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American Journal of Respiratory Cell and Molecular Biology. Vol. 28, pp. 528-537, 2003
© 2003 American Thoracic Society
DOI: 10.1165/rcmb.2002-0141OC

Pseudomonas aeruginosa Elastase Degrades Surfactant Proteins A and D

William I. Mariencheck*, John F. Alcorn*, Scott M. Palmer and Jo Rae Wright

Departments of Medicine and Cell Biology, Duke University Medical Center, Durham, North Carolina

Address correspondence to: Jo Rae Wright, Ph.D., Department of Cell Biology, Box 3709, Duke University Medical Center, Durham, NC 27710. E-mail: J.Wright{at}cellbio.duke.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Both in vitro and in vivo studies provide evidence that surfactant protein (SP)-A and SP-D have an important role in the innate immune response to Pseudomonas aeruginosa. In preliminary experiments characterizing the binding of SP-A to this bacteria, we observed the appearance of apparent degradation products of SP-A, and therefore we hypothesized that P. aeruginosa produces an enzyme that degrades SP-A. Incubation of SP-A with P. aeruginosa organisms from several clinical isolates resulted in concentration- and temperature-dependent degradation of SP-A that was inhibited by a metalloproteinase inhibitor, phosphoramidon. The degradative enzyme was purified by anion exchange chromatography and identified by ion trap mass spectroscopy as Pseudomonas elastase, which was shown to directly degrade SP-A and SP-D. Incubation of P. aeruginosa or purified elastase with cell-free bronchoalveolar lavage (BAL) resulted in degradation of SP-A. Furthermore, SP-A degradation fragments were detectable in BAL from lung transplant patients with cystic fibrosis. We speculate that degradation of SP-A and SP-D is a virulence mechanism in the pathogenesis of chronic P. aeruginosa infection.

Abbreviations: bronchoalveolar lavage, BAL • cystic fibrosis, CF • colony-forming units, cfu • collagenase-resistant fragment, CRF • high-performance liquid chromatography, HPLC • surfactant protein, SP • Tris-buffered saline, TBS


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
A growing number of independent investigations have provided evidence that both surfactant protein (SP)-A and SP-D mediate a variety of events in lung innate immunity (reviewed in Refs. 1, 2). The innate immune response uses germline-encoded proteins, which recognize molecular patterns common to pathogens, to initiate host defense before the antigen-specific response of the adaptive immune system (3). Several experimental observations suggest that SP-A and SP-D have important roles in the innate immune response to Pseudomonas aeruginosa. For example, intratracheal instillation of P. aeruginosa in mice made deficient in SP-A by targeted disruption of the gene results in higher bacterial loads and an earlier and more severe neutrophil infiltrate than in wild-type mice that express SP-A (4). In addition, in vitro studies demonstrate that SP-A and SP-D stimulate alveolar macrophage phagocytosis of P. aeruginosa by both opsonic and nonopsonic mechanisms (5–7).

Chronic infection of the lung with P. aeruginosa affects over 80% of adults with cystic fibrosis (CF) (8). This pathogen is a major cause of morbidity and mortality in patients with CF or other causes of bronchiectasis, but rarely affects those without underlying pulmonary or immune disease (9). In healthy individuals, inhaled bacteria are rapidly cleared by the mucociliary system, or are engulfed and killed by phagocytes of the innate immune system. Factors predisposing patients with structural lung disease to P. aeruginosa colonization include impaired mucociliary clearance, altered epithelial cell surfaces that facilitate binding by bacterial adhesins, and diminished innate killing of the bacteria by epithelial cells and macrophages (10–12).

It has been observed that the composition and quantity of surfactant is altered in the lavage fluid of patients with CF. For example Griese and coworkers reported that the concentration of SP-A in the bronchoalveolar lavage (BAL) of patients with CF is reduced compared with that of healthy control subjects (13). Further, that group reported detection of degradation fragments of SP-A in the BAL of patients with CF colonized with P. aeruginosa (14). Griese and coworkers also reported decreased levels of both SP-A and SP-D in BAL of patients with CF; interestingly, the levels of phospholipids were not different, suggesting that a global suppression of surfactant synthesis is not responsible for the observed changes. There are several factors that may contribute to decreased SP-A levels in the lungs of these patients. During chronic P. aeruginosa infection, neutrophils are attracted into lung airways by both host and bacterial chemotactic factors. These activated neutrophils produce reactive oxygen molecules and enzymes that damage the epithelial cells, which synthesize and secrete SP-A and other surfactant components (15, 16). Reactive oxygen species and neutrophil elastase damage and degrade SP-A, inhibiting SP-A–mediated surfactant lipid aggregation and adsorption speed in vitro (17–19). In addition, P. aeruginosa releases many virulence factors, including degradative enzymes. While examining binding interactions between SP-A and P. aeruginosa, we observed decreased levels of intact SP-A and the appearance of proteins smaller than intact SP-A that reacted with an anti–SP-A polyclonal antibody. In this work, we tested the hypothesis that P. aeruginosa produces an enzyme that degrades SP-A and SP-D, and speculate that this action is a virulence mechanism of P. aeruginosa lung infection.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents
All chemicals, except where noted, were obtained from Sigma Chemical Co. (St. Louis, MO).

Bacteria
Multiple strains of P. aeruginosa isolated from the sputum of patients with CF were obtained from the Clinical Microbiology Laboratories at Duke University (Durham, NC) or from the American Type Culture Collection (Rockville, MD); PAO1 and Escherichia coli S17–1 were kindly provided by Dr. Paul Phibbs (Eastern Carolina University, Greenville, NC). Individual colonies of these isolates were grown on nutrient agar plates (Difco, Detroit, MI) for 18 h at 37°C. The bacteria were then suspended in nutrient broth (Difco) with 20% glycerol and frozen in aliquots at -80°C. Immediately before each assay, aliquots of the bacteria were grown on nutrient agar plates for 18 h at 37°C, then suspended in Tris-buffered saline (TBS), pH 7.4. Colony-forming units (cfu) were determined by optical density at 660 nm, which was correlated to direct determination of cfu by serial dilution for each strain.

SP-A Isolation
SP-A was purified from BAL of patients with alveolar proteinosis as previously described (20). SP-A preparations were then treated with polymyxin agarose to reduce endotoxin contamination (21), dialyzed against 5 mM Tris, and centrifuged at 100,000 x g for 30 min before storage in 5 mM Tris, pH 7.4. Aliquots of each preparation were tested for the presence of endotoxin by the Limulus amebocyte lysate assay (Bio-Whittaker, Walkersville, MD), and only samples containing < 0.5 pg endotoxin/µg protein were used (21).

Iodination of SP-A
SP-A purified as above was labeled with Na125I (DuPont-NEN, Boston, MA) using Iodo Beads (Pierce, Rockford, IL) as previously described (22). Fractions with > 85% trichloroacetic acid precipitable counts were pooled and assayed for protein concentration by the bicinchoninic assay (Pierce), and counts per minute per microgram SP-A were determined by a {gamma} counter. The specific activity of the 125I-SP-A used ranged from 40,000 to 60,000 cpm/µg.

Isolation of SP-D
Recombinant rat SP-D was purified by maltose affinity chromatography from the media supernatant of cultured Chinese hamster ovary cells stably transfected with a full-length rat SP-D cDNA clone as previously described (23).

BAL
BAL from normal human (nonsmoking) volunteers was generously provided by Andrew Ghio, M.D. (Environmental Protection Agency, Chapel Hill, NC). Aliquots of BAL from lung transplant recipients were obtained during surveillance or diagnostic fiberoptic bronchoscopy at Duke University. The bronchoscope was positioned in a subsegmental orifice of the right middle lobe or lingula, and BAL performed with four aliquots of 25 ml 0.9% sterile saline. Aliquots of the pooled BAL were centrifuged at 250 x g for 10 min, and the cell-free supernatant was stored at –80°C until use. The study protocol was approved by the Duke University Medical Center Institutional Review Board (# 1115–98–7), and informed consent was obtained from all patients before the procedure.

Degradation Assay
For initial studies, SP-A purified from patients with alveolar proteinosis was used with a single strain of mucoid P. aeruginosa (S470) that was found to degrade SP-A. 125I-SP-A was incubated with 108 cfu of P. aeruginosa in 250 µl TBS with 2 mM CaCl2 for 16 h at 37°C. A volume corresponding to 10 ng of 125I-SP-A (from the concentration originally used) was resolved on a 15% SDS-PAGE gel under reducing conditions. The gel was dried and exposed to film. Unlabeled SP-A was then incubated in the presence or absence of increasing concentrations of P. aeruginosa for varying times at 4°C and 37°C. After incubation, the bacteria were collected by centrifugation at 7,000 x g in a Beckman 12 microfuge. Aliquots of the supernatants (or pellets) were resolved under reducing conditions by 15% SDS-PAGE and transferred to a nitrocellulose membrane. Immunoreactive SP-A (intact and degradation fragments) was visualized using a well characterized polyclonal antibody to SP-A by chemiluminescence (ECL; Amersham, Arlington Heights, IL) (24). Similar studies were performed with recombinant rat SP-D using a polyclonal anti-rat SP-D antibody that we have previously described (25). The migration pattern of elastase digested SP-D was also analyzed by Coomassie Blue staining of 7.5% SDS-PAGE under nonreducing conditions. Studies were conducted to characterize the SP-D elastase degradation fragment by digesting SP-D sequentially with elastase and collagenase (Worthington Biochemical Corp., Freehold, NJ). SP-D (20 µg/ml) was incubated with elastase (112 µg/ml) for 24 h at 37°C. Then collagenase was added at a final concentration of 5 U/µg of SP-D and the incubation was continued for 24 h at 37°C (26). The digestion products were resolved under reducing conditions by 15% SDS-PAGE and stained by Coomassie Blue.

For experiments designed to test the effect of the metalloproteinase inhibitor, phosphoramidon, on the degradation of SP-A by P. aeruginosa, a range of phosphoramidon (0.1–10 mM) was included in the incubations of SP-A with bacteria. For experiments to test whether normal human SP-A in association with surfactant phospholipids is susceptible to degradation by P. aeruginosa, 250 µl of cell-free BAL from normal human volunteers was incubated with purified elastase. Immunoreactive SP-A (intact and degraded) was detected from these reactions as described for the proteinosis SP-A above.

Analysis of Lung Transplant BAL
BAL from patients with CF who had received bilateral lung allografts were analyzed for the presence of SP-A and possible degradation fragments. Samples from the CF lung transplant recipients were separated under reducing conditions by SDS-PAGE and transferred to nitrocellulose. Immunoreactive SP-A was detected as described above.

Purification of P. aeruginosa Proteases
Proteases secreted by P. aeruginosa were isolated following the protocol of Coin and coworkers (27). A mucoid strain of P. aeruginosa that was found to degrade SP-A was grown in a 2-liter culture in nutrient broth (Difco) for 18 h at 37°C, shaking at 250 rpm. Alginate was precipitated from the cell-free culture supernatant with 0.25 M CaCl2 for 2 h at 4°C and removed by centrifugation. This product was then concentrated by ultrafiltration with tangential flow using the Minitan system (Millipore, Marlborough, MA) with a 10-kD cutoff membrane to a volume of 150 ml. The culture concentrate was then passed through a DEAE Sepharose CL-6B column (1.6 x 50 cm) in 30 mM Tris-HCl, pH 8.3, followed by a continuous linear gradient of NaCl (0–0.5 M) in the same buffer. Eluates were monitored by absorbance at 280 nm. Pooled and individual fractions, along with aliquots of the culture supernatant at each point in this process, were resolved by SDS-PAGE (15%) under reducing and nonreducing conditions and visualized by staining with Coomassie Blue. Varying concentrations of the eluates were then incubated with purified alveolar proteinosis SP-A and aliquots of that reaction were analyzed by Western blot with the polyclonal anti–SP-A antibody as described above. A polyclonal rabbit anti-elastase antibody was kindly provided by Dr. Efrat Kessler, Sheba Medical Center, Tel Aviv University (Tel Aviv, Israel).

Amino Acid Analysis and Protein Sequencing
Protein sequence analysis was performed at the Harvard Microchemistry Facility as previously described (28, 29). In brief, two purified proteins from column fractions exhibited degradative activity toward SP-A. These two proteins were separated by SDS-PAGE and treated in-gel by reduction, S-carboxyamidomethylation, and tryptic digestion. The resulting mixture was analyzed by microcapillary reverse-phase high-performance liquid chromatography (HPLC) nano-electrospray tandem mass spectrometry (µLC/MS/MS) on a Finnigan LCQ quadrupole ion trap mass spectrometer. The MS/MS spectra were correlated with known sequences in the NCBI nr and dbest databases using the algorithm Sequest (30) and programs developed at the Harvard Microchemistry Facility (28, 29) and confirmed by manual inspection.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
SP-A Is Degraded during Incubation with P. aeruginosa
In preliminary experiments characterizing the binding of 125I-SP-A to P. aeruginosa organisms, we observed loss of radioactive signal from the labeled SP-A during incubation with this organism. Incubation of 125I-SP-A in the presence of P. aeruginosa resulted in complete loss of detectable radioactivity from the monomeric and dimeric isoforms of 125I-SP-A after 16 h at 37°C. There was no change in the appearance of the 125I-SP-A after this incubation in the absence of bacteria (Figure 1).



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  		Figure 1. 125I-SP-A is degraded during incubation with P. aeruginosa. 125I-SP-A (25 µg/ml) was incubated in the presence or absence of 108 cfu P. aeruginosa in TBS + 2 mM CaCl2 for 16 h at 37°C. A volume corresponding to 10 ng of 125I-SP-A (from the starting concentration) was resolved by 15% SDS-PAGE under reducing conditions. The gel was then dried and exposed to film.

 
P. aeruginosa Degrades SP-A in a Time-, Concentration-, and Temperature-Dependent Manner
To determine whether the loss of signal from 125I-SP-A during incubation with P. aeruginosa was due to specific loss of 125I label at sites of SP-A iodination or from degradation of SP-A by a product of this bacteria, unlabeled SP-A was incubated with increasing concentrations of P. aeruginosa at 4°C and 37°C. At different time intervals, the bacteria were collected by centrifugation and samples of the supernatant were assayed by Western blot with a polyclonal antibody against SP-A. Incubating increasing concentrations of SP-A with P. aeruginosa organisms resulted in appearance of immunoreactive degradation fragments of SP-A (Figure 2A) corresponding in relative molecular mass to degradation products of SP-A incubated with purified human neutrophil elastase (18). Incubation of SP-A with increasing concentrations of P. aeruginosa resulted in qualitatively decreased concentrations of SP-A dimer and monomer, and increased concentrations of their degradation products at 37°C (Figure 2B), but not at 4°C. The polyclonal antibody used in these experiments does not react with supernatant from incubations of P. aeruginosa in the absence of SP-A.



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  		Figure 2. Degradation of SP-A by P. aeruginosa is temperature- and concentration-dependent. (A) Unlabeled SP-A (2.5–40 µg/ml) was incubated in the presence or absence of 108 cfu P. aeruginosa for 1 h at 37°C. The bacteria were then collected by centrifugation, and a sample of the supernatant was resolved by SDS-PAGE and transferred to nitrocellulose. Immunoreactive SP-A was visualized by chemiluminescence. (B) Unlabeled SP-A (25 µg/ml) was incubated with increasing concentrations of P. aeruginosa organisms (10-7 x 10-8 cfu) at 37°C or 4°C. At various time intervals (1 h is shown), bacteria were collected by centrifugation, and anti–SP-A immunoreactive products were detected as above.

 
P. aeruginosa Degradation of SP-A Is Inhibited by a Metalloproteinase Inhibitor
Given the similarity in molecular mass of the degradation fragments of SP-A produced by incubation with P. aeruginosa and purified neutrophil elastase, we hypothesized that the metalloproteinase, elastase, produced by P. aeruginosa degrades SP-A. Increasing concentrations of a potent metalloproteinase inhibitor, phosphoramidon, were added to reactions containing unlabeled SP-A and P. aeruginosa. The reactions were performed for 3 h, the bacteria were collected by centrifugation, and immunoreactive SP-A was detected by Western blot. Increasing concentrations of phosphoramidon inhibited degradation of SP-A by P. aeruginosa (Figure 3).



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  		Figure 3. A metalloproteinase inhibitor, phosphoramidon, inhibits P. aeruginosa degradation of SP-A. Increasing concentrations of phosphoramidon (0.1–10 mM) were added to reactions containing SP-A (25 µg/ml) and 108 cfu P. aeruginosa. Incubations were performed for 1 h at 37°C, and immunoreactive SP-A was detected in aliquots of the supernatants by Western blot.

 
Purification of P. aeruginosa Proteinases that Degrade SP-A
To test the hypothesis that the elastase produced by P. aeruginosa degrades SP-A, concentrated P. aeruginosa culture supernatant was passed through an anion exchange column and eluted with a continuous linear NaCl gradient (Figure 4). Coomassie staining of the pooled fractions demonstrated separation of the proteins present in the concentrated starting material (Figure 5A). The culture concentrate starting material was found to completely degrade SP-A, as were two pooled fractions (37–45 and 64–72). Partial degradation of SP-A was also noted in fractions 55–63 (Figure 5B).



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  		Figure 4. Purification of P. aeruginosa proteinases. The alginate-free supernatant of an 18 h culture of a mucoid strain of P. aeruginosa was concentrated and dialyzed against 30 mM Tris-HCl, pH 8.3. The concentrate was passed through a DEAE Sepharose CL-6B column (1.6 x 50 cm) followed by a continuous linear gradient of NaCl (0–0.5 M). Eluates were monitored by absorbance at 280 nm. Arrows indicate fractions containing degradative activity.

 


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  		Figure 5. Analysis of pooled fractions of P. aeruginosa culture concentrate. Proteins from the culture concentrate starting material (C) and eluates (fraction #'s) of the above column, were resolved by SDS-PAGE (15%) under reducing conditions and stained with Coomassie blue (A). Aliquots of the pooled fractions were incubated with SP-A (25 µg/ml) for 3 h at 37°C, and the products of this reaction were resolved by SDS-PAGE and transferred to nitrocellulose. Degradation of SP-A by pooled fractions is shown by loss of immunoreactive SP-A as assessed by Western blot (B).

 
Because degradation in fractions 64–72 correlated well with the presence of an ~ 36-kD protein, this protein was excised from SDS-PAGE of fraction 69 and processed for protein sequence analysis. The 36-kD protein was identified by microcapillary reverse phase HPLC nano-electrospray tandem mass spectrometry as Pseudomonas aeruginosa elastase (E.C.3.4.24.26; zinc metalloprotease, accession number 442,880) (31). The seventeen peptide sequences obtained are shown in Table 1. We were unable to identify definitively the protein responsible for degradation in fractions 37–45.


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  		TABLE 1 Tryptic peptide fragment sequences identified by ion trap mass spectrometry from Pseudomonas elastase

 
Several Different Isolates of P. aeruginosa Degrade SP-A
Distinct clinical isolates obtained from the sputum of patients with CF or laboratory isolates of mucoid and nonmucoid strains of P. aeruginosa were incubated with SP-A for 3 h at 37°C, and the bacteria collected by centrifugation. Immunoreactive SP-A was detected in aliquots of the supernatants of these reactions by Western blot (Figure 6A). Of eight distinct P. aeruginosa isolates tested, four were found to degrade SP-A, and these four strains all were positive for elastase by Western blot (Figure 6B). E. coli S17–2 did not degrade SP-A.



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  		Figure 6. Isolates of different strains of P. aeruginosa degrade SP-A. Isolates of laboratory or clinical strains of P. aeruginosa recovered from the sputa of patients with CF were incubated with SP-A (25 µg/ml SP-A, supernatants collected by overnight incubation of 108 cfu bacteria) for 3 h at 37°C. The bacteria were then collected by centrifugation, and immunoreactive SP-A was detected in the supernatant of this reaction by Western blot (A). Immunoreactive elastase was detected by Western blot using an anti-elastase antibody (B). Samples in A, by lane number, are: lane 1, purified SP-A; lanes 2–10, purified SP-A incubated with isolates as follows (PA indicates P. aeruginosa): lane 2, PA1 = PAO1; lane 3, PA2 = ATCC 27853; lane 4, E. coli S17–1; lane 5, PA3 = N66679; lane 6, PA4 = S2385; lane 7, PA5 = A55093; lane 8, PA6 = S472; lane 9, PA7 = S470a; lane 10, PA8 = S470. (B) Samples by lane number are: lane 1, purified elastase; lanes 2–10, supernatants from cultures of: lane 2, PA1 = PAO1; lane 3, PA2 = ATCC 27853; lane 4, E. coli; lane 5, PA3 = N66679; lane 6, PA4 = S2385; lane 7, PA5 = A55093; lane 8, PA6 = S472; lane 9, PA7 = S470a; lane 10, PA 8 = S470.

 
Purified Elastase Degrades SP-A and SP-D
Highly purified elastase isolated by anion exchange chromatography as described above was tested for its ability to degrade SP-A and SP-D. The identity of the elastase was confirmed by immunoblotting with anti-elastase antibody (data not shown). As shown in Figure 7, elastase degraded both human SP-A as well as rat SP-D. Interestingly, a degradation product of {bsim} 35 kD was consistently obtained when SP-D was incubated with elastase.



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  		Figure 7. Purified elastase degrades purified SP-A and SP-D. Increasing concentrations of purified human SP-A isolated from lavage of patients with alveolar proteinosis or purified recombinant rat SP-D isolated by maltose affinity chromatography was incubated for 3 h at 37°C with 60 µg/ml of elastase purified as described in Figure 4. Immunoreactive products were detected by Western blot of SP-A (A) or SP-D (B).

 
P. aeruginosa Elastase Degrades SP-A in the Presence of Surfactant Lipids
The vast majority of SP-A recovered by BAL is associated with surfactant phospholipids, primarily dipalmitoylphosphatidylcholine, particularly in the form of tubular myelin (32). To determine if SP-A in this environment is susceptible to degradation by P. aeruginosa, we obtained bronchoalveolar lavage from rats or from normal human volunteers. Cells were removed from the BAL by centrifugation, aliquots of this cell-free lavage were incubated with or without supernatant from 108 cfu P. aeruginosa organisms or purified elastase at 37°C for 3 h, and immunoreactive SP-A was detected in aliquots of the supernatant by Western blot. As shown in Figure 8, SP-A in human BAL is degraded by incubation with elastase. Similar results were obtained with rat BAL (data not shown).



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  		Figure 8. P. aeruginosa elastase degrades normal human SP-A in the presence of surfactant lipids. BAL was obtained from normal human volunteers and cells were removed by centrifugation (250 x g for 10 min), and the resulting cell-free lavage was assayed for protein concentration. Aliquots of this lavage were incubated with increasing concentrations of purified elastase for 3 h at 37°C. Immunoreactive SP-A was detected in an aliquot of the supernatant of this reaction by Western blot.

 
P. aeruginosa Elastase Cleaves the C-Terminal Domain of SP-D
Degradation of SP-D by P. aeruginosa elastase results in the formation of a major cleavage product of {bsim} 35 kD. To determine whether elastase cleaves SP-D in the N-terminal or the C-terminal region, SP-D was digested with elastase and analyzed under nonreducing conditions or sequentially digested with elastase and collagenase. Intact SP-D migrates as trimer of {bsim} 165 kD, whereas elastase-degraded SP-D still forms a presumptive trimer structure of {bsim} 110 kD (Figure 9A). Note that this degradative product migrates at {bsim} 35 kD reduced (see Figure 7). These data suggest that the N-terminus, which is required for multimerization via disulfide bonding (33), remains intact. To determine if the C-terminal region is degraded, the elastase degradation product was digested with collagenase. Bacterial collagenase degrades intact SP-D to an 18-kD collagenase-resistant fragment (CRF) containing the carbohydrate recognition domain of the protein (26, 33). When the elastase degradation product was digested with collagenase, no CRF was evident, thus suggesting that elastase is cleaving the CRF (Figure 9B). A similar analysis of SP-A digested with elastase was not performed because multiple cleavage products are detectable by Coomassie Blue staining of SDS-PAGE analysis.



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  		Figure 9. P. aeruginosa elastase degrades SP-D in the carbohydrate recognition domain. Purified recombinant rat SP-D isolated by maltose affinity chromatography was incubated at 37°C with 100 µg/ml of elastase purified as described in Figure 4. Degradation products were detected by Coomassie Blue staining of SP-D under nonreducing conditions (A). Recombinant rat SP-D was incubated with 112 µg/ml of P. aeruginosa elastase for 24 h at 37°C, followed by incubation with Collagenase (5 U/µg of SP-D) for 24 h at 37°C. SP-D degradation products were visualized by Coomassie Blue staining of SDS-PAGE gel (B). The collagenase-resistant fragment of SP-D is indicated by the arrow.

 
Evidence that P. aeruginosa Infection Is Associated with Degradation and Decreased Concentration of SP-A, In Vivo
Analysis of BAL from patients with CF who have undergone bilateral lung transplantation demonstrates degradation fragments of SP-A, as well as decreased concentrations of SP-A (Figure 10). Immunoreactive SP-A was detected by Western blot. These samples were obtained from a prospective, pilot study in which the investigators were blinded to the clinical outcomes of the patients undergoing lavage until after results of the Western blots were obtained. Three of six patients had apparent degradation fragments of SP-A; two patients, (Patients 2 and 4) had P. aeruginosa infections, and one of them had degradation fragments. Although we attempted to identify SP-D degradation products in these BAL samples, our interpretations were confounded by cross-reactivity of the anti–SP-D antibody with SP-A.



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  		Figure 10. BAL from patients with CF who had undergone bilateral lung transplant contain SP-A degradtion fragments. BAL from patients with CF who had undergone bilateral lung transplantation were obtained and immunoreactive SP-A was detected by Western blot analysis of equal aliquots of cell-free BAL from each patient. Patients 2 and 4 were positive for P. aeruginosa infection.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this article, we demonstrate that several isolates of P. aeruginosa organisms degrade purified SP-A from alveolar proteinosis patients, purified SP-D, as well as SP-A in the cell-free human BAL in a concentration- and temperature-dependent mechanism. Of the isolates studied in these experiments, {bsim} 50% (4 of 8) are capable of degrading SP-A, in vitro. This degradation is inhibited by phosphoramidon, a metalloproteinase inhibitor. A protein isolated from a concentrate of P. aeruginosa culture supernatant that degrades SP-A was purified and identified by microcapillary reverse phase HPLC nano-electrospray tandem mass spectrometry as P. aeruginosa elastase (E.C.3.4.24.26; zinc metalloprotease, accession number 442,880) (31). Identity of this protein as elastase was confirmed by Western blotting using an anti-Pseudomonas elastase antibody. In addition to degrading SP-A, P. aeruginosa elastase degraded SP-D in its C-terminal domain.

P. aeruginosa produces several extracellular virulence factors that are important in the pathogenesis of infections caused by this organism, including the Las A protease, alkaline protease, a lysine-specific protease, and the las B gene product, elastase. Over 75% of strains of P. aeruginosa isolated from clinical lung infections produce elastase (34), one of the extracellular and cell-associated virulence factors that contributes to tissue damage in Pseudomonas infections (35). Elastase is a zinc metalloprotease of {bsim} 33 kD that degrades elastin (36), collagen, several cytokines (reviewed in Ref. 35) as well as several complement components, including the opsonin C3 and the chemotactic peptide C5 (36–38). Elastase also inactivates immunoglobulin G (38). In a recent intriguing study, Vandivier and coworkers (39) reported that cleavage of the phosphatidylserine receptor by elastase impairs clearance of apoptotic cells in patients with CF and bronchiectasis; they speculate that the resultant defective clearance in apoptotic cells may result in ongoing airway inflammation.

To the best of our knowledge, the ability of P. aeruginosa elastase to degrade SP-A and SP-D has not been previously reported, although it has been shown that neutrophil elastase degrades SP-A (18). Interestingly, incubation of purified surfactant with neutrophil elastase resulted in an inhibition of the adsorption speed of the surfactant to the air–liquid interface. This inhibition of activity was attributed to degradation of SP-A, although the possibility that the hydrophobic proteins of surfactant, SP-B and SP-C, were also degraded or inactivated could not be excluded. Subsequently, Liau and colleagues (40) reported that neutrophil elastase degraded SP-A, SP-B, and SP-C, and resulted in decreased adsorption speed and increased surface tension of the surfactant. It is likely that these decreases in surfactant function are mainly due to the degradation of SP-B and SP-C. In our studies with P. aeruginosa elastase, SP-B in rat BAL was not degraded (data not shown). Interestingly, Lema and coworkers (41) found that incubation of surfactant with the supernatant of P. aeruginosa cultures isolated from patients with CF resulted in inhibition of surfactant function. Thus, these studies show that exposure of surfactant to elastase affects the surface tension reducing activity of surfactant.

Our findings that SP-A is degraded in the lavage fluid of transplant patients with CF colonized with Pseudomonas is consistent with published reports that SP-A levels are decreased in the BAL of patients with CF. For example, Postle and coworkers (42) reported that SP-A levels were decreased by {bsim} 5-fold in patients with CF, whereas SP-D levels were decreased by {bsim} 50-fold, a finding consistent with our in vitro observations that both SP-A and SP-D are degraded by elastase. In contrast, phospholipid levels and content were unaffected. Likewise, Griese and coworkers also reported diminished levels of SP-A in lavage fluid of CF patients with lung infections (13) and degradation products of SP-A were detected (43). Furthermore, Meyer and colleagues reported decreased association of SP-A with lipid components of surfactant in patients with CF, although total SP-A levels were comparable in patients with CF and in normal volunteers (44). In contrast, Hull and colleagues reported increased SP-A in lavage of patients with CF with lung infections (45). Although the reasons for the differences are not known, the fact that the patients in the studies by Hull and coworkers were very young (with a mean age of 22.7 mo) and the fact that the samples were collected by a smaller volume of lung lavage may have impacted the outcome.

These studies, in conjunction with our findings of SP-A degradation products in lung transplant patients with CF and Pseudomonas infections, do suggest that in the cases where SP-A levels are decreased, degradation by Pseudomonas may be a contributing factor to susceptibility to infection. This possibility is supported by the finding that SP-A null mice are more susceptible to P. aeruginosa infection than are wild-type mice. It should be noted that we did not see degradation of SP-A in lavage fluid of all patients with Pseudomonas infections, a finding consistent with our observation that not all clinical isolates degrade SP-A in vitro. In addition, we did observe SP-A degradation products in patients without Pseudomonas infections, suggesting that other factors may also contribute to degradation of SP-A.

Degradation of SP-D by P. aeruginosa elastase results in the formation of a 35-kD fragment. Our findings suggest that SP-D is cleaved in the C-terminal carbohydrate recognition domain. This finding is consistent with the hypothesis that degradation of SP-D by P. aeruginosa elastase affects normal SP-D innate immune function. The absence of an intact collagenase-resistant fragment of SP-D indicates that the lectin-binding ability of the SP-D fragment is likely altered, an intriguing possibility which requires further study. Similar studies with SP-A could not be conducted due the complete degradation of SP-A by P. aeruginosa elastase after 3 h of incubation with elastase and the multiple degradation products present after shorter incubation periods.

We also observed that fractions of Pseudomonas culture supernatants that were eluted from the anion exchange column before elastase degraded SP-A and SP-D. Although we have attempted to identify the degradative protein in these fractions, we have not been successful to date in obtaining sufficient quantities of pure protein for unambiguous identification. These studies are ongoing.

What are the possible functional consequences of the degradation of SP-A and SP-D by elastase produced by Pseudomonas? Based upon the data discussed above, it appears that degradation of surfactant proteins with elastase results in inhibition of surface tension–reducing activity. In addition, we speculate that degradation of SP-A and SP-D will result in impaired host defense in the lung. In vitro, we have shown that SP-A and SP-D facilitate the phagocytosis of Pseudomonas by alveolar macrophages. In addition, SP-A–deficient mice are more susceptible to Pseudomonas infections and inflammation (4). Thus, in patients with CF, who are colonized with Pseudomonas, degradation of SP-A and SP-D by elastase or other enzymes would result in diminished levels of these proteins, and consequently an enhanced susceptibility to further infection. In a recent review (46) of pulmonary collectin (SP-A and SP-D) function in host defense, Crouch posed the question, "Why haven't microorganisms developed effective ways to avoid ‘static’ collectin defenses?" We propose that, at least in the case of P. aeruginosa, microorganisms have developed strategies for evading the SP-A– and SP-D–mediated host response, and that this strategy contributes to the enhanced susceptibility of certain patient populations to infection with P. aeruginosa.


   Acknowledgments
 
This work was supported by RO1 HL-51134 from the National Heart, Lung and Blood Institute. The authors thank Hollie Garner for purification of SP-A; Eric Walsh for purification of recombinant SP-D; Dr. Efrat Kessler, Sheba Medical Center, Tel Aviv University for kindly providing the anti-elastase antibody; Dr. Paul Phibbs, East Carolina University (Greenville, NC) for providing PAO1 and E. coli S17-1; and Dr. Andrew J. Ghio, Environmental Protection Agency, Chapel Hill, NC, for providing normal human lavage.


   Footnotes
 
* These authors contributed equally to the work presented in this article. Back

Received in original form July 30, 2002

Received in final form October 25, 2002


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Crouch, E. C. 1998. Collectins and pulmonary host defense. Am. J. Respir. Cell Mol. Biol. 19:177–201.[Abstract/Free Full Text]
  2. Wright, J. R. 1997. Immunomodulatory functions of surfactant. Physiol. Rev. 77:931–962. (Review)[Abstract/Free Full Text]
  3. Medzhitov, R., and C. A. Janeway, Jr. 1997. Innate immunity: impact on the adaptive immune response. Curr. Opin. Immunol. 9:4–9.[CrossRef][Medline]
  4. LeVine, A. M., K. E. Kurak, M. D. Bruno, J. M. Stark, J. A. Whitsett, and T. R. Korfhagen. 1998. Surfactant protein-A–deficient mice are susceptible to Pseudomonas aeruginosa infection. Am. J. Respir. Cell Mol. Biol. 19:700–708.[Abstract/Free Full Text]
  5. Mariencheck, W. I., J. Savov, Q. Dong, M. J. Tino, and J. R. Wright. 1999. Surfactant protein A enhances alveolar macrophage phagocytosis of a live, mucoid strain of P. aeruginosa. Am. J. Physiol. 277:L777–L786.[Abstract/Free Full Text]
  6. Manz-Keinke, H., H. Plattner, and J. Schlepper-Schafer. 1992. Lung surfactant protein A (SP-A) enhances serum-independent phagocytosis of bacteria by alveolar macrophages. Eur. J. Cell Biol. 57:95–100.[Medline]
  7. Restrepo, C. I., Q. Dong, J. Savov, W. I. Mariencheck, and J. R. Wright. 1999. Surfactant protein D stimulates phagocytosis of Pseudomonas aeruginosa by alveolar macrophages. Am. J. Respir. Cell Mol. Biol. 21:576–585.[Abstract/Free Full Text]
  8. FitzSimmons, S. C. 1993. The changing epidemiology of cystic fibrosis. J. Pediatr. 122:1–9.[Medline]
  9. Govan, J. R., and V. Deretic. 1996. Microbial pathogenesis in cystic fibrosis: mucoid Pseudomonas aeruginosa and Burkholderia cepacia. Microbiol. Rev. 60:539–574.[Abstract/Free Full Text]
  10. Prince, A. 1992. Adhesins and receptors of Pseudomonas aeruginosa associated with infection of the respiratory tract. Microb. Pathog. 13:251–260.[CrossRef][Medline]
  11. Ramphal, R., S. K. Arora, and B. W. Ritchings. 1996. Recognition of mucin by the adhesin-flagellar system of Pseudomonas aeruginosa. Am. J. Respir. Crit. Care Med. 154:S170–S174.
  12. Smith, J. J., S. M. Travis, E. P. Greenberg, and M. J. Welsh. 1996. Cystic fibrosis airway epithelia fail to kill bacteria because of abnormal airway surface fluid. Cell 85:229–236. [Published erratum appears in Cell 87(2):355.][CrossRef][Medline]
  13. Griese, M., P. Birrer, and A. Demirsoy. 1997. Pulmonary surfactant in cystic fibrosis. Eur. Respir. J. 10:1983–1988.[Abstract]
  14. Bredow, C. H., G. Obermaier, and M. Griese. 1998. Structure of surfactant protein A (SP-A) isoforms and other bronchoalveolar lavage fluid proteins in patients with cystic fibrosis (CF). 5th Marburg Surfactant Symposium: surfactant and alveolar biology:A65.
  15. Venaille, T. J., G. Ryan, and B. W. Robinson. 1998. Epithelial cell damage is induced by neutrophil-derived, not pseudomonas-derived, proteases in cystic fibrosis sputum. Respir. Med. 92:233–240.[CrossRef][Medline]
  16. Baltimore, R. S., C. D. Christie, and G. J. Smith. 1989. Immunohistopathologic localization of Pseudomonas aeruginosa in lungs from patients with cystic fibrosis: implications for the pathogenesis of progressive lung deterioration. Am. Rev. Respir. Dis. 140:1650–1661.[Medline]
  17. Haddad, I. Y., J. P. Crow, P. Hu, Y. Ye, J. Beckman, and S. Matalon. 1994. Concurrent generation of nitric oxide and superoxide damages surfactant protein A. Am. J. Physiol. 267:L242–L249.[Abstract/Free Full Text]
  18. Pison, U., E. K. Tam, G. H. Caughey, and S. Hawgood. 1989. Proteolytic inactivation of dog lung surfactant-associated proteins by neutrophil elastase. Biochim. Biophys. Acta 992:251–257.[Medline]
  19. Ross, G. F., J. Sawyer, T. O'Connor, and J. A. Whitsett. 1991. Intermolecular cross-links mediate aggregation of phospholipid vesicles by pulmonary surfactant protein SP-A. Biochemistry 30:858–865.[CrossRef][Medline]
  20. Wright, J. R., R. E. Wager, S. Hawgood, L. Dobbs, and J. A. Clements. 1987. Surfactant apoprotein Mr=26,000–36,000 enhances uptake of liposomes by type II cells. J. Biol. Chem. 262:2888–2894.[Abstract/Free Full Text]
  21. Wright, J. R., D. F. Zlogar, J. C. Taylor, T. M. Zlogar, and C. I. Restrepo. 1999. Effects of endotoxin on surfactant protein A and D stimulation of NO production by alveolar macrophages. Am. J. Physiol. 276:L650–L658.[Abstract/Free Full Text]
  22. Tino, M. J., and J. R. Wright. 1996. Surfactant protein A stimulates phagocytosis of specific pulmonary pathogens by alveolar macrophages. Am. J. Physiol. 14:L677–L688.
  23. Dong, Q., and J. R. Wright. 1998. Degradation of surfactant protein D by alveolar macrophages. Am. J. Physiol. 18:L97–105.
  24. Tenner, A. J., S. L. Robinson, J. Borchelt, and J. R. Wright. 1989. Human pulmonary surfactant protein (SP-A), a protein structurally homologous to C1q, can enhance FcR- and CR1-mediated phagocytosis. J. Biol. Chem. 264:13923–13928.[Abstract/Free Full Text]
  25. McIntosh, J. C., A. H. Swyers, J. H. Fisher, and J. R. Wright. 1996. Surfactant proteins A and D increase in response to intratracheal lipopolysaccharide. Am. J. Respir. Cell Mol. Biol. 15:509–519.[Abstract]
  26. Ogasawara, Y., and D. R. Voelker. 1995. The role of the amino-terminal domain and the collagenous region in the structure and the function of rat surfactant protein D. J. Biol. Chem. 270:19052–19058.[Abstract/Free Full Text]
  27. Coin, D., D. Louis, J. Bernillon, M. Guinand, and J. Wallach. 1997. LasA, alkaline protease and elastase in clinical strains of Pseudomonas aeruginosa: quantification by immunochemical methods. FEMS Immunol. Med. Microbiol. 18:175–184.[Medline]
  28. Chittum, H. S., W. S. Lane, B. A. Carlson, P. P. Roller, F. D. Lung, B. J. Lee, and D. L. Hatfield. 1998. Rabbit beta-globin is extended beyond its UGA stop codon by multiple suppressions and translational reading gaps. Biochemistry 37:10866–10870.[CrossRef][Medline]
  29. Zhang, Y., G. LeRoy, H. P. Seelig, W. S. Lane, and D. Reinberg. 1998. The dermatomyositis-specific autoantigen Mi2 is a component of a complex containing histone deacetylase and nucleosome remodeling activities. Cell 95:279–289.[CrossRef][Medline]
  30. Eng, J. K., A. L. McCormick, and J. R. I. Yates. 1994. An approach to correlate tandem mass spectral data of peptides with amino acid sequences in a protein database. J. Am. Soc. Mass Spectrom. 5:976–989.[CrossRef]
  31. Thayer, M. M., K. M. Flaherty, and D. B. McKay. 1991. Three-dimensional structure of the elastase of Pseudomonas aeruginosa at 1.5-A resolution. J. Biol. Chem. 266:2864–2871.[Abstract/Free Full Text]
  32. Weaver, T. E., and J. A. Whitsett. 1991. Function and regulation of expression of pulmonary surfactant-associated proteins. Biochem. J. 273:249–264.
  33. Crouch, E., A. Persson, D. Chang, and J. Heuser. 1994. Molecular structure of pulmonary surfactant protein D (SP-D). J. Biol. Chem. 269:17311–17319.[Abstract/Free Full Text]
  34. Hamood, A. N., J. A. Griswold, and C. M. Duhan. 1996. Production of extracellular virulence factors by Pseudomonas aeruginosa isolates obtained from tracheal, urinary tract, and wound infections. J. Surg. Res. 61:425–432.[CrossRef][Medline]
  35. Galloway, D. R. 1991. Pseudomonas aeruginosa elastase and elastolysis revisited: recent developments. Mol. Microbiol. 5:2315–2321.[CrossRef][Medline]
  36. Morihara, K. 1964. Production of elastase and proteinase by Pseudomonas aeruginosa. J. Bacteriol. 88:745[Abstract/Free Full Text]
  37. Schad, P. A., R. A. Bever, T. I. Nicas, F. Leduc, L. F. Hanne, and B. H. Iglewski. 1987. Cloning and characterization of elastase genes from Pseudomonas aeruginosa. J. Bacteriol. 169:2691–2696.[Abstract/Free Full Text]
  38. Schultz, D. R., and K. D. Miller. 1974. Elastase of Pseudomonas aeruginosa: inactivation of complement components and complement-derived chemotactic and phagocytic factors. Infect. Immun. 10:128–135.[Abstract/Free Full Text]
  39. Vandivier, R. W., V. A. Fadok, P. R. Hoffmann, D. L. Bratton, C. Penvari, K. K. Brown, J. D. Brain, F. J. Accurso, and P. M. Henson. 2002. Elastase-mediated phosphatidylserine receptor cleavage impairs apoptotic cell clearance in cystic fibrosis and bronchiectasis. J. Clin. Invest. 109:661–670.[CrossRef][Medline]
  40. Liau, D. F., N. X. Yin, J. Huang, and S. F. Ryan. 1996. Effects of human polymorphonuclear leukocyte elastase upon surfactant proteins in vitro. Biochim. Biophys. Acta 1302:117–128.[Medline]
  41. Lema, G., D. Dryja, I. Vargas, and G. Enhorning. 2000. Pseudomonas aeruginosa from patients with cystic fibrosis affects function of pulmonary surfactant. Pediatr. Res. 47:121–126.[Medline]
  42. Postle, A. D., A. Mander, K. B. Reid, J. Y. Wang, S. M. Wright, M. Moustaki, and J. O. Warner. 1999. Deficient hydrophilic lung surfactant proteins A and D with normal surfactant phospholipid molecular species in cystic fibrosis. Am. J. Respir. Cell Mol. Biol. 20:90–98.[Abstract/Free Full Text]
  43. Griese, M., C. von Bredow, and P. Birrer. 2001. Reduced proteolysis of surfactant protein A and changes of the bronchoalveolar lavage fluid proteome by inhaled alpha 1-protease inhibitor in cystic fibrosis. Electrophoresis 22:165–171.[CrossRef][Medline]
  44. Meyer, K. C., A. Sharma, R. Brown, M. Weatherly, F. R. Moya, J. Lewandoski, and J. J. Zimmerman. 2000. Function and composition of pulmonary surfactant and surfactant-derived fatty acid profiles are altered in young adults with cystic fibrosis. Chest 118:164–174.[Abstract/Free Full Text]
  45. Hull, J., M. South, P. Phelan, and K. Grimwood. 1997. Surfactant composition in infants and young children with cystic fibrosis. Am. J. Respir. Crit. Care Med. 156:161–165.[Abstract/Free Full Text]
  46. Crouch, E. C. 1999. Modulation of host-bacterial interactions by collectins. Am. J. Respir. Cell Mol. Biol. 21:558–561.[Free Full Text]



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