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Published ahead of print on November 4, 2004, doi:10.1165/rcmb.2004-0276OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 32, pp. 128-134, 2005
© 2005 American Thoracic Society
DOI: 10.1165/rcmb.2004-0276OC

Pseudomonas aeruginosa Degrades Pulmonary Surfactant and Increases Conversion In Vitro

Alexis L. Beatty, Jaret L. Malloy and Jo Rae Wright

Department of Cell Biology, Duke University Medical Center, Durham, North Carolina

Correspondence and requests for reprints should be addressed to Jo Rae Wright, Box 3709, Department of Cell Biology, 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
 
Although it is known that surfactant lipids and proteins are altered in patients with Pseudomonas aeruginosa infections, the mechanisms and implications of these alterations are not clear. In this study, the effects of P. aeruginosa on the surfactant large aggregate fraction were examined using an in vitro surface area cycling model. Large aggregates were isolated from porcine bronchoalveolar lavage fluid and incubated with supernatants from P. aeruginosa cultures (PAO1, parent strain; PAO1-A1, lasA-negative mutant; PAO1-B1, elastase-negative mutant) or purified elastase. Amounts of surfactant protein (SP)-A and SP-B, phospholipid content, and large aggregate conversion were assessed. In addition, lipid degradation was assessed by incubating a mixture of radiolabeled phospholipids with P. aeruginosa supernatants. The results demonstrated that SP-A was degraded by PAO1 and PAO1-A1 supernatants, and by purified elastase. SP-B was degraded by PAO1 and PAO1-B1 supernatants, but not by elastase. P. aeruginosa supernatants degraded phospholipids, a process inhibited by ZnCl2. P. aeruginosa supernatants and elastase increased conversion. The data suggest that protein degradation facilitates increased conversion, and that phospholipid degradation and conversion enhance degradation of surfactant proteins. In conclusion, P. aeruginosa secretes multiple virulence factors that cooperate to result in degradation of surfactant components and alteration of large aggregate conversion.

Key Words: surfactant proteins • surfactant phospholipids • surfactant aggregates • protease • elastase


   Introduction
Top
 Abstract
 Introduction
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Pulmonary surfactant is comprised of lipids (90% by weight) and proteins (10%). Surfactant is synthesized by alveolar type II cells, stored in lamellar bodies, and ultimately secreted into the hypophase of the alveolar space. Within the alveolar space, lamellar bodies rearrange to form tubular myelin, a structured array of surfactant lipids and proteins. Lipids adsorb to the air—liquid interface and lower alveolar surface tension, thereby preventing alveolar collapse during exhalation. Surfactant proteins contribute significantly to the structure and function of surfactant. Surfactant protein (SP)-A and SP-D are hydrophilic proteins belonging to the collectin family, and SP-B and SP-C are hydrophobic proteins. SP-A is essential for the formation of tubular myelin, enhances adsorption of lipids to the surface film, and contributes to the surface tension–lowering properties of surfactant (1–3). SP-A and SP-D both mediate innate host defense in the lung by recognizing nonself patterns (4). SP-B and SP-C facilitate rapid adsorption of lipids to the surface film and are essential for the surface tension–lowering properties of surfactant (3). In addition, it is thought that SP-A and SP-B work synergistically in forming tubular myelin and promoting lipid adsorption (3).

Surfactant can be separated by density centrifugation into two fractions—large aggregates (LA) and small aggregates (SA). LA, which consist of tubular myelin and multilamellar lipid structures, are the metabolic precursors of SA, which consist of small lipid vesicles (5, 6). Whereas LA reduce surface tension, SA have minimal ability to reduce surface tension (7). In vivo, LA are "converted" to SA during the normal respiratory cycle. LA generate a surface film, and as the surface area of the alveoli decreases with each exhalation, SA are squeezed out of the surface film. This process can be modeled in vitro by a method called surface area cycling, which turns partially filled tubes containing LA end-over-end to model the surface area change of the lung (6, 8).

The components of surfactant—lipids and proteins—are integral to the proper functioning of surfactant. Decreasing the concentration of surfactant phospholipid causes an increase in conversion to surface-inactive forms during in vitro cycling (8). Alterations in surfactant proteins perturb LA and produce changes in conversion. SP-A and SP-B interact to preserve LA forms during cycling (7). Degradation of SP-A with trypsin produces an increase in conversion and decreased surface activity (9). When the components of surfactant are altered, the consequences can impact the clinical status of patients. Decreased proportions of LA and decreased amounts of SP-A occur in the setting of pneumonia, inflammation, and lung injury (10, 11).

Pseudomonas aeruginosa is the second most prevalent pathogen causing pneumonia in hospitalized patients, representing 20% of cases (12). Additionally, chronic P. aeruginosa infection plagues many individuals with cystic fibrosis (CF). Patients with CF have decreased amounts of SP-A (13, 14) and SP-D (14), as well as altered amounts of phospholipid species (15). Previous studies have shown that P. aeruginosa secretes virulence factors that degrade surfactant proteins and lipids. For example, elastase, a protease secreted by P. aeruginosa, degrades SP-A and SP-D (16). P. aeruginosa extracts also degrade surfactant lipids (17). However, the impact of P. aeruginosa on LA conversion has not been investigated. In this study, it was hypothesized that P. aeruginosa virulence factors would degrade surfactant components and that this degradation would correlate with increased conversion to inactive SA. The effects of P. aeruginosa proteases on surfactant conversion were examined using an in vitro surface area cycling assay and were correlated with amounts of SP-A and SP-B. Additionally, the effect of P. aeruginosa on degradation of surfactant-like lipids was investigated. The results demonstrated that P. aeruginosa degraded surfactant phospholipids, degraded surfactant proteins, and increased conversion of functional LA to nonfunctional SA.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 References
 
Reagents
Reagents were obtained from Sigma Chemical Co. (St. Louis, MO), except where otherwise noted.

LA Preparation
The Division of Laboratory Animal Resources (DLAR) at Duke University (Durham, NC) provided the discarded lungs of a killed pig without systemic disease or lung injury. The lungs were lavaged twice with 0.15 M NaCl to total lung capacity. Bronchoalveolar lavage fluid (BALF) was centrifuged at 150 x g for 10 min to remove cells. The cell-free BALF was centrifuged at 40,000 x g for 15 min at 4°C to pellet LA. LA were resuspended in 0.15 M NaCl. The amount of phospholipid in the LA was determined by performing a lipid extraction (18) and phosphorus assay (19). LA were resuspended to a concentration of 2.5 mg phospholipid/ml with 0.15 M NaCl and stored at –20°C in 400-µl aliquots.

Bacterial Cultures
Pseudomonas aeruginosa strains PAO1 (kindly provided by Dr. Paul Phibbs, East Carolina University, Greenville, NC), PAO1-A1 (lasA-negative mutant), and PAO1-B1(elastase-negative mutant; mutants kindly provided by B. Iglewski, University of Rochester, Rochester, NY) were grown for 24 h at 37°C in nutrient broth (Difco, Detroit, MI). Cultures were analyzed for density by spectrophotometry at 660 nm and were ~ 2 x 107 cfu/ml. Bacteria were removed by centrifugation at 8,000 x g for 15 min at 4°C. Supernatants were aliquoted and stored at 4°C.

Surface Area Cycling
LA aliquots were resuspended in cycling buffer (150 mM NaCl, 10 mM Tris, 1 mM CaCl 2, 1 mM MgCl 2, 0.1 mM EDTA, pH 7.4) to a phospholipid concentration of 250 µg/ml. Polystyrene tubes (Falcon #2054, 5 ml capacity, 12 x 75 mm; BD Biosciences, Bedford, MA) were filled to a volume of 2 ml. Experiments were conducted at 37°C and paired with one sample "cycled" by rotating end-over-end at 40 rpm and one sample "noncycled" with only slight agitation to maintain suspension.

For PAO1 strain supernatant experiments, 0.5 ml nutrient broth was added to control groups and 0.5 ml bacterial supernatant was added to experimental groups. PAO1 strain supernatant experiments were conducted for 9 h. Purified elastase was obtained as previously described (16). Elastase was added at a concentration of 25 µg/ml. Phosphoramidon, a known inhibitor of elastase (16), was added at a concentration of 1 mM. Additional volume was offset by commensurately decreasing amount of cycling buffer added. Purified elastase experiments were conducted for 24 h.

After the cycling period, samples were centrifuged at 40,000 x g for 15 min at 4°C to separate SA and LA fractions. The supernatant containing the SA was decanted, and the LA pellet was resuspended in a volume of cycling buffer equal to that of supernatant, such that there were equal volumes of SA and LA fractions. Phospholipid content of the samples was determined by lipid extraction (18) followed by Duck-Chong phosphorus assay on the lipid phase (19).

Assessment of Surfactant Protein Amounts
Aliquots of surface area cycling products (whole samples before separation by centrifugation, as well as separated LA and SA) were resolved with 15% SDS-PAGE and transferred to a nitrocellulose membrane. SP-A was examined under reducing conditions, and SP-B was examined under non-reducing Western blotting conditions. Immunoreactive SP-A and SP-B were detected using anti-recombinant human SP-A antibody (gift from ALTANA Pharma AG, Konstanz, Germany) and anti-sheep SP-B antibody, respectively. Westerns were developed using chemiluminescence (ECL; Amersham, Arlington Heights, IL).

Surfactant-Like Liposomes
Dipalmitoylphosphatidylcholine (DPPC), phosphatidylcholine (PC), dipalmitoylphosphatidylglycerol (DPPG), phosphatidylglycerol (PG), and cholesterol (all from Avanti Polar Lipids, Alabaster, AL) were combined in proportions similar to those found in vivo: 41:41:4:8:6 (20). DPPC (choline-methyl-3H; American Radiolabeled Chemicals, St. Louis, MO) and DPPC (1-palmitoyl-14C; NEN Life Science Products, Boston, MA) were added at < 0.01 mol%. Lipids were dried under nitrogen gas and diluted to a final concentration of 1 mg/ml with 0.15 M NaCl. Glass beads were placed in the bottom of the flask, and the flask was heated at 37°C for 1 h with swirling every 10 min to resuspend the lipids. The synthetic lipid mixture was diluted in cycling buffer to a final volume of 2 ml with a phospholipid concentration of 250 µg/ml and incubated for 9 h at 37°C. Experimental conditions included addition of 0.5 ml nutrient broth, 24 h PAO1 cultured supernatant, or 24 h PAO1-B1 cultured supernatant. ZnCl2 (1 mM) was added for inhibition experiments. Lipid extraction was performed. Samples of lipid mixture, aqueous phase, and lipid phase were analyzed with a Minaxi B Tri-Carb 400 Liquid Scintillation Counter. A Duck-Chong phosphorus assay was performed on the lipid phase (19).

Statistical Analysis
All statistical analyses were performed using a two-tailed, nonpaired t test. A probability level of P < 0.05 was considered statistically significant.


   RESULTS
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 References
 
P. aeruginosa Supernatants Degraded SP-A and SP-B
To investigate the effect of surface area cycling with P. aeruginosa supernatants on the degradation of SP-A and SP-B, LA were incubated for 9 h at 37°C, and then analyzed by Western blot immunostaining. All experimental groups were paired to evaluate noncycled versus cycled samples, and control conditions consisted of incubating LA with nutrient broth rather than P. aeruginosa supernatants. Figure 1A displays SP-A levels in whole surfactant samples (both noncycled and cycled) before aggregate separation. SP-A was degraded when incubated with PAO1 and PAO1-A1 (las A negative strain) supernatants compared with controls, and in these samples the degree of SP-A degradation was greater in the presence of surface area cycling. There was no degradation of SP-A detected in aggregates incubated with PAO1-B1 (elastase negative strain). Whole samples were also separated into LA and SA fractions by centrifugation. Analysis of the separated material by Western blot immunostain confirmed that there was less SP-A present after incubation with PAO1 compared with control. This difference was noted in both LA and SA fractions, and in both noncycled and cycled samples (Figure 1B). In the cycled control, there was a decrease in the amount of SP-A in the LA fraction, and a commensurate increase in the amount of SP-A in the SA fraction. However, in the LA cycled with PAO1 supernatant, the decrease in SP-A in the LA fraction was present, but the amount of SP-A in the SA fraction was barely detectable. Loss of SP-B was observed in whole samples after incubation with PAO1 and PAO1-B1 supernatants (Figure 2). There was a small decrease in immunoreactive SP-B in the cycled control.



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  		Figure 1. Degradation of SP-A in large aggregates incubated with P. aeruginosa supernatants. LA were incubated with P. aeruginosa supernatants for 9 h at 37°C both with (C) and without (NC) surface area cycling. (A) Aliquots were taken immediately after incubation and prior to separation of LA and SA by centrifugation. The same volume of sample was loaded into each lane. Products were resolved by SDS-PAGE (reducing conditions), transferred to nitrocellulose, and probed with antibodies against SP-A. Degradation of SP-A was detected in LA incubated with PAO1 and PAO1-A1. The degradation occurred to a greater degree in cycled samples. (B) Aliquots were taken after separation of LA and SA by centrifugation. LA and SA were in the same volume of cycling buffer, and the same volume of sample was loaded in each lane. Less SP-A in all fractions was present in PAO1 groups compared with control groups. Less SP-A was detected in the LA fraction when cycling was present in both control and PAO1. While more SP-A was present in cycled control SA fraction, there was very little SP-A detected in the SA fraction for the PAO1 group. Immunoblots shown are representative of three independent experiments.

 


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  		Figure 2. Degradation of SP-B in large aggregates incubated with P. aeruginosa supernatants. LA were incubated with P. aeruginosa supernatants for 9 h at 37°C both with (C) and without (NC) surface area cycling. Aliquots were taken immediately after incubation and before separation of LA and SA by differential centrifugation. The same volume of sample was loaded into each lane. Products were resolved by SDS-PAGE (nonreducing conditions), transferred to nitrocellulose, and immunostained with antibodies against SP-B. Loss of SP-B was detected in LA incubated with PAO1 and PAO1-B1 supernatants. This loss was virtually complete for PAO1 incubation. For the PAO1-B1 group, the loss was apparent in the noncycled sample, and complete in the cycled sample. Less SP-B was detected in the cycled control compared with the noncycled control. Immunoblot shown is representative of three independent experiments.

 
P. aeruginosa Supernatants Degraded Surfactant Phospholipid
Degradation of surfactant lipids by P. aeruginosa in the setting of surface area cycling was also examined. After LA were incubated with P. aeruginosa supernatants for 9 h at 37°C, a decreased amount of phospholipid was measured. Total phospholipid in the control group (306 ± 63 µg) was greater than total phospholipid after incubation with PAO1 (159 ± 90 µg) (P < 0.05). There was no significant difference in amount of total phospholipid in noncycled and cycled LA (P > 0.25). Thus, the variable of cycling did not impact the amount of total phospholipid recovered.

To evaluate further phospholipid degradation, liposomes comprised of surfactant-like lipids containing both choline-methyl-3H-DPPC and 1-palmitoyl-14C-DPPC were incubated with P. aeruginosa supernatants. After 9 h of incubation at 37°C, the amount of 3H remaining in the organic phase of the control was 92 ± 1%. There was significantly less radiolabel in the organic phase for PAO1 (50 ± 12%) and PAO1-B1 (65 ± 6%) compared with the control group. Similar differences were noted for 14C, with controls retaining 88 ± 1% of radiolabel in the organic phase and PAO1 and PAO1-B1 groups had significantly lower retentions at 60 ± 10% and 71 ± 5%, respectively (Figure 3). When phospholipid-phosphorus was measured, it was found that there was significantly less phosphorus in the PAO1 (256 ± 38 µg) and PAO1-B1 (310 ± 12 µg) groups compared with control (507 ± 47 µg) (Figure 4).



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  		Figure 3. P. aeruginosa supernatants degrade phospholipids. Radiolabeled surfactant lipids were incubated with P. aeruginosa supernatants for 9 h at 37°C. Lipid extraction was performed, and the lipid and aqueous phases were analyzed for radioactivity (dark gray bars, 3H; light gray bars, 14C). A decrease in the amount of radioactivity detected in the lipid phase relative to the total radioactivity in both lipid and aqueous phase was used as a measure of degradation. Significant degradation of lipid was detected in PAO1 and PAO1-B1 groups compared with control ({dagger}P < 0.01; *P < 0.05). Degradation by PAO1 was inhibited by 1 mM ZnCl 2 (*P < 0.05). A small, though statistically significant, difference in lipid-associated radiolabel was detected between control and control with 1 mM ZnCl 2 (*P< 0.05).

 


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  		Figure 4. Degradation of phospholipid by P. aeruginosa supernatants, as detected by phosphorus assay. Surfactant lipids were incubated with P. aeruginosa supernatants for 9 h at 37°C. Lipid extraction was performed, and the lipid phase was assayed for phosphorus content, with loss of phosphorus corresponding to loss of phospholipid. Significant degradation of phospholipid was seen in the PAO1 and PAO1-B1 groups, compared with control ({dagger}P < 0.01). Phospholipid degradation by PAO1 was inhibited with ZnCl 2 ({dagger}P < 0.01). Less lipid-associated phosphorus was also seen in the control plus 1 mM ZnCl 2 group, compared with control (*P < 0.05).

 
To evaluate the possibility that lipid degradation was mediated by Pseudomonas phospholipase, we tested the ability of ZnCl2, a known inhibitor, to block degradation. The addition of ZnCl2 to liposomes incubated with PAO1 supernatants abrogated the loss of recovery of radiolabel in the organic phase (Figure 3). A small but statistically significant decrease in radiolabel was detected in the control samples (without PAO1 supernatant) incubated with ZnCl2, relative to control samples incubated without ZnCl2 (P < 0.05). Similar results were obtained when analyzing lipid phosphorus; that is, zinc abrogated the loss of phospholipid phosphorus in PAO1 treated samples and slightly, but significantly (P < 0.05), decreased the phosphorus recovery in control samples without PAO1 (Figure 4). Zinc did not significantly affect the total recovery of radioactive counts in the organic and aqueous phases of liposomes treated with PAO1 supernatants or their controls. Comparisons of control + 1 mM ZnCl2 and PAO1 + 1 mM ZnCl2 revealed that the amount of radiolabel was not significantly different between the two groups and that lipid phosphorus was greater in the PAO1 + 1 mM ZnCl2 group (P < 0.01). The amount of phospholipid in the PAO1 + 1 mM ZnCl2 group was not different than the control (without zinc) and was significantly greater than PAO1 (P < 0.01). To determine if ZnCl2 interfered with phosphorous measurements, DPPC or PC were assayed in the presence of or absence of ZnCl2 and resulted in no difference in phosphorous measured (data not shown). The reason for the decrease in amount of phospholipid in control samples incubated with ZnCl2 remains unknown.

Surface Area Cycling with P. aeruginosa Supernatants Decreased the Relative Fraction of LA
To assess the effect of P. aeruginosa on conversion of surfactant aggregates, aliquots of LA were cycled with P. aeruginosa supernatants for 9 h. All experimental groups were paired to evaluate noncycled versus cycled samples, and control conditions included incubating LA with nutrient broth rather than P. aeruginosa supernatant. The percentage of LA in noncycled controls after the incubation period was 98 ± 1% and cycled controls was 71 ± 2%, indicating LA conversion occurred with cycling. LA that were surface area–cycled in the presence of supernatants from 24-h cultures of P. aeruginosa strains PAO1 and PAO1-B1 resulted in significantly lower percentage of LA (61 ± 4% and 56 ± 4%, respectively) compared with the cycled control group (P < 0.05) (Figure 5). Cycling with strain PAO1-A1 also yielded a lower percentage of LA than controls (57 ± 11%), but the difference did not reach statistical significance. The small disparity in conversion between PAO1 and PAO1-B1 groups was not significant. To test if phospholipases contributed to the PAO1 mediated increase in conversion, the ability of zinc to inhibit this process was tested. However, we found that zinc alone (in the absence of PAO1 supernatants) inhibited conversion. Thus, it appears that zinc has multiple effects in our system and we cannot attribute its effects solely to inhibition of PAO1 phospholipases.



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  		Figure 5. Surface area cycling with P. aeruginosa supernatants decreases the proportion of LA. LA were incubated (dark gray bars, noncycled; light gray bars, cycled end-over-end at 40 rpm) for 9 h at 37°C, then separated into LA and SA fractions by centrifugation. Fractions were assayed for lipid phosphorus and data are expressed as the percentage of phosphorus recovered in large aggregates relative to sum of phosphorus in both LA and SA. Surface area cycling produced a decrease in the percentage of LA. Incubation with supernatants from PAO1 and PAO1-B1 cultures produced a greater decrease in the percentage of LA compared with cycled controls without P. aeruginosa supernatants (*P < 0.05).

 
P. aeruginosa Elastase Degraded SP-A, but Not SP-B, in the Setting of Surface-Area Cycling
The effect of P. aeruginosa elastase on degradation of surfactant proteins was also investigated. Purified P. aeruginosa elastase (25 µg/ml) was incubated with LA for 24 h in the setting of surface-area cycling. Western Blot immunostain of SP-A in whole samples before aggregate separation revealed similar amounts of SP-A in noncycled controls, cycled controls, and noncycled elastase groups, but less SP-A in samples cycled with purified P. aeruginosa elastase (Figure 6A). Inhibition of elastase with 1 mM phosphoramidon blocked this degradation. When the SA and LA fractions were separated by centrifugation (Figure 6B), the amount of SP-A in the LA fraction was decreased in both control and elastase cycled samples. In the cycled control group this decreased level of SP-A in the LA fraction was associated with a reciprocal increase of SP-A in the SA fraction. However, this reciprocal increase of SP-A levels in the SA fraction was not seen with the samples incubated with elastase. There was also a small decrease in the amount of SP-A detected in LA fraction in the sample cycled with elastase, when compared with cycled control. When SP-B was evaluated by Western blot (data not shown), there was less SP-B found in cycled samples compared with noncycled samples, but no differences among control, elastase, and elastase plus phosphoramidon groups. These findings indicate that P. aeruginosa elastase degraded SP-A, but not SP-B, and that the majority of the SP-A degradation occurred in the SA fraction.



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  		Figure 6. Degradation of SP-A in large aggregates cycled with P. aeruginosa elastase. Western blot immunostain of SP-A from LA incubated with purified P. aeruginosa elastase for 24 h at 37°C (NC = noncycled and C = cycled). (A) Aliquots were taken immediately after incubation and before separation of LA and SA by differential centrifugation. Purified P. aeruginosa degraded SP-A in the cycled sample, and the degradation was inhibited by 1 mM phosphoramidon. (B) Aliquots were taken following separation of LA and SA by density centrifugations. SP-A is lost from LA with surface area cycling. For cycled control samples, there is a corresponding increase in SP-A in the small aggregate fraction. This increase was not detected in samples cycled with elastase. There is also less SP-A in the large aggregate fraction from samples cycled with elastase, compared with cycled control, though the difference is not as dramatic as the difference seen in the SA fraction.

 
Surface Area Cycling with P. aeruginosa Elastase Slightly Increased LA Conversion
To examine the effect of P. aeruginosa elastase on conversion, LA were cycled with purified P. aeruginosa elastase (25 µg/ml). When LA were cycled for 6 h, conversion was present in all cycled groups, and there was no difference between groups incubated under control conditions and in the presence of elastase (data not shown). After 24 h of incubation, conversion was present in all cycled groups, and a greater amount of conversion occurred in the group that was surface area–cycled with 25 µg/ml of purified P. aeruginosa elastase (47 ± 3% LA), compared with control (57 ± 2% LA) (Figure 7). LA incubated in the presence of elastase and its inhibitor phosphoramidon (1 mM) did not show a significant difference in conversion from either control or elastase alone (51 ± 2% LA). The enhancement of the conversion of LA to SA by purified elastase is not due to phosphoramidon-inhibitable elastase activity. Total phospholipid levels were comparable between control and elastase groups (data not shown).



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  		Figure 7. P. aeruginosa elastase produces an increase in LA conversion with surface area cycling. LA incubated (dark gray bars, noncycled; light gray bars, cycled) for 24 h at 37°C, then separated into LA and SA fractions by density centrifugation. Fractions were assayed for lipid phosphorus. Cycled samples display conversion of LA, indicated by a decreased percentage of LA. A greater degree of conversion occurred with LA cycled with purified P. aeruginosa elastase, compared with cycled control (*P < 0.05). The amount of conversion when elastase was inhibited with phosphoramidon was not statistically different from either cycled control or elastase. The enhancement of the conversion of LA to SA by purified elastase is not due to phosphoramidon-inhibitable elastase activity.

 

   DISCUSSION
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
References
 
This study demonstrated that factors secreted by P. aeruginosa degrade surfactant proteins and phospholipids and impact LA conversion in the setting of in vitro surface area cycling. P. aeruginosa supernatants degraded SP-A, SP-B, and phospholipid, and increased aggregate conversion, an indirect measure of surfactant inactivation. P. aeruginosa strains lacking elastase (PAO1-B1) did not degrade SP-A, but still degraded phospholipid, degraded SP-B, and increased conversion. Purified P. aeruginosa elastase degraded SP-A and produced a small increase in conversion.

P. aeruginosa Degrades Surfactant Proteins
The finding that SP-A was degraded by P. aeruginosa is consistent with previous studies with P. aeruginosa elastase and neutrophil elastase (16, 21). Degradation of SP-A has been seen in BALF from patients with CF, and previous work has shown degradation of SP-A in the presence of P. aeruginosa supernatants (13, 14, 16). Examination of the patterns of SP-A loss yields insight into possible mechanisms for SP-A degradation. In LA incubated with elastase, the loss of SP-A occurred only in LA cycled with elastase (Figure 6A). Separation of the cycling products into SA and LA fractions revealed that this loss occurred almost exclusively in the SA fraction (Figure 6B). This suggests that degradation of SP-A is impeded by association with the structured lipid arrays of LA and is facilitated by either the adsorption to the air–liquid interface or association with the lipid vesicles of SA. A comparison of these data with the degradation profiles from incubation with PAO1 (Figure 1A), demonstrates not only that SP-A was degraded in cycled samples, but also that SP-A was degraded in noncycled samples. This implies that factors other than elastase within the supernatants facilitated the degradation of SP-A within the LA structures. Even in the presence of these other factors, degradation of SP-A was still greater in cycled LA. There was less SP-A in the LA fraction (Figure 6B), but there was also a smaller fraction of LA. Still, there is not a commensurate increase in SP-A in the SA fraction, as is seen with the cycled control, suggesting that additional SP-A may be degraded from the SA fraction. These observations of degradation of SP-A in SA after a period of cycling in the presence of elastase and degradation of SP-A in LA only in the presence of elastase and other factors, suggest that SP-A is not as accessible to proteolytic degradation in the structured arrays of large aggregates as it is in small aggregates.

SP-B was degraded by P. aeruginosa supernatants, but not by purified elastase. It is important to note that upon cycling, SP-B is lost in all control and experimental groups. Previous investigators witnessed decreased amounts of SP-B after surface area cycling (22, 23), and reported that much of the lost SP-B could be recovered from the walls of cycling tubes (22). These previous findings led to the conclusion that some of the apparent losses of SP-B observed in cycled samples were due to association of SP-B with the walls of the tubes, and not due to degradation. In the present study, loss of SP-B was observed in both cycled and noncycled samples in LA incubated with PAO1 and PAO1-B1 supernatants. This phenomenon cannot be solely explained by adherence of SP-B to tubes because loss was observed in the noncycled samples as well. Degradation of SP-B has not been reported in BALF from patients with CF or in incubation with P. aeruginosa (13). Several other proteases secreted by P. aeruginosa may be responsible for this degradation: protease IV, LasA, and alkaline protease. Recent work from this lab implicates protease IV in the degradation of SP-B in BALF (24). Losses of SP-B with cycling in the presence of P. aeruginosa supernatants may have an important impact on conversion and function. SP-B associates tightly with surfactant lipids, helps maintain surfactant structure, and contributes to the surface tension–lowering properties of surfactant. Inherited SP-B deficiency causes respiratory failure in newborn infants, and SP-B knockout mice die of respiratory failure immediately after birth (25, 26). Loss of SP-B may result in diminished ability of surfactant to maintain its surface active LA forms and diminished ability of surfactant to lower surface tension within the alveolus.

P. aeruginosa Supernatants Degrade Phospholipids
P. aeruginosa supernatants degraded phospholipids in both LA and in a surfactant-like mixture of lipids. These findings are consistent with a previous report that P. aeruginosa supernatants degraded phospholipids in calf lung surfactant extract (CLSE) (17). In previous analyses of BALF of patients with CF, it is unclear whether amounts of phospholipids are consistently decreased. Postle and colleagues reported negligible differences in surfactant phospholipids in BALF of individuals with CF (14). However, other investigators found decreased amounts of PC and PG relative to other phospholipids (13, 15). P. aeruginosa secretes a number of enzymes capable of degrading phospholipids. Both hemolytic and nonhemolytic phospholipase C (PLC-H and PLC-N) degrade PC. PLC-H also hydrolyzes sphingomyelin, and PLC-N degrades phosphatidylserine (27). The combination of PLC and lipase from P. aeruginosa has been reported to result in degradation of DPPC, producing palmitic acid and dipalmitoylglycerol, though lipase alone produced no degradation (28).

In the present study, incubation with PAO1 strain supernatants resulted in radiolabels from both the palmitate and choline moieties becoming dissociated from the lipid phase, suggesting that DPPC was degraded by more than one enzyme. The degradation of surfactant phospholipids by P. aeruginosa likely occurred due to the combined action of PLC enzymes and lipase. However, the contributions of the individual enzymes and the characteristics of their impact on surfactant need further study. Additionally, inhibiting the action of the phospholipases and lipases may provide insights into their role in degradation and what impact degradation of lipids might have on large aggregate conversion. In the present study, degradation of phospholipids by PAO1 was inhibited by 1 mM ZnCl2. Manganese, copper, cobalt, nickel, and zinc (but not PLC inhibitor D609), have all been reported to inhibit P. aeruginosa PLC-H, but these ions are nonspecific and may also have effects on other processes related to surfactant function (29). Preliminary data reveal that addition of 1 mM ZnCl2 to cycling experiments inhibits conversion, thus making it difficult to explore the contribution of phospholipases to conversion using zinc as an inhibitor. Still, examining the effects of loss of phospholipase activity on conversion via other methods may provide a valuable tool for determining the contribution of PLC to conversion and for isolating the effects of P. aeruginosa proteases.

The physiologic implications of phospholipid degradation by P. aeruginosa supernatants are not clear, but previous work suggested that degradation results in altered surfactant function as degradation of PC in vitro correlated with impaired surface tension lowering (17). Additionally, higher surface tension was witnessed in BALF of patients with CF that had decreased ratios of PC (15). Further investigation is needed to explore the functional impact of the degradation of surfactant phospholipids by P. aeruginosa.

P. aeruginosa Affects Aggregate Conversion
In this study, increases in large to SA conversion for LA incubated with PAO1 strains were observed. These changes in conversion occurred in the setting of surfactant protein degradation and phospholipid degradation. Cycling with PAO1 strains revealed that elastase was not essential for producing an increase in conversion. PAO1-B1 (elastase-negative strain) produced an increase in conversion without degradation of SP-A. Indeed, these data suggest that multiple factors influence LA conversion.

It must be noted that the in vitro cycling system used as a model of conversion is an imperfect model and differs from physiologic conditions, particularly with respect to phospholipid and protein concentrations and extreme surface area changes (6). Although this in vitro model points to changes in surfactant composition and function, it is merely a starting point for future investigation into the physiologic implications of P. aeruginosa on surfactant degradation and functional alteration.

Summary and Conclusions
P. aeruginosa impacts surfactant in a variety of ways, through a variety of different mechanisms. P. aeruginosa degrades both surfactant proteins and phospholipids, and increases LA conversion. Although the effects of any single one of these actions may not be great, this study suggests that the various mechanisms work synergistically to produce changes in surfactant. Within the structured array of LA, SP-A and SP-B are not readily degraded by P. aeruginosa proteases. However, surface area cycling and degradation of phospholipid in LA facilitate surfactant protein degradation by P. aeruginosa. The combination of degraded phospholipids and proteins promotes increased conversion. Within the small lipid vesicles of small aggregates or in the process of conversion into SA, surfactant proteins are likely more susceptible to degradation by proteases, accounting for the observation that SP-A is totally degraded in SA fractions.

The surface tension–lowering abilities of SA are known to be inferior to LA. Thus, any increase in conversion may have a negative impact on the surface tension–lowering properties of surfactant. Investigating the surface tension properties of surfactant cycled with P. aeruginosa would be a valuable step in further defining the functional consequences of P. aeruginosa infection. However, it is uncertain whether the small changes observed here will have a definite impact on surface tension or whether the changes in LA integrity will be physiologically significant.

The observation that SP-A was virtually absent within the SA fraction of LA cycled with P. aeruginosa or elastase may have important potential implications with regard to lung host defense. Indeed, it was observed that SP-A–null mice were more susceptible to P. aeruginosa infection, with decreased phagocytosis of bacteria by alveolar macrophages and increased inflammatory cytokine production (30).

In summary, P. aeruginosa produces virulence factors that cooperate to degrade surfactant phospholipids, degrade SP-A and SP-B, and increase conversion of surface-active LA to surface-inactive SA in the setting of in vitro surface area cycling. Indeed, P. aeruginosa is capable of attacking pulmonary surfactant on many different fronts. Further study is needed to elucidate the mechanisms and consequences of P. aeruginosa damage to the surfactant, including its impact on function in the in vivo setting. Greater understanding may yield new strategies for defending against pulmonary infections with P. aeruginosa and other pathogens.


   Acknowledgments
 
J.L.M. is a Parker B. Francis fellow.


   Footnotes
 
This study was funded by NIH grant HL-30923 (J.R.W.).

Conflict of Interest Statement: A.L.B. has no declared conflicts of interest; J.L.M. has no declared conflicts of interest; and J.R.W. has no declared conflicts of interest.

Received in original form August 26, 2004

Received in final form November 1, 2004


   References
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 

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