Elastase-producing Pseudomonas aeruginosa degrade plasma proteins and extracellular products of human skin and fibroblasts, and inhibit fibroblast growth

Microbial Pathogenesis 34 (2003) 47–55 www.elsevier.com/locate/micpath

Elastase-producing Pseudomonas aeruginosa degrade plasma proteins and extracellular products of human skin and fibroblasts, and inhibit fibroblast growth Artur Schmidtchena,*, Elisabet Holstb, Hans Tapperc, Lars Bjo¨rckc a

Section for Dermatology, Department of Medical Microbiology, Dermatology and Infection, Biomedical Center B14, Lund University, Tornava¨gen 10, S-22184 Lund, Sweden b Section for Medical Microbiology, Department of Medical Microbiology, Dermatology and Infection, Lund University, Lund, Sweden c Section for Molecular Pathogenesis, Department of Cell and Molecular Biology, Lund University, Lund, Sweden Received 5 October 2001; received in revised form 6 November 2002; accepted 11 November 2002

Abstract Leg ulcers of venous origin represent a disease affecting 0.1 – 0.2% of the population. It is known that almost all chronic ulcers are colonized by different bacteria, such as staphylococci, enterococci and Pseudomonas aeruginosa. We here report that P. aeruginosa, expressing the major metalloproteinase elastase, induces degradation of complement C3, various antiproteinases, kininogens, fibroblast proteins, and proteoglycans (PG) in vitro, thus mimicking proteolytic activity previously identified in chronic ulcer fluid in vivo. Elastase-producing P. aeruginosa isolates were shown to significantly degrade human wound fluid as well as human skin proteins ex vivo. Elastase-containing conditioned P. aeruginosa medium and purified elastase inhibited fibroblast cell growth. These effects, in conjunction with the finding that proteinase production was detected in wound fluid ex vivo, suggest that bacterial proteinases play a pathogenic role in chronic ulcers. q 2003 Elsevier Science Ltd. All rights reserved. Keywords: Bacteria; Connective tissue; Proteoglycan; Wound healing

Underlying conditions, such as venous or arterial insufficiency, diabetes, and immunological disease, predispose for the development of non-healing leg ulcers [1,2]. Ulcers due to venous insufficiency comprise 50 –60% of all ulcers and affect 0.1 –0.2% of the total population [3]. At the molecular level, chronic leg ulcers fail to follow the normal pattern of wound repair involving inflammation, granulation and reepithelialization, but instead remain in a persistent inflammatory state characterized by complement degradation and ongoing proteolysis, as indicated by increased levels of neutrophil elastase and gelatinases ([4 – 8], for a review see Ref. [9]). Proteinase inhibitors such as a2macroglobulin (a2-M), a1-antitrypsin (a1-AT) and inter-ainhibitor (I-a-I) are degraded in chronic ulcers [5,8 – 10] and hence, may not counterbalance proteolytic enzymes [5]. The resulting degradation of matrix molecules, such as * Corresponding author. Tel.: þ 46-46-2224522; fax: þ 46-46-157756. E-mail address: [email protected] (A. Schmidtchen).

fibronectin (FN), laminin, tenascin, and various collagens [5 –10], hinders the formation of proper contacts between keratinocyte integrins and the underlying matrices in chronic ulcers. Furthermore, various growth factors, such as platelet-derived growth factor, are degraded, thus retarding cell-proliferation. These findings, together with the observation that keratinocytes display an altered, nonmigratory fenotype in venous ulcers, leads to a failure of reepithelialization [9]. There is no single unifying theory as to the etiology of chronic ulcers, thus, it is likely to be multifactorial. Chronic ulcers are all colonized by bacteria and recent clinical and laboratory evidence supports the view that bacteria contribute to non-healing [11]. Pseudomonas aeruginosa occur in about 20 – 30% of all ulcers [12,13]. This observation prompted us to define the proteinases produced by leg ulcer-derived P. aeruginosa, and to investigate the mechanisms by which P. aeruginosa may influence the connective tissue and thus, affect the wound healing

0882-4010/03/$ - see front matter q 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0882-4010(02)00197-3

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process. Here, we demonstrate that the production of a highly conserved metalloproteinase, elastase, by ulcerderived P. aeruginosa induces degradation of wound fluid and human skin proteins during infection ex vivo. Furthermore, elastase-producing P. aeruginosa degraded various antiproteinases, complement C3, kininogens (KIN), fibroblast proteins and the proteoglycan decorin, and inhibited fibroblast growth.

1. Results 1.1. Identification of P. aeruginosa proteinases P. aeruginosa was cultured in Todd – Hewitt (TH) medium, or in 50% acute wound fluid (collected after mastectomy), and the proteinase production was assessed by gelatin zymography (Fig. 1, ELA þ TH and ELA þ WF). Two P. aeruginosa proteinases, migrating at positions corresponding to molecular weights of , 150 and , 50 kDa (Fig. 1), were detected. Acute wound fluid, which was included as a control (Fig. 1, WF), did not contain these proteinases, whereas endogenous gelatinases were identified. Interestingly, these proteinases were not detected in the wound fluid infected with P. aeruginosa (Fig. 1, ELA þ WF). Electrophoresis (of conditioned TH-medium) followed by immunoblotting with antibodies specific against P. aeruginosa elastase detected a , 150 kDa component (Fig. 1, ELA native), which when boiled in SDS –PAGE sample buffer, migrated as a , 35 kDa protein (Fig. 1, ELA denat.). Boiling abolished the enzyme activity (not shown). This indicated that native elastase migrates as a multimeric , 150 kDa form on zymograms, and that denaturation by boiling generates monomeric elastase.

Fig. 1. Zymographic and immunoblotting analysis of P. aeruginosa proteinases. Degradation of gelatin was analyzed by zymography on 10% polyacrylamide gels containing gelatin (1 mg/ml). 0.1 ml of sterile-filtered conditioned medium from P. aeruginosa grown at 37 8C in TH medium (ELA þ TH) or 50% wound fluid (ELA þ WF) was analyzed. Wound fluid (WF) was included as control. Ten microliters of conditioned TH medium was electrophoresed in parallel, and immunoblotted with polyclonal antibodies against elastase. ELA native; unboiled material, ELA denat; boiled material. Antibodies specific for P. aeruginosa alkaline proteinase detected a ,50 kDa protein (AP). Molecular weight markers are indicated to the left.

The two proteinases were separated by anion-exchange chromatography at pH 8.0. The flow-through fraction contained elastase as shown by zymographic analysis as well as immunoblotting (not shown). The sequence of the first 12 amino acids of this proteinase, Ala-Glu-AlaGly-Gly-Pro-Gly-Gly-Asn-Gln-Lys-Ile, was identical to the amino terminus of the mature form of elastase [14]. Sequence analysis was performed on the (PCR-generated) DNA coding for the mature form of P. aeruginosa elastase. When compared with a previously published sequence (GenBank accession number M24531, strain IFO 3455) [14], few alterations were noted; 1115 C ! T, 1286 G ! A, 1299 A ! G, 1391 T ! C and 1555 A ! G. Only the 1299 A ! G and the 1555 A ! G transitions resulted in amino acid substitutions, and both were Asp ! Gly. The other changes were missense mutations. No changes occurred in the active site of the enzyme. The second, minor, proteinase eluted at a position corresponding to about 0.5– 0.6 M NaCl (not shown). This , 50 kDa proteinase co-migrated perfectly with purified alkaline proteinase (a kind gift of Dr H. Maeda) as assessed by zymography, and was recognized by polyclonal antibodies against P. aeruginosa alkaline proteinase (Fig. 1, AP). Parts of the gene coding for alkaline proteinase [15] were amplified from P. aeruginosa DNA by PCR and all fragments yielded the expected molecular sizes (see Section 3). 1.2. Ex vivo experiments using human wound fluid, skin biopsies and plasma Zymography, in combination with protein/DNA sequencing and immunoblotting, thus demonstrated that the herein used P. aeruginosa isolate produced a major enzyme, elastase. Recently, using zymographic detection, we showed that , 50% of chronic ulcer-derived P. aeruginosa isolates produced a major 150 kDa proteinase similar to the herein described elastase [16]. To investigate the effect of elastase on human wound fluid and skin, various ulcerderived P. aeruginosa, producing either undetectable levels of elastase or expressing high levels of elastase (three isolates each) [16] were grown in TH medium, human wound fluid, or in minimal essential medium in the presence of human skin biopsies. P. aeruginosa elastase was only detected in supernatants from cultures of the previously characterized elastase-expressing bacteria. For all the three elastase-expressing isolates, the elastase expression was similar in TH, wound fluid, and in the presence of human skin, as judged by zymographic analysis (one representative isolate in each group is shown on the zymogram, see inset Fig. 2A and B) and immunoblotting (not shown). In all isolates, a minor , 50 kDa enzyme (position of alkaline proteinase, AP) was detected on the zymograms (Fig. 2A and B, inset). Bacterial growth was similar in all overnight cultures. A significant degradation of wound fluid components was only noted in the wound fluids containing elastase-producing P. aeruginosa (ELA þ , Fig. 2A). Slight

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Fig. 2. Effects of various P. aeruginosa isolates on human wound fluid and skin biopsies. Elastase-producing P. aeruginosa (ELA þ ) and isolates devoid of elastase (ELA 2 ) (three each) were grown to stationary phase in human wound fluid (panel A) and in presence of skin biopsies (immersed in DMEM) (panel B and C). The wound fluids and culture medium from the skin biopsies were centrifuged, and 5 and 20 ml of the supernatant, respectively, analyzed on 3–12% SDS–PAGE (panel A and B). In C, biopsies were extracted with 10% SDS, centrifuged, and 20 ml of the supernatant was analyzed on 3–12% SDS–PAGE. Molecular markers are indicated to the left and ELA and AP (indicated to the right of the insets) indicate the positions of elastase and alkaline proteinase, respectively. Elastase expression was demonstrated by zymography (ELA þ ). 1 and 2.5 mg of PAELA was included together with the skin biopsies (PAELA, 1 and 2, respectively).

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Fig. 3. Elastase-expressing P. aeruginosa degrade human plasma a1-AT, I-a-I, a2-M, KIN and C3. One microliter human plasma (,70 mg protein) was incubated for 6 h with PBS alone (C) (incubation with TH medium yielded the same result) or P. aeruginosa conditioned medium (1; 10 ml, 2; 50 ml, 3; 100 ml). Phosphate-buffered saline was supplemented to 100 ml. An amount corresponding to 10 mg of plasma-proteins was then electrophoresed (3–12% SDS–PAGE), and the separated proteins were transferred onto nitrocellulose and immunostained by the corresponding polyclonal antiserum. a1-AT, a1-antitrypsin; I-a-I, inter-a-inhibitor; a2-M, a2-macroglobulin; KIN, kininogen, and C3, C3. The prestained molecular weight markers are indicated to the left.

was also affected; the a-chain was partially degraded and additional fragments of molecular weights 40 – 50 kDa were detected (Fig. 3, lanes 1 – 3). 1.3. Effects on extracellular matrix components

differences were detected between the control incubation (C) and those wound fluids not containing P. aeruginosa elastase (ELA 2 ). Similar findings were obtained when the P. aeruginosa isolates were cultured together with human skin biopsies. Zymographic analysis verified elastase production (Fig. 2B, inset ELA þ ). There was a clear association between elastase production and degradation of protein components of the human skin biopsies. Only elastase-producing P. aeruginosa degraded human skin (Fig. 2B and C, ELA þ ). The finding that addition of purified elastase (PAELA) resulted in identical degradation patterns of the skin biopsies when compared with those generated by elastase-producing bacteria (compare PAELA with ELA þ , Fig. 2B and C), confirmed that the effects on protein degradation were due to the production of P. aeruginosa elastase. Next, we determined the effects of elastase-producing P. aeruginosa on isolated proteins that are known to be degraded in chronic infected ulcers. Since wound fluid may itself contain variably processed forms of these proteins [8,9], plasma was chosen in these experiments. Thus, different amounts of conditioned medium from elastaseproducing P. aeruginosa cultures were incubated with human plasma and the effects on various plasma components were analyzed by immunoblotting (see Section 3 and legend to Fig. 3). The proteinase inhibitors a1-AT, I-a-I, a2-M, and KIN, were all degraded by P. aeruginosa growth medium (Fig. 3, lanes 1 –3). Complement factor C3

To examine the effect of elastase-producing P. aeruginosa on extracellular matrix, 35 S-methionine and 35 S-sulphate labelled fibroblast components (Fig. 4B, proteins; prot, and PG), purified human FN, and decorin

Fig. 4. Elastase-expressing P. aeruginosa degrade fibroblast extracellular proteins and PG, purified FN, and Dec. 35S-methionine labelled proteins (Prot) or 35S-sulphate labelled PG (10 ml) were incubated for 6 h at 37 8C with conditioned medium (10 ml) from P. aeruginosa cultures (þ ). Analysis was performed on SDS– PAGE (3– 12%) and radioactivity visualized by radioimaging (Fuji). Two micrograms purified human FN was incubated with 10 ml of medium from P. aeruginosa cultures for the same period of time (FN; þ) (analysis by 4–15% SDS–PAGE, protein staining). The PG Dec, was incubated with P. aeruginosa products (Dec; þ ) and glycosaminoglycans were visualized by the dye Azur A (3–12% SDS–PAGE). In all experiments, unconditioned medium was used as control (lanes indicated by C). Molecular weight markers (in kDa) are indicated to the left.

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(Dec), were incubated with TH-medium (C) or medium from elastase-expressing P. aeruginosa (þ ). In the experiments below, the elastase-producing isolate described in Fig. 1 was used. The addition of medium from elastaseproducing P. aeruginosa, resulted in extensive degradation of extracellular fibroblast proteins as well as PG. The , 100 kDa PG produced by fibroblasts, previously identified as Dec [17], was completely degraded, and 40 –50 kDa 35 S-labelled material, corresponding to free and/or peptide linked glycosaminoglycan chains [18], was identified (Fig. 4, Dec; þ ). Degradation was also identified after incubation of P. aeruginosa medium with purified FN and Dec (Fig. 4; FN and Dec), or boiled fibroblast products (for inactivation of endogenous MMPs). Likewise, degradation was seen after addition of PAELA or alkaline proteinase to purified FN and Dec (not shown). This demonstrates a direct effect of bacterial proteinases on fibroblast proteins and PG. Growth media from radiolabelled fibroblast cultures, cocultivated with P. aeruginosa products for 48 h, contained similarly degraded proteins and PG (not shown). Analysis of the cell-density, assayed with crystal violet, was performed. Addition of P. aeruginosa conditioned medium to cell cultures significantly inhibited fibroblast cell-growth (Fig. 5A). Insets (Fig. 5A) show fibroblasts stained with crystal violet after addition of 15% (v/v) TH-medium (C) or conditioned medium (15%). Next, we analyzed whether proteinase was responsible for the observed effects on fibroblast cell growth. In growth assays, the inhibition exerted by P. aeruginosa medium (containing 0.08 U of proteinase activity) [19], was compared with PAELA of the same activity. 40 – 50% growth inhibition was observed, indicating that the bacterial proteinase was responsible for most/all of the growth inhibitory effect exerted by this particular elastase-producing P. aeruginosa isolate. Finally, we investigated whether apoptosis was induced by the P. aeruginosa conditioned medium. Fibroblasts were incubated with P. aeruginosa medium (at 15%, v/v) and the generation of nucleosomal ladders was studied (Fig. 5B). Indomethacin and staurosporine both induced DNA fragmentation (Fig. 5B, d– f), but no such effect was identified after treatment with products from the P. aeruginosa isolate (Fig. 5B, b). Furthermore, by fluorescence microscopy, externalization of phosphatidylserine and intense DAPI staining of nuclei were readily observed in cultures treated with the apoptosis-inducing drug camptothecin (not shown). However, treatment with 15% (v/v) P. aeruginosaconditioned medium for up to 72 h did not trigger these apoptotic changes, in conjunction with the finding that the studied isolate was not expressing exotoxin A (not shown).

2. Discussion Release of various proteinases, such as elastase and alkaline proteinase of P. aeruginosa [14,15,20], degrade various host factors; kallikreins, kininogen, coagulation

Fig. 5. Fibroblast growth is inhibited by P. aeruginosa extracellular products. Panel A: confluent human skin fibroblasts were trypsinized and plated on 96-well plates (about 3000 cells/well) in minimal essential medium containing 10% serum. After plating for 4 h, the medium was changed to the same medium without serum and cells were incubated for 24 h. The experiments were then performed in F12 medium supplemented with insulin, transferrin and epidermal growth-factor (20 ng/ml) with the addition (5–15%, v/v) of unconditioned TH medium or conditioned sterilefiltered P. aeruginosa medium. After 5 days, cell density was assayed with the crystal violet method (see Section 3). The growth inhibition (in %) relative the control medium is indicated. The numbers indicate the mean of 3 experiments (each using 5 wells/point). SD is indicated. The photographs show representative sections of fibroblast cultures after 5 days of incubation, after staining with crystal-violet (C, control; 15%, addition of medium of elastase-expressing P. aeruginosa). In Panel B, proliferating fibroblast cultures were treated with P. aeruginosa medium (15%, v/v), or the apoptosis inductors indomethacine and staurosporine. The extent of DNA-degradation was analyzed by Ligation Mediated PCR followed by agarose gel electrophoresis (1.5%). (a) control; (b) 15% P. aeruginosa medium; (c) DMSO (solvent for the apoptosis inductors); (d) indomethacine; (e) staurosporine (adherent cells); (f) staurosporine (floating cells); (g) positive control (calf thymus DNA). The inset shows a less exposed gel (d– g).

factors, complement, cytokines, and antiproteinases (for review see Refs. [21 –24]), transferrin [25], the matrix components laminin and collagen [26,27] and may delay airway epithelial wound repair [28], increase lung epithelial permeability [29] and induce inflammation in a rat airpouch model [30]. Various exotoxins, phospholipase C, lipopolysaccharide, and alginate, represent additional virulence factors [22,31,32], which allow this versatile pathogen to colonize and infect various human tissues. P. aeruginosa

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causes clinically relevant diseases including bacteriemia in burn victims [33], chronic lung infection in cystic fibrosis patients [34], nosocomial pneumonia among critically ill patients [35], and ulcerative keratitis [36]. P. aeruginosa is also frequently found in chronic venous leg ulcerations, a disease which afflicts about 300,000 patients in US only. This disease, characterized by excessive tissue proteolysis, is aggravated by the presence of high bacterial loads [11]. This fact emphasizes the need of characterizing bacterial proteinases and their potential effects when released into chronic ulcers during P. aeruginosa infection. The major proteinase produced by the herein studied ulcer-derived strain was shown to be identical to previously characterized P. aeruginosa elastase, as judged by sequence analysis and immunoblotting. As presented here, several lines of evidence indicate a role of P. aeruginosa elastase in chronic ulcers. First, ex vivo experiments demonstrated that elastase-producing P. aeruginosa induced extensive degradation of human wound fluid and human skin extracellular products. Second, major proteins/glycoproteins and PG secreted by human fibroblasts were extensively degraded by elastase-producing P. aeruginosa in vitro. Third, P. aeruginosa, in vitro, induced strikingly similar changes in antiproteinases and complement as those previously identified in chronic wound fluid in vivo [8]. Finally, P. aeruginosa elastase inhibited the growth of human skin fibroblasts. Thus, P. aeruginosa acts at multiple molecular targets that exert crucial functions during the wound healing process. For example, Dec, a ubiquitous PG of connective tissues (such as dermis), regulates, the assembly of collagen fibers, but also binds TGF-b [37]. Dec degradation release free dermatan sulphate chains of Dec which bind to and inhibit the function of antibacterial peptides, such as defensin and LL-37, thus enabling persistence in skin ulcers [38,39]. Analogous findings demonstrated that P. aeruginosa metalloproteinase lasA, releases the heparan sulphatecontaining PG syndecan, compromising innate immunity [40]. P. aeruginosa elastase also degrades and inactivates the antibacterial peptides LL-37 and a-defensin [39], leading to enhanced survival in wound fluid. The finding that elastase, in vitro, inhibited fibroblast growth was unexpected. P. aeruginosa exotoxin A, among other exotoxins, induce apoptosis [41 – 43]. However, the strain studied here exerted most of its effect on cell-growth by an apoptosis-independent, proteinase-mediated, mechanism. The molecular mechanisms underlying the growth arrest were not studied, but could be related to P. aeruginosa elastase-mediated degradation of growth factors and matrix proteins or degradation or release of cell-associated receptors. It is interesting to note that another P. aeruginosa virulence factor, the metalloproteinase lasA, has been shown to induce shedding of syndecan-1, a PG involved in growth factor activation and cell adhesion [44]. We have previously shown that , 50% of all chronic ulcer-derived P. aeruginosa isolates express elastase [16].

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From a clinical point of view, this finding could perhaps underlie the observation that not all chronic ulcers deteriorate during P. aeruginosa infection, a finding that has caused some debate as to whether this pathogen causes wound delay or not. Our data, together with previous findings on variation of other P. aeruginosa virulence factors, certainly underline the necessity of incorporating bacterial genotype/phenotype data in future wound healing studies. From a therapeutical point of view, it is highly interesting that proteinase inhibitors have been shown to reduce P. aeruginosa induced tissue damage in corneal infections [45], and that immunization with a P. aeruginosa elastase peptide reduced the severity of experimental lung infections caused by this pathogen [46]. A hydroxamate inhibitor of lasA blocked syndecan shedding, and led to restoration of innate immunity and less bacterial propagation during lung infections [40]. Thus, specific inhibitors of P. aeruginosa elastase may reduce inactivation of endogenous antibacterial peptides as well as tissue destruction in chronic skin ulcers infected by P. aeruginosa.

3. Materials and methods 3.1. Preparation of P. aeruginosa conditioned medium and wound fluid P. aeruginosa was isolated by routine procedures from patients with chronic venous ulcers. The venous insufficiency was determined by a hand-held doppler. For preparation of P. aeruginosa conditioned medium, TH medium (Difco) was inoculated and bacteria grown over night to stationary phase. Bacteria were pelleted by centrifugation and supernatants sterile-filtered (0.3 mm) and stored at 2 20 8C. Sterile wound fluid was obtained from surgical drainages after mastectomy. Collection was for 24 h after operation. Wound fluids were centrifuged, aliquoted, and stored at 2 20 8C. The use of this material was approved by the Ethics Committee at Lund University (LU 509-01, LU 708-01). Informed consent was obtained from the patients. For preparation of infected wound fluids, wound fluid from surgical wounds (1 ml diluted 1:1 with TH, denoted 50% WF in the text) was inoculated with 20 ml of overnight cultures of P. aeruginosa. Bacteria were pelleted by centrifugation and supernatants were stored at 2 20 8C. 3.2. Preparation of radiolabelled fibroblast products Human foreskin fibroblasts (passage 10– 20) were grown to confluency in minimal essential medium (Gibco BRL) supplemented with 10% (v/v) donor calf serum, 2 mM L -glutamine, penicillin (100 units/ml) and streptomycin (100 mg/ml) (Life Technologies). The cells were then incubated 2 h with medium supplemented with 2% serum

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followed by no serum for 2 h. Incorporation of radioactive precursors was performed under serum-free conditions. Incorporation of 35S-sulphate (50 mCi/ml, Amersham) was performed in sulphate-deficient medium, where MgSO4 was replaced with MgCl2. 35S –methionine labelling was performed in methionine-free medium supplemented with 35 S – methionine (50 mCi/ml, Amersham). Labelling was for 48 h. The medium was decanted and cell-debris was spun down (1000 g, 10 min). The supernatants were aliquoted and stored at 2 20 8C. 3.3. Separation of P. aeruginosa proteinases and protein sequencing For preparation of P. aeruginosa proteinase [47], proteins in the growth medium were precipitated by ammonium sulphate (70% saturation). After dialysis (10 mM Tris, pH 8.0), separation was performed on a high Q anion exchange column (Bio-Rad, USA) using a gradient of 0 –1.0 M NaCl in 10 mM Tris, pH 8.0. Aliquots were analyzed by zymography, and fractions containing a 150 kDa proteinase, which was identified as a multimeric form of elastase (, 35 kDa) by immunoblotting (see Section 1) and a 50 kDa protease, recognized by antibodies against alkaline proteinase (see Section 1), were pooled. For sequencing of the , 150/35 kDa proteinase, material was precipitated with 9 volumes of ethanol, separated on a 4 – 15% Bio Rad minigel (see above) and proteins were transferred to PVDF membranes. A protein band corresponding to the molecular size of P. aeruginosa elastase (33 kDa) was cut out. Amino terminal sequencing was performed by Innovagen AB, Lund, Sweden. 3.4. PCR analysis of genes coding for P. aeruginosa elastase and alkaline proteinase and sequencing of P. aeruginosa elastase Genomic DNA was prepared essentially as previously described [48] with the addition that bacteria were treated with 1000 U mutanolysin (Sigma) at 37 8C for 2 h, followed by lysis by 1% SDS –0.2% Tween-20 at 0 8C. PCR-reactions (50 ml) were performed in buffer containing 1 £ Taq polymerase buffer (Life Technologies, USA), 1.5 mM NaCl, 0.5 mM of each primer, 0.05 U/ml Taq platinum polymerase (Life Technologies), 0.5 ml P. aeruginosa genomic DNA template and 0.1 mM each of dATP, dCTP, dGTP and dTTP (Boehringer Mannheim, Germany). PCR samples were heated to 95 8C for 5 min before amplification. Parameters used were; 30 s at 95 8C (denaturation), 45 s at 56 8C (annealing) and 90 s at 72 8C (extension). Amplified DNA was analyzed by 1% agarose gel electrophoresis. For sequencing, primers amplifying the mature form of elastase were selected. These primers contained nucleotides enabling ligation into pGEX-5X-3. 50 -TGGGAAGGCCGGATCCACGCCGAGGCG, 50 -CAGCCGGGACCGAATTCCTTACAACGCGCT; 942 bp. The selected DNA fragment

was purified with Magice PCR Preps DNA Purification System (Promega, USA). The final PCR-product was ligated into pGEX-5X-3, which was used for transformation of E. coli. Sequencing was performed by Innovagen AB, Lund, Sweden. Oligonucleotides (synthesis by Innovagen AB, Lund) for the amplification of the sequence coding for the mature form of elastase [14] and alkaline proteinase [15] were selected and three sets of primer pairs each, amplifying different parts of the elastase and alkaline proteinase genes yielded expected fragment sizes (see Ref. [16] for information). 3.5. Incubation of P. aeruginosa products with fibroblast extracellular glycoproteins and proteoglycans, purified human decorin, or fibronectin 10 ml of radiolabelled fibroblast products (35S-methionine or 35S-sulphate) were incubated with 10 ml sterilefiltered conditioned medium of elastase-expressing P. aeruginosa. For analysis of effects on purified human Dec and FN, 10 mg Dec, isolated from cervix (a generous gift from Dr A. Malmstro¨m), and 5 mg FN (Sigma) were separately incubated with 10 ml of P. aeruginosa products. In all cases, incubations were for 6 h at 37 8C, and addition of TH medium to reactions was used as control. For electrophoresis, SDS – sample buffer (5% SDS, 20% glycerol, 4 mM EDTA, 0.04% bromophenol blue, 125 mM Tris – HCl, pH 6.8, and 10% b-mercaptoethanol) was added. 3.6. Metabolic labeling of fibroblast cultures in the presence of P. aeruginosa secreted products Human skin fibroblasts were plated onto 12-well plates in minimal essential medium supplemented with 10% (v/v) donor calf serum, 2 mM L -glutamine, penicillin (100 units/ml), and streptomycin (100 mg/ml). After 4 h, the newly plated proliferating cells were radiolabelled with 35 SO4 or 35S-methionine in MgSO4-free F12 medium or methionine-free medium, respectively, supplemented with epidermal growth factor (20 ng/ml) (Life Technologies), insulin, and transferrin, and 10% of sterile-filtered conditioned medium of elastase-expressing P. aeruginosa. For control, the same amount of unconditioned TH medium was added. After a labelling period of 48 h, the medium (kept on ice) was decanted, centrifuged (2000 rpm, 5 min, 4 8C), and the supernatants were aliquoted and stored at 2 20 8C. 3.7. Degradation of plasma components by P. aeruginosa secreted products One microliter (, 70 mg) of plasma (4 mM EDTA – plasma) was incubated with 10, 50 or 100 ml of conditioned medium of elastase-expressing P. aeruginosa for 6 h at 37 8C. When needed, PBS was added to a final volume of 100 ml. As control, PBS or TH medium was added. The final

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concentration of EDTA (0.04 mM) in these experiments was significantly below the required inhibitory concentration (1 –10 mM). Fourteen microliters of this material (, 10 mg plasma protein) was then supplemented with SDS sample buffer and analyzed by electrophoresis. 3.8. Infection of human skin biopsies by P. aeruginosa Human skin was obtained in connection with skin transplant surgery, and kept in PBS in the cold (, 2 h) before use. Four millimeter punch biopsies were made, and immersed in 24-well plates (Falcon) in 200 ml minimal essential medium according to a previously published procedure [49]. Inoculation was made with 5 ml of overnight cultures of P. aeruginosa. After 18 h, the supernatants were gently removed, bacteria were pelleted by centrifugation and supernatants were stored at 2 20 8C. Skin biopsies were extracted by boiling for 10 min in 10% SDS (in water). 3.9. Zymography Substrate gel zymography was performed essentially as described [50] with 1 mg bovine gelatin per ml gel. P. aeruginosa products were mixed with sample buffer (0.4 M Tris – HCl, 20% glycerol, 5% SDS, 0.03% bromophenol blue, pH 6.8) and electrophoresed on 10% polyacrylamide gels. To remove SDS, gels were incubated with 2.5% Triton X-100. Incubation was then performed for 18 h at 37 8C in buffer containing 50 mM Tris – HCl, 200 mM NaCl, 5 mM CaCl2, 1 mM ZnCl2 (pH 7.5). Gels were stained with Coomassie blue G-250 in 30% methanol, 10% acetic acid for 1 h and destained in the same solution without the dye. Gelatinase containing bands were visualized as clear bands against a dark background. 3.10. SDS – polyacrylamide gel electrophoresis, immunoblotting and radioimaging SDS – polyacrylamide gel electrophoresis (SDS –PAGE) was performed on 3– 12% (Hoefer system, Pharmacia, Sweden), 10% or 4 –15% (Ready-Gel system, Bio-Rad, USA) polyacrylamide gradient gels. Samples were dissolved in 25 ml of 5% (w/v) SDS, 20% (v/v) glycerol, 4 mM EDTA, 0.04% bromophenol blue, 125 mM Tris/HCl, pH 6.8. b-mercaptoethanol was added to a final concentration of 10% (v/v). Samples were boiled for 3 min and electrophoresed for approx. 30 –40 min (Bio-Rad system) or 16 h (Pharmacia system). For detection of proteins, the gels were developed with GELCODE blue stain reagent (Pierce, USA). For detection of PG, gels were fixed (30 min) and transferred to water (2 £ 1 h). Polyanionic material was visualized by incubation (1 – 3 h) with 0.08% Azur A (aqueous solution, Sigma) and subsequent destaining in water. For immunodetection of different plasma proteins, proteins were transferred to nitrocellulose membranes

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(Hybond NC) by diffusion for at least 48 h and immunoblotting was performed as previously described [8]. The dilutions for the primary antibodies were as follows, a1-AT; 1:10,000; a2-M, I-a-I, C3; 1:1000, polyclonals (goat) against KIN; 1:6000. Dilution for all antirabbit polyclonals (HRP) was 1:1000 and for antigoat polyclonals 1:3000. For detection of P. aeruginosa elastase and alkaline proteinase, specific rabbit polyclonals (a generous gift of Dr J. Fukushima) were used (1:500 and 1:8000, respectively). The membranes were developed using the ECL system (Boehringer, Germany) [8]. For radioimaging, gels were dried on paper, and development was performed using a radioimaging system (Bas 2000, Fuji). 3.11. Cell proliferation assay This was performed as previously described [51]. Briefly, trypsinized cells were plated onto 96-well plates (Costar, Cambridge) in minimal essential medium supplemented with 10% donor calf serum. After a period of 4 h, the medium was changed to serum-free MEM. After 24 h, serum free F12, supplemented with insulin and transferrin and EGF (20 ng/ml) and varying amounts of conditioned P. aeruginosa medium, was added. For control, TH medium was used. The cells were grown for 5 days and the celldensity was subsequently assayed by use of crystal violet. 3.12. Apoptosis detection by Ligation Mediated-PCR Cells were plated onto petri dishes (5 £ 105 cells/dish) and essentially treated as for the cell proliferation experiments (see above) with the exception that P. aeruginosa conditioned medium was added after 72 h and incubation was for 48 h. For induction of apoptosis, cells were incubated with indomethacine (400 mM) (Sigma) and staurosporine (0.1 mM) (Sigma) for 24 h before DNA preparation. Only dishes containing sturosporine-treated fibroblasts contained non-adherent cells in the culture medium. These were centrifuged (2000g for 5 min) before DNA preparation. DMSO (solvent for the apoptosis inducers) and TH medium were included as control. The ApoAlerte Ligation Mediated-PCR Ladder Assay Kit (Clontech, USA) was used for detection of nucleosomal ladders in DNA obtained from fibroblast cultures. Briefly, fibroblast DNA (, 0.25 mg) was ligated to adaptors (12 and 24-mers) and the adaptor-ligated DNA (100 ng) was subsequently subjected to PCR (30 cycles). The material was then analyzed by agarose gels (1.5%). SYBR gold was used for visualization of bands. 3.13. Fluorescence microscopy Human fibroblasts were grown on methanol-cleansed glass cover slips for 48 h with different supplements. After culture, the fibroblast cultures were washed with ice-cold calcium-containing PBS, and the cells were stained with

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A. Schmidtchen et al. / Microbial Pathogenesis 34 (2003) 47–55

FITC-labeled annexin (Trevigen) and DAPI (100 nM) (Molecular probes) for 20 min at 4 8C. Thereafter, the cells were carefully washed prior to fixation with 1% paraformaldehyde (Becton Dickinson). Fixation was initiated on ice for 15 min and then continued at room temperature for 45 min. Cover slips were then mounted using ProLong AntiFade Reagent (Molecular probes). Visual inspection and recording of images was performed using a Nicon Eclipse TE300 inverted fluorescence microscope equipped with a Hamamatsu C4742-95 cooled CCD camera, using a Plan Apochromat 60 £ objective and a high N.A. oil-condenser.

[11]

[12]

[13]

[14]

Acknowledgements [15]

This work was supported by grants from the Swedish Medical Research Council (projects 13471, 7480, 12182 and 12613), the Royal Physiographic Society in Lund, the Welander-Finsen, Magnus Bergvall, Thelma-Zoegas, Cra˚ hle´n and Kock ¨ sterlund, Groschinsky, A foord, Alfred O Foundations, and Hansa Medical AB. We thank Professor Jun Fukushima for the P. aeruginosa elastase and alkaline proteinase antibodies, Prof. Hiroshi Maeda for the purified alkaline proteinase enzyme and Professor Anders Malmstro¨m for the human decorin. We also wish to thank Mina Davoudi, Monica Heidenholm, and Victoria Rydenga˚rd for expert technical assistance.

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