TY - JOUR
AU - Wallach, Jean
AB - Abstract Thirty Pseudomonas aeruginosa strains were isolated from the sputa of cystic fibrosis patients. In each culture supernatant, the amount of three exoproteases (LasA, alkaline protease and elastase) was determined using immunochemical procedures. These assays used selected peptide-MAP (multiple antigen peptide) strategy as antigen for animal immunisation. The method appeared to be reproducible, simple, sensitive and specific without cross-reactivity between the antisera. The resulting values differed from one strain to another mostly for elastase production. Despite the fact that four genes (lasA, lasB, las R and rhl R) were shown to be necessary for full elastolytic activity, it was obvious that if LasA was not secreted in a naturally non-elastase-producing strain, in return in an elastase-producing strain, there were no apparent relationships between LasA and elastase production and between LasA and alkaline protease secretion. Furthermore, in vitro, the secretion of the three exoproteases seemed to be independent of the mucoid or non-mucoid phenotype of the bacteria. Pseudomonas aeruginosa, Multiple antigen peptide, LasA, Alkaline protease, Elastase 1 Introduction Pseudomonas aeruginosa is an opportunistic pathogen which can cause severe and lethal infections in vulnerable hosts due to its ability to secrete many extracellular factors that are associated with the virulence [1]. Among these are several proteases: alkaline protease, elastase and LasA, the latter being purified as a by-product during elastase purification [2]. The role of elastase as a virulence factor is supported by the variety of biologically important substrates that it may inactivate or degrade [3–8]. Full elastolytic activity of elastase depends on at least four genes: lasA, lasB, lasR[9] and recently rhlR[10]. The expression of both lasA and lasB genes is under the control of lasR[11, 12] and rhlR[10], members of two autoinducer-responsive regulatory systems in P. aeruginosa. The lasB gene is described as the elastase structural gene [13]. Expression of the lasA gene in Escherichia coli resulted in an about 40 kDa protein previously named LasA [14], and now called proLasA because it has very recently been identified as the precursor form of LasA [15]. Peters and Galloway [2] had previously shown that a highly basic active 22 kDa fragment from P. aeruginosa PAO1 and PA220 was the carboxy-terminal moiety of the 40 kDa protein. Gustin et al. [15] have recently shown that this active fragment is the mature LasA. Therefore, the term LasA is used in this paper for the 22 kDa polypeptide. LasA was shown to enhance the elastolytic activity of elastase but not the proteolytic activity [16, 17]. Kessler et al. [18] emphasised that LasA possesses all the properties of the bacteriolytic proteases. Furthermore, the amino acid sequence of LasA shows about 40% identity with those of the β-lytic proteases from Lysobacter enzymogenes and Achromobacter lyticus[18]. Therefore, LasA and the staphylolytic protease of P. aeruginosa, an endoprotease that lyses Staphylococcus aureus cells by cleaving the pentaglycine bridges within the peptidoglycan [19], were supposed to be the same protease [18]. The authors also suggested that LasA may enhance elastolysis by cleaving Gly-Gly peptide bonds abundant in elastin. In fact, the mechanism and biological role of LasA remain unsolved. The third protease involved in the virulence of the bacteria is alkaline protease [3], of which the specific targets in vivo are not clearly understood. This enzyme may play a key role in infection through the inactivation of various physiological factors including C1q and C3 proteins of serum complement and many protease inhibitors [3]. Moreover, it hydrolyses natural substrates such as fibrin and fibrinogen [20]. Expression of alkaline protease depends on the apr genes [21]; surprisingly, one of these genes, aprA, described as the structural gene of alkaline protease [22], requires the same positive regulator lasR gene product as elastase and LasA [23]. In this report, immunochemical methods were developed to quantify elastase, alkaline protease and LasA in 30 culture supernatants from P. aeruginosa clinical strains. The strains were obtained from sputum from cystic fibrosis (CF) patients, so some were mucoid strains. For elastase detection a direct ELISA assay has been previously described [24]. A radioimmunoassay for elastase and alkaline protease detection has also been developed [24, 25] but the main disadvantage of this procedure was the use of radiolabelled materials. A direct ELISA assay for LasA detection based upon a multiple antigen peptide strategy for immunisation and a double sandwich ELISA method for alkaline protease detection were therefore developed. The aim was not only to quantify selectively each protease secreted in culture supernatants but also to select several clinical strains that produce a high level of LasA. Indeed, to date LasA secretion has been only studied from a reference strain (PAO1) or modified clinical isolates (PA220) [2], never from clinical samples. Such a selective procedure will be useful to locate the proteases secreted in small amounts in P. aeruginosa infections and will contribute to our understanding of how the proteases contribute to virulence. 2 Materials and methods 2.1 Bacterial strains and growth conditions Thirty P. aeruginosa clinical strains, isolated from the sputum of CF patients, were kindly provided by Dr J.P. Flandrois (Centre Hospitalier Lyon-Sud, Université Lyon 1, France). Strains were maintained on L-broth medium supplemented with 50% glycerol at −70°C and were routinely grown in the same culture medium at 37°C for 24 h with vigorous shaking for maximum aeration. 2.2 Treatment of culture supernatant Each supernatant of a 50–100 ml culture of P. aeruginosa was precipitated with 0.25 M calcium chloride for 2 h at 4°C with gentle stirring. The precipitate, containing putative alginate secreted by the mucoid strains, was removed by centrifugation. The supernatant was then filtered on a 0.22 µm membrane (Millipore) and stored at −20°C in the presence of 0.02% thimerosal. 2.3 Purification of LasA The protein was purified from a 2 litre culture of P. aeruginosa IFO 3455 following the slightly modified procedure of Peters and Galloway [2]. The culture supernatant was treated with calcium chloride as described above then concentrated by ultrafiltration with tangential flow on a 10 kDa cut-off membrane using a Minitan® system (Millipore). The final preparation was obtained by anion exchange chromatography on DEAE-Sepharose® CL-6B (Pharmacia Biotech) in 30 mM Tris-HCl buffer pH 8.3. The purity of the LasA preparation was assessed by comparison with LasA from P. aeruginosa PAO1 (generous gift from Dr D.R. Galloway, Department of Microbiology, Ohio State University, Columbus, OH, USA). 2.4 Synthesis of peptides and multiple antigen peptide (MAP) system The hydropathic pattern of LasA was determined according to Kyte and Doolittle [26] using the amino acid sequence determined by Darzins et al. [27]. Putative antigenic amino acid sequences containing more than 10 amino acids were then subjected to the predictive analysis of Milton et al. [28] in order to discard the sequences liable to set some problem during the solid-phase synthesis. A heptadecapeptide, SNTGSGYPYSSFDASYD, was then selected, which corresponds to residues 24–40 of LasA. In the alkaline protease amino acid sequence described by Duong et al. [21], a tridecapeptide, EEQNTGQDFKGAY, was selected, corresponding to residues 218–230. Peptides were synthesised by the solid-phase method (KH resin, 0.1 mEq g−1) with Fmoc derivatives as described by Rutault et al. [29]. A glycyl residue was added at the C-terminal end of these peptides to avoid peptide racemisation during synthesis. The purity of the protected peptides was estimated by HPLC on a C18 reversed-phase column with a continuous gradient of acetonitrile in 0.1% trifluoroacetic acid. In order to control the synthesis, a part of the (24–40) peptide was deprotected and a total identity between the selected and the synthesised peptide was demonstrated by peptide sequencing; a part of the (218–230) peptide was deprotected and analysed by FAB+-MS and a total agreement between the calculated theoretical mass value and the found mass value was observed. Core synthesis was performed according to the procedure of Bernillon and Wallach [30] by successive couplings of Fmoc-Lys(Fmoc)-OPfp and Fmoc-β-Ala-OPfp, alternately. Each protected peptide (excess 2.5) was conjugated to the core in the presence of diisopropylcarbodiimide and 1-hydroxybenzotriazole (excess 2.5) with gentle stirring for 18 h at room temperature. The final concentration of the reagents was 0.2 M. The spectrophotometrically followed Fmoc deprotection permitted calculation of the coupling yield. 2.5 Preparation of antisera Production of antiserum against P. aeruginosa elastase has been previously described by Saulnier et al. [24]. Antiserum against P. aeruginosa alkaline protease was produced in two guinea pigs immunised subcutaneously with 250 µg of commercial alkaline protease (Nagase Biochemicals Ltd, Fukuchiyama City, Kyoto, Japan) first in Freund's complete adjuvant and then in Freund's incomplete adjuvant on days 14, 42 and 70. They were bled 1 week after the last booster dose and the serum was decomplemented (56°C, 30 min), filter-sterilised (0.45 µm) and stored at −20°C. According to the procedure described by Coin et al. [31], New Zealand white rabbits were immunised for each injection with 150 µg and 300 µg of the MAP (24–40) conjugate and the MAP (218–230) conjugate, respectively, and the antisera were used for LasA and alkaline protease detection. 2.6 Enzyme-linked immunoassays The quantification of elastase and LasA in culture supernatants was achieved by a direct ELISA method based upon the procedure of Bourdenet et al. [32]. All assays were performed in triplicate. Microtitre plates (MicroElisa Dynatech M124B) were coated with 100 µl of various dilutions of culture supernatants in 50 mM carbonate buffer pH 9.6. They were incubated for 2 h at 37°C then overnight at 4°C. The plates were washed three times with phosphate buffered saline (PBS) pH 7.2 (bioMérieux, France), blocked with 1% BSA in PBS for 1 h at 37°C, then rinsed three times with PBS supplemented with 0.05% Tween 20 (PBST). Sera (100 µl) diluted 1:100 in PBST containing 1% BSA were added to the coated wells. After a 2 h incubation at 37°C, the plates were washed five times before addition of 100 µl of a 1000-fold dilution of peroxidase-labelled goat anti-rabbit IgG antibody (Sigma) in PBST containing 1% BSA. The plates were incubated for 2 h at 37°C and after three washes with PBST, 100 µl aliquots of o-phenylenediamine were added to develop the ELISA. After 10 min at 28°C, the reaction was stopped by addition of 50 µl of 10% SDS. The absorbance was measured at 490 nm (Dynatech Minireader II) and the elastase or LasA concentrations were evaluated from the respective standard curves, which were obtained in a range of 0–40 ng ml−1 of commercial P. aeruginosa elastase (Nagase Biochemicals Ltd, Japan) and in a range of 0–150 ng ml−1 of purified LasA. For the immunochemical reaction with LasA as antigen, the anti-MAP (24–40) antiserum was used. The quantification of alkaline protease in the culture supernatant was achieved by a double sandwich ELISA procedure. The first antibody used was guinea pig anti-alkaline protease (whole molecule) partially purified on Affi-gel blue column (Bio-Rad) and the second antibody was rabbit anti-MAP (218–230). Microtitre plates were coated with 100 µl of anti-alkaline protease antiserum diluted 100-fold in 0.1 M phosphate buffer pH 7.2 and incubated for 24 h at room temperature, then overnight at 4°C. The plates were washed as described above and saturated with 3% gelatin in PBS for 90 min at 37°C. The plates were washed again, then various dilutions of culture supernatants or commercial alkaline protease in PBST containing 0.1% gelatin were added and incubated for 2 h at 37°C. The assay was then performed essentially as described for the direct procedure except that a 250-fold dilution of the rabbit anti-MAP (218–230) antibody was used and the detecting antiserum was a 2500-fold dilution of donkey anti-rabbit IgG antibody conjugated to peroxidase (Jackson Immuno Research) in PBST containing 0.1% gelatin. The standard alkaline protease curve was used in a range of 0–60 ng ml−1. 2.7 Immunoblot assays The culture supernatants were subjected to SDS-PAGE on 12% or 15% gels according to the method of Laemmli [33]. The gel was soaked in transfer buffer (192 mM glycine, 25 mM Tris buffer pH 8.2–8.3 with 15% methanol) and the proteins were electrotransferred on a polyvinylidenedifluoride membrane (Westran®, Schleicher & Schuell) for 2 h at 0.8 mA cm−2 on a Novablot electrophoretic transfer apparatus (Pharmacia-LKB). Excess protein binding capacity of the membrane was blocked by application of a 3% gelatin in Tris buffered saline (TBS: 500 mM NaCl, 20 mM Tris-HCl buffer pH 7.5). The membrane was then washed in TBS containing 0.05% Tween 20 (TTBS) and reacted with appropriate antisera overnight at room temperature. A 1000-fold dilution of anti-elastase antiserum in TTBS containing 1% gelatin for elastase detection, a 500-fold dilution of anti-MAP (24–40) antiserum for LasA detection and a 250-fold dilution of anti-MAP (218–230) antiserum for alkaline protease detection were used. The bands were detected with an immunoassay kit (Bio-Rad) using a 3000-fold dilution of a goat anti-rabbit IgG antibody conjugated to alkaline phosphatase in TTBS containing 1% gelatin and were stained purple red with p-nitroblue tetrazolium chloride and 5-bromo-4-chloro-3-indolyl phosphate, toluidine salt. 2.8 Other procedures Protein concentration was determined with the bicinchoninic acid (Pierce) assay [34] or the Lowry procedure [35] with BSA as standard. Peptide sequencing was performed in a protein microsequencer (Applied Biosystems, model 407 A). FAB+-MS spectra were recorded with a VG ZAB2-SEG spectrometer. 3 Results 3.1 Purification of LasA According to the procedure described in Section 2, LasA was recovered in the column pass-through fractions after a one step anion exchange chromatography. Elastase was eluted at 0.2 M sodium chloride by a continuous linear gradient of 0–0.5 M NaCl in 30 mM Tris-HCl buffer pH 8.3 (Fig. 1). LasA protein was essentially pure as compared to a LasA standard purified from strain PAO1. Purified LasA (10 mg) was stored at −20°C in 30 mM Tris-HCl buffer pH 8.3. Elastase (132 mg) was stored at 4°C as a suspension in a solution containing 3 M ammonium sulfate, 10 mM sodium acetate, 2 mM calcium chloride, and 0.05 mM zinc chloride, pH 7.5. Figure 1 View largeDownload slide Purification of P. aeruginosa IFO 3455 LasA and SDS-PAGE analyses of the pooled fractions. A concentrated 24 h culture filtrate was passed through a DEAE Sepharose column (1.6×50 cm) in 30 mM Tris-HCl buffer pH 8.3 followed by a continuous linear gradient of 0–0.5 M sodium chloride in the same buffer. Proteins were detected by absorbance at 280 nm. SDS-PAGE (15%) of the DEAE Sepharose-purified enzymes, previously boiled for 5 min in denaturing buffer in the presence of 5%β-mercaptoethanol, is shown in the insert; lane 1: LasA standard from P. aeruginosa PAO1; lane 2: purified LasA; lane 3: commercial elastase; lane 4: purified elastase. Protein bands were visualised by Coomassie blue staining. Figure 1 View largeDownload slide Purification of P. aeruginosa IFO 3455 LasA and SDS-PAGE analyses of the pooled fractions. A concentrated 24 h culture filtrate was passed through a DEAE Sepharose column (1.6×50 cm) in 30 mM Tris-HCl buffer pH 8.3 followed by a continuous linear gradient of 0–0.5 M sodium chloride in the same buffer. Proteins were detected by absorbance at 280 nm. SDS-PAGE (15%) of the DEAE Sepharose-purified enzymes, previously boiled for 5 min in denaturing buffer in the presence of 5%β-mercaptoethanol, is shown in the insert; lane 1: LasA standard from P. aeruginosa PAO1; lane 2: purified LasA; lane 3: commercial elastase; lane 4: purified elastase. Protein bands were visualised by Coomassie blue staining. 3.2 Anti-peptide antisera analysis The antiserum raised in a rabbit against the selected MAP (24–40) was examined for its antigenic specificity. Using LasA as coating antigen (5 µg ml−1) or MAP (24–40) (5 µg ml−1), specific anti-MAP (24–40) antibodies were titrated by ELISA. The titres, defined as the reciprocal of the dilution giving half-maximum absorbance, were 200 and 2000, respectively (Fig. 2). This result was expected since the heptadecapeptide sequence was less frequent in the whole molecule than in the MAP (24–40). This antiserum was also tested using elastase (5 µg ml−1) or alkaline protease (5 µg ml−1) as coating antigens and no reaction occurred. Figure 2 View largeDownload slide Titration of the antisera with the ELISA method. The titre was defined as the reciprocal of the dilution giving half-maximum absorbance. Anti-MAP (24–40) antiserum with LasA as coating antigen (○) or MAP (24–40) (●) and anti-MAP (218–230) antiserum with alkaline protease as coating antigen (□). Figure 2 View largeDownload slide Titration of the antisera with the ELISA method. The titre was defined as the reciprocal of the dilution giving half-maximum absorbance. Anti-MAP (24–40) antiserum with LasA as coating antigen (○) or MAP (24–40) (●) and anti-MAP (218–230) antiserum with alkaline protease as coating antigen (□). The titre of the specific anti-MAP (218–230) antibody raised in a rabbit was 450 using alkaline protease (5 µg ml−1) as coating antigen (Fig. 2). There was no reaction between this serum and LasA or elastase when they were used as coating antigens (5 µg ml−1 each protease). 3.3 Qualitative determination of each protease Culture supernatants in denaturing conditions were analysed by Western blotting. This procedure was applied especially for detection of LasA and alkaline protease which were secreted in smaller amounts than elastase. It should be noted that the anti-elastase antiserum was used mostly to identify the putative partially degraded form of elastase if there was ambiguity among a few bands. Otherwise, elastase was identified using the classic Coomassie blue staining step. Concerning LasA, a major band at 22 kDa migrated at the same level as the protein from strain PAO1 (Fig. 3A). In one strain, in addition to the major band, the presence of another band located at approximately 40 kDa was noted (Fig. 3A, lane 5). Figure 3 View largeDownload slide Immunoreactivity of antisera with P. aeruginosa supernatants. All samples were solubilised in SDS-PAGE sample buffer at 100°C for 5 min then subjected to SDS-PAGE 15% (A) or 10% (B) before Western blotting. A: The immunoreaction was achieved with a 500-fold dilution of anti-MAP (24–40) antiserum. Lane 1: LasA from P. aeruginosa PAO1; lane 2: purified LasA from P. aeruginosa IFO 3455; lane 3: supernatant I 655; lane 4: supernatant I 7105; lane 5: supernatant I 932. B: The immunoreaction was achieved with a 250-fold dilution of anti-MAP (218–230) antiserum. Lane 1: commercial alkaline protease; lane 2: supernatant I 723; lane 3: supernatant I 883; lane 4: supernatant I 7101. Figure 3 View largeDownload slide Immunoreactivity of antisera with P. aeruginosa supernatants. All samples were solubilised in SDS-PAGE sample buffer at 100°C for 5 min then subjected to SDS-PAGE 15% (A) or 10% (B) before Western blotting. A: The immunoreaction was achieved with a 500-fold dilution of anti-MAP (24–40) antiserum. Lane 1: LasA from P. aeruginosa PAO1; lane 2: purified LasA from P. aeruginosa IFO 3455; lane 3: supernatant I 655; lane 4: supernatant I 7105; lane 5: supernatant I 932. B: The immunoreaction was achieved with a 250-fold dilution of anti-MAP (218–230) antiserum. Lane 1: commercial alkaline protease; lane 2: supernatant I 723; lane 3: supernatant I 883; lane 4: supernatant I 7101. Concerning alkaline protease, the presence of two major bands was observed, one slightly above 51 kDa and the other at 49 kDa at the migration level of the commercial enzyme (Fig. 3B). 3.4 Quantitative determination of each protease The procedure for elastase quantification was according to Saulnier et al. [24] with slight modifications. Due to the different sensitivity of the antibodies to their specific targeted proteases, a suitable range of each sample protease was defined for the standard curves. For instance, Fig. 4A, B shows the standard curves of elastase and LasA obtained by a direct ELISA procedure using the same dilution of specific antiserum (1/100). The amount of coated LasA was increased by a factor of 4 to obtain an absorbance range as high as with elastase because only a small part of LasA was recognised using the anti-peptide (24–40) antiserum, whereas more elastase antigenic sites were recognised with the anti-elastase antiserum. Figure 4 View largeDownload slide ELISA standard curves. Assays were performed as described in Section 2. The culture medium has no influence on the assays as has been previously published [24]. Commercial elastase concentrations were kept between 0 and 40 ng ml−1 (A), purified LasA concentrations were kept between 0 and 150 ng ml−1 (B) and commercial alkaline protease concentrations were kept between 0 and 60 ng ml−1 (C). Figure 4 View largeDownload slide ELISA standard curves. Assays were performed as described in Section 2. The culture medium has no influence on the assays as has been previously published [24]. Commercial elastase concentrations were kept between 0 and 40 ng ml−1 (A), purified LasA concentrations were kept between 0 and 150 ng ml−1 (B) and commercial alkaline protease concentrations were kept between 0 and 60 ng ml−1 (C). In the case of alkaline protease, the double sandwich ELISA procedure was chosen because this protease was produced in the lowest range when compared to elastase and, to an extent, LasA. Interestingly, this method increased by a factor of approximately 2.5 the level of protein detection even when an anti-peptide antiserum against the whole alkaline protease was used. The useful range for the alkaline protease standard curve (0–60 ng ml−1) was then near that of elastase (0–40 ng ml−1), even if the dilution of peroxidase-conjugated antibody used did not lead to the same range of absorbance in each case (Fig. 4A, C). The amount of alginate, the exopolysaccharide specifically produced by the mucoid strains of P. aeruginosa, was also determined as described elsewhere [31]. The results of the measurements are listed in Table 1. Each value was the mean of three independent assays. When produced, alkaline protease levels were 0.04–1.3 µg ml−1, elastase 9–80 µg ml−1 and LasA 0.3–3.1 µg ml−1. Table 1 Alginate, elastase, LasA and alkaline protease concentrations of culture supernatants of 30 P. aeruginosa clinical strains Strain  Alginate (µg ml−1)  Elastase (µg ml−1)  LasA (µg ml−1)  Alkaline protease (µg ml−1)  I 3101  530  15  0.3  0.04  I 7101  300  0  0  0.05  I 685  300  34  2.3  0.15  I 695  330  35  3.1  0.06  I 782  550  0  0  0.09  I 7105  450  23  2.2  0.1  I 883  500  19  0.9  0  I 886  580  22  0.7  1  I 925  500  12  1.3  –  I 926  500  9  0.4  0.09  I 946  560  0  0  –  I 632  340  58  1.8  1.3  I 273  70  37  0  1.2  I 533  0  41  0.3  0.7  I 534  1200  37  0  –  I 616  0  32  0.2  0  I 624  780  40  0.3  0  I 626  560  80  0  0.95  I 634  610  36  0  0.3  I 655  0  39  0  0.1  I 662  0  62  0  0.15  I 774  0  57  0  0.1  I 792  80  11  0  0.07  I 811  830  54  0.4  0  I 883  540  39  0.3  0.1  I 885  0  42  0  0.04  I 931  0  17  1.1  –  I 932  0  20  2.4  0.6  I 7103  0  25  0.8  0.5  I 7104  0  41  2.7  0.04  Strain  Alginate (µg ml−1)  Elastase (µg ml−1)  LasA (µg ml−1)  Alkaline protease (µg ml−1)  I 3101  530  15  0.3  0.04  I 7101  300  0  0  0.05  I 685  300  34  2.3  0.15  I 695  330  35  3.1  0.06  I 782  550  0  0  0.09  I 7105  450  23  2.2  0.1  I 883  500  19  0.9  0  I 886  580  22  0.7  1  I 925  500  12  1.3  –  I 926  500  9  0.4  0.09  I 946  560  0  0  –  I 632  340  58  1.8  1.3  I 273  70  37  0  1.2  I 533  0  41  0.3  0.7  I 534  1200  37  0  –  I 616  0  32  0.2  0  I 624  780  40  0.3  0  I 626  560  80  0  0.95  I 634  610  36  0  0.3  I 655  0  39  0  0.1  I 662  0  62  0  0.15  I 774  0  57  0  0.1  I 792  80  11  0  0.07  I 811  830  54  0.4  0  I 883  540  39  0.3  0.1  I 885  0  42  0  0.04  I 931  0  17  1.1  –  I 932  0  20  2.4  0.6  I 7103  0  25  0.8  0.5  I 7104  0  41  2.7  0.04  Each value was the mean of three independent assays, all assays were performed in triplicate. View Large Table 1 Alginate, elastase, LasA and alkaline protease concentrations of culture supernatants of 30 P. aeruginosa clinical strains Strain  Alginate (µg ml−1)  Elastase (µg ml−1)  LasA (µg ml−1)  Alkaline protease (µg ml−1)  I 3101  530  15  0.3  0.04  I 7101  300  0  0  0.05  I 685  300  34  2.3  0.15  I 695  330  35  3.1  0.06  I 782  550  0  0  0.09  I 7105  450  23  2.2  0.1  I 883  500  19  0.9  0  I 886  580  22  0.7  1  I 925  500  12  1.3  –  I 926  500  9  0.4  0.09  I 946  560  0  0  –  I 632  340  58  1.8  1.3  I 273  70  37  0  1.2  I 533  0  41  0.3  0.7  I 534  1200  37  0  –  I 616  0  32  0.2  0  I 624  780  40  0.3  0  I 626  560  80  0  0.95  I 634  610  36  0  0.3  I 655  0  39  0  0.1  I 662  0  62  0  0.15  I 774  0  57  0  0.1  I 792  80  11  0  0.07  I 811  830  54  0.4  0  I 883  540  39  0.3  0.1  I 885  0  42  0  0.04  I 931  0  17  1.1  –  I 932  0  20  2.4  0.6  I 7103  0  25  0.8  0.5  I 7104  0  41  2.7  0.04  Strain  Alginate (µg ml−1)  Elastase (µg ml−1)  LasA (µg ml−1)  Alkaline protease (µg ml−1)  I 3101  530  15  0.3  0.04  I 7101  300  0  0  0.05  I 685  300  34  2.3  0.15  I 695  330  35  3.1  0.06  I 782  550  0  0  0.09  I 7105  450  23  2.2  0.1  I 883  500  19  0.9  0  I 886  580  22  0.7  1  I 925  500  12  1.3  –  I 926  500  9  0.4  0.09  I 946  560  0  0  –  I 632  340  58  1.8  1.3  I 273  70  37  0  1.2  I 533  0  41  0.3  0.7  I 534  1200  37  0  –  I 616  0  32  0.2  0  I 624  780  40  0.3  0  I 626  560  80  0  0.95  I 634  610  36  0  0.3  I 655  0  39  0  0.1  I 662  0  62  0  0.15  I 774  0  57  0  0.1  I 792  80  11  0  0.07  I 811  830  54  0.4  0  I 883  540  39  0.3  0.1  I 885  0  42  0  0.04  I 931  0  17  1.1  –  I 932  0  20  2.4  0.6  I 7103  0  25  0.8  0.5  I 7104  0  41  2.7  0.04  Each value was the mean of three independent assays, all assays were performed in triplicate. View Large 4 Discussion Two of the three proteases of P. aeruginosa, elastase and alkaline protease, have previously been extensively studied. On the other hand, most of LasA's biological characteristics remain unknown probably because of the low level of LasA secretion in comparison to elastase production and also because the involvement of LasA in diseases is not as obvious as that of elastase or exotoxin A [36, 37]. In order to easily detect the proteases of P. aeruginosa, especially those secreted in small amounts, the present work developed a quantitative sensitive immunochemical assay providing some information about P. aeruginosa clinical strains and the secretion of the different proteases. A strategy using peptides as antigens was chosen to generate specific antibodies against alkaline protease and LasA. Antibody specificity and the absence of cross-reactivity between the different antibodies were demonstrated, which validates the use of an enzyme immunoassay procedure to detect the presence of LasA and alkaline protease in mixtures of the other proteins secreted by the bacteria. Several strains of P. aeruginosa secreted elastase, LasA and alkaline protease simultaneously but protease concentrations varied greatly from one strain to another, especially for elastase and alkaline protease. Three of the 30 strains were devoid of both elastase and LasA secretion, which agrees with a previous estimate that 15% of P. aeruginosa clinical isolates are devoid of elastolytic activity [9]. Contrary to the potential relationships which should exist between the elastase and LasA secretion if the elastolytic activity is fully expressed when lasB and lasA gene products are encoded, there was no evident correlation between the two proteases except that 70% of elastase-producing strains were also able to secrete LasA. Strains of P. aeruginosa isolated from CF patients are well known for their ability to be spontaneously converted into mucoid strains. These strains secrete alginate, a polysaccharide constituted of mannuronic and guluronic acids, which plays a role in the adhesion of the bacteria to tracheal cells and respiratory mucins [38]. It also protects P. aeruginosa from phagocytosis [39]. A relationship between the production of alginate and that of elastase or LasA might indicate the different steps of colonisation by firstly non-mucoid strains and afterwards mucoid strains, in cystic fibrosis. Unfortunately, in vitro, the secretion of LasA or elastase was apparently not related to the mucoid or non-mucoid phenotype of the bacteria. LasA was produced by 50% of non-mucoid strains as opposed to 65% of mucoid strains. The immunochemical procedures developed in this report measured the total elastase and/or alkaline protease and/or LasA present in culture supernatants, irrespective of whether proteases were active or inactive. In a previous study, Saulnier et al. [24] have demonstrated the elastase concentration with both immunochemical and enzymatic assays. The latter assays used conductimetric procedures using elastin as natural substrate, or tetraalanine as synthetic elastase-specific substrate. Linear relationships were demonstrated between both immunochemical and enzymatic procedures showing that the methodologies were therefore complementary. Contrary to the case of elastase, natural and synthetic alkaline protease and LasA substrates are not well known. However, the alkaline protease activity was recently estimated using a synthetic N-carbobenzyloxyarginylarginylparanitroanilide (Z-Arg-Arg-pNA) substrate [40]. It was also possible to determine the LasA activity with pentaglycine which is a specific synthetic substrate for this protease (Vessillier, Besson, Saulnier and Wallach, personal communication). The use of all these methodologies should give information about the quantity of each active protease in future studies. It would be interesting to investigate the production of the proteases in biological secretions of patients infected with P. aeruginosa, especially those colonised by mucoid strains. The ELISA procedures described above should be a useful tool, sensitive and reproducible in such identification. 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TI - LasA, alkaline protease and elastase in clinical strains of Pseudomonas aeruginosa: quantification by immunochemical methods
JF - Journal of the Endocrine Society
DO - 10.1111/j.1574-695X.1997.tb01043.x
DA - 1997-07-01
UR - https://www.deepdyve.com/lp/oxford-university-press/lasa-alkaline-protease-and-elastase-in-clinical-strains-of-pseudomonas-1uT7Hrd738
SP - 175
EP - 184
VL - 18
IS - 3
DP - DeepDyve
ER -