell, the full-length protease could be processed into the intermediate form of 45 kDa that represents a form of protease IV free of the signal sequence. The 45-kDa intermediate could undergo a conformational change that activates the protease activity, triggering the cleavage of the propeptide from the mature protease domain. The mature protease IV could then be secreted through the outer membrane.



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  		FIGURE 6. The proposed model of maturation of protease IV. This diagram illustrates the proposed biogenesis of protease IV. SS, signal sequence; P, propeptide domain; M, mature protease IV; P1, a 48-kDa protease IV precursor; P2, a 45-kDa intermediate precursor with no signal sequence.

 
The fate and function(s) of the propeptide are still unknown. After proteolytic cleavage, the propeptide could be secreted from the cell or degraded within the cell. An antibody specific for the propeptide domain is needed to clarify this point. The propeptide of protease IV, similar to elastase B, could act as an inhibitor preventing intracellular proteolysis.22 23 24 An additional function of the propeptide could be to function as a chaperone for the proper folding of the mature protease before secretion of the enzyme.25 A chaperone function for the propeptide domain is consistent with the fact that cloning of the mature protease domain without the propeptide failed to result in an active enzyme. In fact, the mature protease accumulated inside the cell and was not secreted extracellularly (Caballero A, unpublished findings, 1998). In the present study, the cloned full-length protease IV was properly expressed and secreted by P. putida, suggesting that the propeptide may be involved in the proper folding and facilitate the secretion of protease IV.

In the rabbit intrastromal injection model of keratitis, we demonstrated an important role of protease IV in corneal virulence. Complementation of P. putida with a plasmid expressing protease IV tripled the extent of ocular inflammation when compared with that of P. putida without protease IV expression. This result suggests that the ocular virulence observed could be attributable only to protease IV.

In summary, the protease IV gene was successfully cloned and expressed in a heterologous host. This finding allows us to gain a better understanding of the biosynthesis of this enzyme. Protease IV is synthesized as a precursor and processed through an intermediate form during the secretion mechanism. This large protein undergoes intracellular proteolytic processing to produce the mature protease IV molecule. The mature protease is then secreted as an active enzyme. In addition, the results confirm the concept that protease IV contributes to ocular pathogenicity as a virulence factor.


   Footnotes
 
Supported by National Eye Institute Grant EY012961.

Submitted for publication May 9, 2002; revised July 2, 2002; accepted July 19, 2002.

Commercial relationships policy: N.

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Corresponding author: Richard J. O’Callaghan, Department of Microbiology, Immunology and Parasitology, LSU Health Sciences Center, 1901 Perdido Street, New Orleans, LA 70112; rocall{at}lsuhsc.edu.


   References
Top
 Abstract
 Methods
 Results
 Discussion
 References
 

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