INFECTION AND IMMUNITY, Mar. 1991, p. 822-828 0019-9567/91/030822-07$02.00/0 Copyright C) 1991, American Society for Microbiology
Vol. 59, No. 3
Interaction of Pseudomonas aeruginosa with A549 Pneumocyte Cells EMIL CHI,' THOMAS MEHL,' DAVID NUNN,2 AND STEPHEN LORY2*
Departments of Pathology1 and Microbiology,2 School of Medicine, University of Washington, Seattle, Washington 98195 Received 4 September 1990/Accepted 30 November 1990
The interaction of Pseudomonas aeruginosa with a human lung pneumocyte cell line (A549) was studied. Wild-type strain PAK adhered efficiently to the A549 cells, while an isogenic mutant, carrying a mutation in the pilin structural gene, adhered at 10 to 20% of the wild-type levels. Another nonpiliated mutant of P. aeruginosa PAK, defective in the pleiotropic regulatory gene rpoN, did not adhere to A549 cells, suggesting the presence of a second, RpoN-controlled adhesin on the bacterial surface. Endocytosis of wild-type P. aeruginosa PAK by A549 cells was also demonstrated. A significant fraction of the internalized bacteria were recovered in a viable form after several hours of residence within the A549 cells. When examined by electron microscopy, intracellular bacteria were located in membranous vesicles, and no evidence of killing by lysosomal mechanisms was observed. These studies raise the possibility that during chronic respiratory tract infections in immunocompromised patients, P. aeruginosa may persist in intracellular compartments and therefore be protected from the defense mechanisms of the host.
Pseudomonas aeruginosa is an important bacterial agent of chronic pneumonia in patients during prolonged hospitalization and is responsible for lethal pulmonary infection in a majority of individuals with cystic fibrosis. Following inhalation or aspiration, the bacteria attach to receptors on the epithelial cells or the mucous coating of the airways. Adherent bacteria are resistant to mucocilliary clearance mechanisms and proliferate, with progressive spread through the respiratory tract (18). The ability of P. aeruginosa to adhere to specific sites on host tissues has been investigated with various cell culture models, such as human buccal epithelial cells (4, 26), rat cornea (9), cells of humans, hamster, and bovine trachea (4, 16, 22), and epidermal cells (23). Specific binding has been also demonstrated to human tracheobronchial mucin (19) and glycolipids (12). These findings demonstrate the complexity of possible host receptors for this microorganism. Several bacterial products have been implicated as potential adhesins during various stages of infection. Purified pili were shown to bind directly to a number of cell types and compete with intact bacteria for binding to such cells (20, 23). Monoclonal antibodies to pili have also been shown to block adhesion of bacteria to cells (3), and antibodies to a peptide corresponding to a C-terminal region of pilin also blocked binding of pili to epithelial cells (13). Binding of P. aeruginosa to tracheobronchial mucin, mediated by alginate exopolysaccharide, has also been demonstrated in vitro (19). This affinity of the alginate-coated bacteria for the lung tissues is believed to be one of several factors that select mucoid variants from the nonmucoid invading pathogens during later stages of infection. To identify the bacterial components that play a role in colonization of tissues, we have explored the possibility of using epithelial cell lines, derived from the human respiratory tract, as in vitro models for studying the initial interaction of the bacterium with a host. The human pulmonary carcinoma cell line A549 (14) was selected for the studies *
described here, as these cells possess the morphological and biochemical characteristics of type II pneumocytes of the intact lung. A well-characterized non-mucoid strain of P. aeruginosa, PAK, was used to study the interaction of bacteria with A549 cells. We also examined isogenic mutants of PAK that have lost the ability to form pili by mutations in either the pilin structural gene or the regulatory gene rpoN for involvement of pili in adherence. In this report, we characterize the kinetic parameters of binding of P. aeruginosa PAK to A549 cells and show reduced binding of the nonpiliated variants. Furthermore, a significant fraction of bound P. aeruginosa PAK were taken up into endocytic vesicles, where they persisted intracellularly without appreciable loss of viability. Intracellular persistence of a fraction of the bacteria colonizing the respiratory tract of cystic fibrosis patients may explain the chronic nature of P. aeruginosa infection and the protection of this pathogen from host defense mechanisms. Furthermore, bacteria living intracellularly are very likely refractory to the killing action of antimicrobial agents that cannot penetrate the protective environment afforded by the host cell. MATERIALS AND METHODS Bacteria and growth conditions. P. aeruginosa PAK was
provided by D. Bradley, Memorial University of Newfoundland. P. aeruginosa PAK-Nl is an rpoN mutant of PAK (10). P. aeruginosa PAK-NP is a mutant of PAK with an insertionally inactivated pilin gene in its chromosome (21). Bacteria were grown statically in Luria broth (1% tryptone, 0.5% yeast extract, 0.5% sodium chloride) supplemented with 5 mM magnesium chloride, at 37°C for 16 h. Prior to their use in adhesion assays, the bacteria were sedimented by centrifugation at 5,000 x g for 5 min, washed once with Hank's balanced salt solution (HBSS; GIBCO, Santa Clara, Calif.) supplemented with 1 mM CaCl2, 2 mM MgCl2, 20 mM HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, pH 7.3) (this solution will be referred to as HBSS with supplements in this report), and resuspended in HBSS with supplements to the desired bacterial concentration before
Corresponding author. 822
VOL. 59, 1991
being added to A549 cells. The bacterial concentration was estimated by measuring the A60. An OD6. of 1.0 corresponds to 6 x 108 viable bacteria, as established from the number of CFU resulting from plating dilutions of bacterial suspension onto L-agar plates and overnight incubation. Cell culture methods. The A549 cell line ATCC CCL 165, passage 77 (13), was obtained from the American Type Culture Collection (Rockville, Md.). The cells were maintained in 10% fetal calf serum in Waymouth MB752/1 medium, supplemented with 2 mM glutamine, 1 mM sodium pyruvate, and a standard antibiotic mixture (100 U of penicillin, 100 ,ug of streptomycin, and 0.5 ,ug of amphotericin B per ml) and incubated at 37°C in 5% CO2. Passage 91 to 99 was used for all experiments in this report. The cells were fed every 3 to 4 days and passaged at 7- to 10-day intervals. Briefly, cells were rinsed with 0.53 mM Na-EDTA in PBS (20 mM sodium phosphate, 150 mM NaCl [pH 7.4]) and treated with 0.25% (wt/vol) trypsin in PBS, followed by addition of 50% fetal calf serum to stop the protease activity. The cells were then plated at 0.5 x 106 per 25-cm2 flask (Corning 25100) or 35-mm-diameter petri dish (Falcon 3001). All tissue culture reagents were obtained from GIBCO. Quantitation of lactate dehydrogenase release, cell recovery, and viability. Lactate dehydrogenase released into the culture fluid from A549 cells incubated with bacteria was determined by the method of Amador and Wacker (1) and compared with the total enzyme activity recovered from monolayers and A549 cells following lysis of cells with 0.1% Triton X-100 in PBS. Bacteria and cellular debris were removed from the lysates by centrifugation at 5,000 x g for 5 min before assaying for the enzyme. Bacterial adherence assay. Approximately 1 x 105 to 2 x 105 A549 cells were seeded into 35-mm dishes and incubated overnight prior to binding assays. To determine the kinetics of bacterial association, the monolayers were first washed three times with warm (37°C) serum-free HBSS with supplements, and the cells were overlaid with 1 ml of this solution. A 0.2-ml aliquot of bacterial suspension was then added to give a final ratio of 50 bacteria per A549 cell. The plates were gently swirled and incubated for 1 to 4 h at 37°C. When constructing binding curves with multiple ratios of bacteria to cultured cells, twofold dilutions of the bacteria in 1-ml aliquots were added directly to washed A549 monolayers and incubated for 1 h at 37°C. At the conclusion of the adherence experiments, the unbound bacteria were removed by washing the monolayers three times with 1 ml of HBSS with supplements. The monolayers were then fixed for 2 h with 2.5% (vol/vol) glutaraldehyde in PBS (pH 7.4) or a fixative composed of 5% (vol/vol) formaldehyde and 5% (vol/vol) glacial acetic acid in 70% (vol/vol) methanol. The monolayers were washed once with water and stained with 10% Giemsa stain for 10 min. The stain was removed by washing three times with water and air-drying the plates. The extent of bacterial adherance to A549 cells was determined by examining the monolayers under a light microscope at 400 x magnification and enumerating the bacterial cells residing over 100 A549 cells. Because this technique does not distinguish internalized from surface-bound bacteria, binding refers to bacteria associated with A549 cells and includes those bacteria located on the surface as well as those internalized by the A549 cells. Determination of uptake of P. aeruginosa. A549 cells grown in 35-mm dishes to 75 to 100% confluency were washed three times with 1 ml of HBSS with supplements, warmed to 37°C. A 1.5-ml suspension of bacteria in HBSS with supplements was added to each monolayer, and the dishes were incubated
P. AERUGINOSA ADHERENCE
823
at 37°C in an air incubator for the indicated periods. The nonadherent bacteria were removed from the monolayer by
washing the monolayers three times with 1 ml of HBSS maintained at 37°C. Fresh HBSS containing gentamicin sulfate (Sigma Chemical Co., St. Louis, Mo.) at 50 jig/ml was added to kill extracellular bacteria. The MIC of gentamicin for P. aeruginosa is 2 to 4 ,ug/ml. The antibiotic was removed at the conclusion of the experiment by washing the monolayers four times with 1 ml of HBSS. The monolayers containing the internalized bacteria were rinsed with 1 ml of 0.53 mM Na-EDTA in PBS, followed by 1 ml of trypsin (0.25%, wt/vol) for 5 min. The trypsinized cells were scraped off the plates with a rubber policeman and
transferred to a sterile centrifuge tube, and 1 ml of soybean trypsin inhibitor (0.005%, wt/vol) was added. The total number of viable A549 cells was determined by hemacytometer counting after addition of 5 RI of 0.4% trypan blue (GIBCO) to 50 RI of cell suspension. To release intracellular bacteria, the A549 cells were sedimented in a Beckman microfuge for 5 min, and the pellet was resuspended in 20 p1 of 1% Triton X-100. After 10 min of incubation at room temperature, the lysate was diluted with 1 ml of Luria broth, serial 10-fold dilutions were made, and aliquots were plated on L-agar plates. After overnight incubation at 37°C, the number of CFU was determined. Electron microscopy. The cells for electron microscopy were fixed with 2% glutaraldehyde in PBS for 2 h, washed with 0.1 M cacodylate buffer (pH 7.4), and postfixed in 1% OS04 in cacodylate buffer, as described previously (2). Following dehydration of samples through a graded alcohol series, the cells were embedded in Epon. Thin sections were cut with a diamond knife, stained with uranyl acetate and lead citrate, and then examined with a JEOL-1OOB electron microscope at 60 kV. Scanning electron microscopy was performed with a JEOL-35C microscope, with the methods of sample preparation described previously (2). RESULTS Adhesion of P. aeruginosa to A549 cells. The interaction of P. aeruginosa PAK and a nonpiliated isogenic mutant, PAK-NP, with A549 cells was studied by incubating a suspension of bacteria with monolayer-grown A549 cells for 1 to 4 h at a ratio of 50 bacteria per A549 cell. Following removal of nonadherent bacteria by extensive washing, the number of P. aeruginosa associated with 100 A549 cells was determined. As shown in Fig. 1, the number of bound PAK cells increased linearly with time during the course of the experiment. The rate of association of PAK-NP cells was significantly slower and showed indications of leveling off after 2 h. The observed binding was dependent on the presence of cells, because no adhesion of either PAK or PAK-NP was observed when bacteria were incubated in tissue culture flasks under identical experimental conditions
(data not shown). Figure 2 shows the dose dependence of bacterial association with A549 cells. At an input ratio of less than 100 bacteria per A549 cell, the binding of wild-type P. aeruginosa was linear in relation to the number of bacteria present in the assay. When higher concentrations of bacteria were added to A549 monolayers, a decreasing fraction of these bacteria bound, indicating an approaching saturation of the sites on the A549 cells available for bacterial adhesion. A Scatchard plot (5, 24) derived from these data (Fig. 2B) was used to calculate the association constant, Ka, as 24 pl per
bacterium.
INFECT. IMMUN.
CHI ET AL.
824
We compared the adherence of two different isogenic, nonpiliated mutants of P. aeruginosa with that of their wild-type parent. Strain PAK-NP, carrying a mutation in the pilin structural gene, and PAK-Nl, containing a mutation in the pilin regulatory gene rpoN, showed binding properties significantly different from those of the parental strain PAK. PAK-NP bound to A549 cells in a dose-dependent manner over a wide range of bacterial concentrations. However, the number of PAK-NP cells associated with A549 cells ranged from 10 to 20% of that observed for the wild-type strain PAK. No significant association of the mutant strain PAK-Ni with the A549 cells was observed even at very high input ratios of bacteria to A549 cells. An association constant of 47 pl per bacterium for the binding of PAK-NP, based on the Scatchard plot shown in Fig. 2C, was similar to that calculated for the wild-type PAK strain. A fraction of the A549 cells, as examined by light microscopy, underwent pronounced rounding following their interaction with P. aeruginosa. At an input ratio of 50 bacteria (PAK or PAK-NP) per A549 cell, approximately 10% of the cells were noticeably rounded in 1 h. After 3 h, 40% of the A549 cells were rounded when associated with PAK, while the fraction of rounded A549 cells remained at 10% following their interaction with the same number of nonpiliated PAKNP. These findings suggest that the observed morphological changes are due to an association of piliated bacteria with the A549 cells or a release of cellular components from these bacteria. No difference in the distribution of adherent bac-
18 --
16-
*
PAK PAK-NP
14ci 01
0
12-
X) 10Q, L.
8-
g
64-
20
0.5
1
2.5
2
1.5
Time (htours)
3
FIG. 1. Kinetics of binding of P. aeruginosa PAK and PAK-NP to A549 cells. The association of P. aeruginosa strains with A549 cells was estimated at bacteria-to-cell ratios of 50:1 as described Materials and Methods. The binding data are expressed as the mean ± standard error of the mean of four experiments (number of bacteria on 100 A549 cells was counted at each time point for each experiment).
PAK
*
-+-. PAK-NP PAK-N1
--
A) 2.5
2
Bacteria (x 108) 80,
200-
180-
,, 60
'J 140:
01
-v.
L* 120-
oz
so-
60:
.t
v
10
20
0
30
_ 20
A
`4 40
B)
50
40
_100-
,;,
PAK-NP
70
PAK
A
;D160-
0
10
20
30
40
6 50 ... 60
B (bacteria per A549 cell)
70
70
80
C)
v
o
v
0
1
2
3
4
5
v
6
7
B (bacteria per A549 cell)
FIG. 2. (A) Dose-response curves of bacterial association with A549 cells. P. aeruginosa PAK (3.6 x 106 to 2.13 x 108 bacteria per ml) and PAK-NP (4.8 x 106 to 3.0 x 108 bacteria per ml) were incubated with 4 x 105 A549 cells per ml for 1 h. After monolayers were washed, 100 stained cells were located and associated bacteria were counted. Each point represents the mean + standard error of the mean for 100 cells counted. Scatchard plots were constructed for PAK (B) and PAK-NP (C) from the binding data; B is the number of bacteria per A549 cell, F is the concentration of bacteria in the assay, Kc, (association constant) is the slope of the least-square regression line, and N is the number of binding sites per cell at saturation.
VOL. 59, 1991
teria on flat versus rounded cells was noticed. The rounded cells were not lysed, since no significant release of a cytoplasmic marker (lactate dehydrogenase) from A549 cells was detected during the course of the experiments (data not shown). Furthermore, the level of viability of the A549 cells, as measured by the exclusion of trypan blue dye, was greater than 90% in cultures used in adherence assays with PAK or PAK-NP as well as in control cultures that were not exposed to bacteria. The observed association of P. aeruginosa PAK was temperature dependent. Following a 1-h incubation of bacteria with A549 cells, only 0.13 adherent bacteria per A549 cell were detected at 4°C, compared with an average of 3.44 bacteria bound in the same time period at 37°C. Heat treatment of bacteria (50°C for 30 min) also resulted in a 10-fold reduction in the number of adherent bacteria, while treatment of P. aeruginosa PAK with gentamicin (100 ,ug/ml for 1 h) prior to the assay resulted in an eightfold reduction in adherence. These findings suggest the involvement of a bacterial adhesive component that is thermolabile and is rapidly turned over in the bacterial cell. Internalization of bacteria following their association with A549 cells. Thin-section transmission electron microscopy was used to examine the interaction of P. aeruginosa PAK with A549 cells. Monolayers were incubated with bacteria for 2 or 3 h at 37°C at a ratio of 50 bacteria per A549 cell, and after extensive washing, the monolayers were fixed and processed for electron microscopy. While many P. aeruginosa PAK cells were seen to be attached to the membranes of A549 cells, frequent intracellular localization of bacteria was also observed (Fig. 3). The intracellular bacteria were always enclosed within a vesicle and in the vicinity of the Golgi apparatus. A peribacillary clear space separated the microorganisms from the surrounding membrane of the enclosing vesicle. Many vesicles were lined with the spherical projections on their luminal side. Occasionally, multiple bacteria were seen in the same vesicle, suggesting that cell division may have occurred while they were internalized (Fig. 3C). Lamellar bodies, the organelles that typify type II alveolar cells such as A549, were also seen closely associated with the vesicles containing the bacteria (Fig. 3B). Both the bacteria and the A549 cells were well preserved, even after a 3-h incubation prior to fixation and sectioning. Survival of bacteria in A549 cells. The significant number of relatively intact bacteria internalized by the A549 cells suggested that these bacteria somehow escaped the normal bactericidal mechanisms of the A549 cell. Monolayers of A549 cells were exposed to PAK and PAK-NP at a ratio of 50 bacteria per host cell for 2 h. Nonadherent bacteria were removed by washing, and those cells that were adherent but not internalized were killed by exposure to gentamicin (50 ,ug/ml). At intervals thereafter, internalized bacteria were released by detergent lysis of A549 cells and viable P. aeruginosa were enumerated as CFU by plating dilutions from cell lysates on L-agar plates and incubating overnight. Figure 4 shows the kinetics of survival of the fraction of bacteria that were internalized by A549 cells during the experiment. While a noticeable decrease in the number of viable bacteria over time was observed for P. aeruginosa PAK, a significant fraction (30%) of the viable bacteria were recovered from A549 cells after 4 h. In contrast, only a small fraction of nonpiliated PAK-NP bacteria were initially internalized (ca. 0.05 per A549 cell), and the viability of these bacteria decreased 20-fold in 4 h. To estimate the efficiency of internalization and survival, we determined the fraction of adherent bacteria that were
P. AERUGINOSA ADHERENCE
825
subsequently internalized. The number of A549-associated bacteria was first estimated following a 2.5-h incubation. In replicate experiments, the fraction of viable bacteria was estimated following exposure to gentamicin during the last half hour of the experiment. While approximately 12% of adherent PAK bacteria were surviving intracellularly, only 1.6% of adherent nonpiliated PAK-NP bacteria were determined to be viable, i.e., not killed by external gentamicin (data not shown). DISCUSSION We have examined the interaction of P. aeruginosa with A549 cells, an established pulmonary epithelial cell line, in order to develop an in vitro assay system for studying the role of bacterial adhesins during the critical early phase of human respiratory tract infection by this organism. The data presented in this article strongly suggest that P. aeruginosa utilizes pili or a component of pili for attachment, confirming the observations from several laboratories with different cell lines. Two nonpiliated mutants of P. aeruginosa PAK showed less adherence to A549 cells than the wild-type parental strain. While the pilin-deficient strain PAK-NP adhered poorly to A549 cells, the few bacteria that adhered showed an affinity for the receptors on the A549 membrane similar to that of the wild-type PAK strain. These findings suggest that P. aeruginosa possesses one major adhesin that is associated with the ability of bacteria to form functional pili, while a different class of adhesin molecules may be found in the bacterial cell envelope. Alternatively, only one pilus-associated adhesin is made by P. aeruginosa, and the observed reduced binding of the pilin-deificient PAK-NP strain might be due to the localization of the adhesin in the outer membrane, where adhesin interaction with the host cell surface receptors is less efficient. Several of the previously described accessory proteins of type 1 pili (8, 11) and pili of uropathogenic Escherichia coli (25) have been implicated in binding of bacteria to cellular receptors. In the absence of pilin subunits, these proteins were membrane localized but still allowed the bacteria to adhere to cell surfaces. Similarly, Paruchuri et al. (17) have demonstrated the presence of a pilus-independent adhesin on the surface of Neisseria gonorrhoeae. We have attempted to determine the kinetic constants for the interaction of P. aeruginosa with the cultured A549 cells under the conditions of the assay. The similar association constants calculated for the interaction of the bacteria with A549 cells (24 pl for PAK and 83 pl for PAK-NP) confirm that the adhesin molecules responsible for binding of the wild-type PAK strain and the nonpiliated mutant PAK-NP are similar or perhaps identical. The use of in vitro adhesion assays for determination of the kinetic parameters of the bacterium-A549 cell association may be influenced by several additional factors, such as modification of receptors on A549 cells by bacterial products or, alternatively, degradation of bacterial adhesins by enzymes released from the epithelial cells. The complete lack of adhesion by P. aeruginosa PAK-N1, carrying a mutation in the pleiotropic regulatory gene rpoN, suggests that the expression of the gene(s) encoding the putative adhesin molecules on the bacterial surface is dependent on the presence of this regulatory polypeptide. RpoN is a sigma factor of RNA polymerase and is responsible for the transcription of a number of genes involved in a variety of metabolic functions, including pilin expression. The complete repertoire of genes in P. aeruginosa that are under
A.
FIG. 3. Morphology of P. aeruginosa within A549 cells. Transmission electron micrographs showing internalized P. aeruginosa PAK in A549 cells after a 3-h incubation. (A) Portion of a cell showing at least 11 bacteria in membrane-bound vacuoles. The bacteria shown are located in the vicinity of the nucleus (N) and the Golgi apparatus (G). The characteristic lamellar bodies (LB) of this cell line are seen. A few dividing bacteria are also seen in the vacuoles (arrows). (B) Micrograph showing lamellar bodies (LB) and a mitochondrion (M) closely associated with the bacteria-containing vacuoles. (C) Cytoplasmic vacuole containing four bacteria. The particles, possibly glycogen, are clearly seen attached to the vacuolar (V) membrane (arrows). 826
P. AERUGINOSA ADHERENCE
VOL. 59, 1991
-+-
0.1
HeLa cells require expression of a number of virulence factors by B. pertussis that are under control of the vir regulatory element (6). Our study has also shown that the association of P. aeruginosa with cultured cells may require the expression of several genes under the control of a common regulatory element, RpoN. The successful colonization of a host, including attachment to and invasion of eukaryotic cells by P. aeruginosa, may also require the coordinate expression of a number of different virulence factors. Environmental modulation of the signals received by such controlling elements may be an important component of the natural host defense mechanism and may present new and effective approaches to treatment of a variety of infections caused by P. aeruginosa.
A549 Cells + PAK
-a- A549 Cells + PAK-NP
I.4 .
0.01
.0b
*;je
0.001
0.5
1
1.5
2
2.5
827
3.5
Time (hiours)
FIG. 4. Kinetics of survival of P. aeruginosa in A549 cells. Monolayers of A549 cells were incubated with P. aeruginosa PAK or PAK-NP. Gentamicin was added at the times indicated to kill adherent or extracellular bacteria, and intracellular survival was determined as described in Materials and Methods. Each point represents the mean standard error of the mean of four different experiments. ±
RpoN control is not known; however, coregulation of adhesion with pilin expression suggests that the adhesin may be responding to the same regulatory stimuli as pilin. It is known that related functions are often coregulated in bacteria. Therefore, it is possible that the synthesis of pilin and the adhesin molecule is coordinated in the bacterium to allow their interaction during pilus assembly. The entry and survival of P. aeruginosa within the A549 cells was a surprising finding, since P. aeruginosa is usually considered an extracellular parasite which generates tissue damage during colonization by producing extracellular toxic substances. There have not been any reports to date of intracellular bacteria in tissues of heavily colonized patients, such as the lungs of cystic fibrosis patients. Since only a minority of adherent bacteria are internalized, the lack of histological evidence may reflect the relatively few epithelial cells containing bacteria at any one time. Our observation that piliated P. aeruginosa is capable of surviving inside A549 cells with minimal loss of viability while the nonpiliated mutant is rapidly killed by the same cells suggests that pili may interfere with killing mechanisms following endocytosis by epithelial cells. Alternatively, the enhanced killing of the nonpiliated mutant may simply reflect the lower numbers of bacteria attached to each A549 cells. Similarly, enhanced survival of piliated PA but not of nonpiliated PAK-NP was observed when a primary cell line derived from the trachea of a fetal monkey was used in the assay (data not shown). This suggests that the ability to harbor viable bacteria intracellularly may be a property of most epithelial cells. The availability of genetically engineered isogenic mutants of P. aeruginosa with mutations in a number of extracellular and cell-associated virulence factors may allow identification of the bacterial components that play a role in invasion. Recently, Ewanowich et al. demonstrated invasion of HeLa cells by Bordetella pertussis (6) and Bordetella parapertussis (7), pathogens normally considered noninvasive. These workers also showed that optimal invasion of and survival in
ACKNOWLEDGMENTS We thank Jean Reding for technical assistance, Arnie Smith and Mark Strom for helpful discussions, and Karyn Ishimoto for construction of strains used in this study. This work was supported by an RDP grant from the Cystic Fibrosis Foundation and grant HL 30542 from NIH. D.N. is a postdoctoral fellow of the Cystic Fibrosis Foundation. S.L. holds a Research Scholar Award from the Cystic Fibrosis Foundation.
REFERENCES 1. Amador, E., and W. E. C. Wacker. 1965. Enzymatic methods used for diagnosis. Methods Biochem. Anal. 13:265-356. 2. Chi, E. Y., and W. R. Henderson. 1984. Ultrastructure of mast cell degranulation induced by eosinophil peroxidase: use of diaminobenzidine cytochemistry by scanning electron microscopy. J. Histochem. Cytochem. 32:332-341. 3. Doig, P., P. A. Sastry, R. S. Hedges, K. K. Lee, W. Paranchych, and R. T. Irvin. 1990. Inhibition of pilus-mediated adhesion of Pseudomonas aeruginosa to buccal epithelial cells by monoclonal antibodies directed against pili. Infect. Immun. 58:124-130. 4. Doig, P., T. Todd, P. A. Sastry, K. K. Lee, R. S. Hodges, W. Paranchych, and R. T. Irvin. 1988. Role of pili in adhesion of Pseudomonas aeruginosa to human respiratory epithelial cells. Infect. Immun. 56:1641-1646. 5. Doyle, R. J., J. D. Oakley, K. R. Murphy, D. McAlister, and K. G. Taylor. 1985. Graphical analyses of adherence data, p. 109-113. In S. E. Mergenhagen and B. Rosen (ed.), Molecular basis of oral microbial adhesion. American Society for Microbiology, Washington, D.C. 6. Ewanowich, C. A., A. R. Melton, A. A. Weiss, R. K. Sherburne, and M. S. Peppler. 1989. Invasion of HeLa 229 cells by virulent Bordetella pertussis. Infect. Immun. 57:2698-2704. 7. Ewanowich, C. A., R. K. Sherburne, S. F. Man, and M. S. Peppler. 1989. Bordetella parapertussis invasion of HeLa 229 cells and human respiratory epithelial cells in primary culture. Infect. Immun. 57:1240-1247. 8. Hanson, M. S., and C. C. Brinton. 1988. Identification and characterisation of the Escherichia coli type 1 pilus tip adhesin protein. Nature (London) 322:265-268. 9. Hazlett, L. D., M. Moon, M. Strejc, and R. S. Berk. 1987. Evidence for N-acetylmannosamine as an ocular receptor for Pseudomonas aeruginosa adherence to scarified cornea. Invest. Ophthalmol. Visual Sci. 28:1978-1985. 10. Ishimoto, K., and S. Lory. 1989. Formation of pilin in Pseudomonas aeruginosa requires the alternative sigma factor (RpoN) of RNA polymerase. Proc. Natl. Acad. Sci. USA 86:1954-1957. 11. Klemm, P., K. A. Krogfelt, L. Hedegaard, and G. Christiansen. 1990. The major subunit of Escherichia coli type 1 fimbriae is not required for D-mannose-specific adhesion. Mol. Microbiol. 4:553-559. 12. Krivan, H. C., V. Ginsburg, and D. D. Roberts. 1988. Pseudomonas aeruginosa and Pseudomonas cepacia isolated from cystic fibrosis patients binds specifically to gangliotetraosylceramide (asialo GM1) and gangliotriaosylceramide (asialo GM2). Arch. Biochem. Biophys. 260:493-496. 13. Lee, K. K., P. Doig, R. T. Irvin, W. Paranchych, and R. D.
828
14.
15.
16. 17.
18. 19.
CHI ET AL.
Hodges. 1989. Mapping the surface region of Pseudomonas aeruginosa PAK pilin: the importance of the C-terminal region for adherence to human buccal epithelial cells. Mol. Microbiol. 3:1493-1499. Lieber, M., B. Smith, A. Szakal, W. Nelson-Rees, and G. Tadaro. 1976. A continuous tumor cell line from human lung carcinoma with properties of type II alveolar epithelial cells. Int. J. Cancer 17:62-70. Lund, B., F. Lindberg, B.-I. Marklund, and S. Normark. 1987. The PapG protein is the alpha-D-galactopyranosyl-(1-4)-J3-Dgalactopyranose-binding adhesin of uropathogenic Escherichia coli. Proc. Natl. Acad. Sci. USA 84:5898-5902. Marcus, H., A. Austria, and N. R. Baker. 1989. Adherence of Pseudomonas aeruginosa to tracheal epithelium. Infect. Immun. 57:1050-1053. Paruchuri, D. K., H. S. Seifert, R. S. Ajioka, K.-A. Karisson, and M. So. 1990. Identification and characterization of a Neisseria gonorrhoeae gene encoding a glycolipid-binding adhesin. Proc. Natl. Acad. Sci. USA 87:333-337. Pier, G. B. 1985. Pulmonary disease associated with Pseudomonas aeruginosa in cystic fibrosis: current status of the hostbacterium interaction. J. Infect. Dis. 151:575-580. Ramphal, R., C. Guay, and G. B. Pier. 1987. Pseudomonas aeruginosa adhesins for tracheobronchial mucins. Infect. Immun. 55:600-603.
INFECT. IMMUN.
20. Ramphal, R., J. C. Sadoff, M. Pyle, and J. D. Silipingi. 1984. Role of pili in adherence of Pseudomonas aeruginosa to injured tracheal epithelium. Infect. Immun. 44:38-40. 21. Saiman, L., K. Ishimoto, S. Lory, and A. Prince. 1990. The effect of piliation and exoproduct expression on the adherence of Pseudomonas aeruginosa to respiratory epithelial monolayers. J. Infect. Dis. 161:541-548. 22. Saiman, L., J. Sadoff, and A. Prince. 1989. Cross-reactivity of Pseudomonas aeruginosa antipilin monoclonal antibodies with heterogeneous strains of P. aeruginosa and Pseudomonas cepacia. Infect. Immun. 57:2764-2770. 23. Sato, H., and K. Okinaga. 1987. Role of pili in the adherence of Pseudomonas aeruginosa to mouse epidermal cells. Infect. Immun. 55:1774-1778. 24. Scatchard, G. 1949. The attraction of proteins for small molecules and ions. Ann. N.Y. Acad. Sci. 51:660-672. 25. Uhlin, B. E., M. Norgren, M. Baga, and S. Normark. 1985. Adhesion to human cells lacking the major subunit of a digalactoside-specific pilus adhesin. Proc. Natl. Acad. Sci. USA 82: 1800-1804. 26. Woods, D. E., D. C. Strauss, W. G. Johanson, Jr., V. K. Berry, and J. A. Bass. 1980. Role of pili in adherence of Pseudomonas aeruginosa to buccal epithelial cells. Infect. Immun. 29:11461151.
Vol. 59, No. 3
Interaction of Pseudomonas aeruginosa with A549 Pneumocyte Cells EMIL CHI,' THOMAS MEHL,' DAVID NUNN,2 AND STEPHEN LORY2*
Departments of Pathology1 and Microbiology,2 School of Medicine, University of Washington, Seattle, Washington 98195 Received 4 September 1990/Accepted 30 November 1990
The interaction of Pseudomonas aeruginosa with a human lung pneumocyte cell line (A549) was studied. Wild-type strain PAK adhered efficiently to the A549 cells, while an isogenic mutant, carrying a mutation in the pilin structural gene, adhered at 10 to 20% of the wild-type levels. Another nonpiliated mutant of P. aeruginosa PAK, defective in the pleiotropic regulatory gene rpoN, did not adhere to A549 cells, suggesting the presence of a second, RpoN-controlled adhesin on the bacterial surface. Endocytosis of wild-type P. aeruginosa PAK by A549 cells was also demonstrated. A significant fraction of the internalized bacteria were recovered in a viable form after several hours of residence within the A549 cells. When examined by electron microscopy, intracellular bacteria were located in membranous vesicles, and no evidence of killing by lysosomal mechanisms was observed. These studies raise the possibility that during chronic respiratory tract infections in immunocompromised patients, P. aeruginosa may persist in intracellular compartments and therefore be protected from the defense mechanisms of the host.
Pseudomonas aeruginosa is an important bacterial agent of chronic pneumonia in patients during prolonged hospitalization and is responsible for lethal pulmonary infection in a majority of individuals with cystic fibrosis. Following inhalation or aspiration, the bacteria attach to receptors on the epithelial cells or the mucous coating of the airways. Adherent bacteria are resistant to mucocilliary clearance mechanisms and proliferate, with progressive spread through the respiratory tract (18). The ability of P. aeruginosa to adhere to specific sites on host tissues has been investigated with various cell culture models, such as human buccal epithelial cells (4, 26), rat cornea (9), cells of humans, hamster, and bovine trachea (4, 16, 22), and epidermal cells (23). Specific binding has been also demonstrated to human tracheobronchial mucin (19) and glycolipids (12). These findings demonstrate the complexity of possible host receptors for this microorganism. Several bacterial products have been implicated as potential adhesins during various stages of infection. Purified pili were shown to bind directly to a number of cell types and compete with intact bacteria for binding to such cells (20, 23). Monoclonal antibodies to pili have also been shown to block adhesion of bacteria to cells (3), and antibodies to a peptide corresponding to a C-terminal region of pilin also blocked binding of pili to epithelial cells (13). Binding of P. aeruginosa to tracheobronchial mucin, mediated by alginate exopolysaccharide, has also been demonstrated in vitro (19). This affinity of the alginate-coated bacteria for the lung tissues is believed to be one of several factors that select mucoid variants from the nonmucoid invading pathogens during later stages of infection. To identify the bacterial components that play a role in colonization of tissues, we have explored the possibility of using epithelial cell lines, derived from the human respiratory tract, as in vitro models for studying the initial interaction of the bacterium with a host. The human pulmonary carcinoma cell line A549 (14) was selected for the studies *
described here, as these cells possess the morphological and biochemical characteristics of type II pneumocytes of the intact lung. A well-characterized non-mucoid strain of P. aeruginosa, PAK, was used to study the interaction of bacteria with A549 cells. We also examined isogenic mutants of PAK that have lost the ability to form pili by mutations in either the pilin structural gene or the regulatory gene rpoN for involvement of pili in adherence. In this report, we characterize the kinetic parameters of binding of P. aeruginosa PAK to A549 cells and show reduced binding of the nonpiliated variants. Furthermore, a significant fraction of bound P. aeruginosa PAK were taken up into endocytic vesicles, where they persisted intracellularly without appreciable loss of viability. Intracellular persistence of a fraction of the bacteria colonizing the respiratory tract of cystic fibrosis patients may explain the chronic nature of P. aeruginosa infection and the protection of this pathogen from host defense mechanisms. Furthermore, bacteria living intracellularly are very likely refractory to the killing action of antimicrobial agents that cannot penetrate the protective environment afforded by the host cell. MATERIALS AND METHODS Bacteria and growth conditions. P. aeruginosa PAK was
provided by D. Bradley, Memorial University of Newfoundland. P. aeruginosa PAK-Nl is an rpoN mutant of PAK (10). P. aeruginosa PAK-NP is a mutant of PAK with an insertionally inactivated pilin gene in its chromosome (21). Bacteria were grown statically in Luria broth (1% tryptone, 0.5% yeast extract, 0.5% sodium chloride) supplemented with 5 mM magnesium chloride, at 37°C for 16 h. Prior to their use in adhesion assays, the bacteria were sedimented by centrifugation at 5,000 x g for 5 min, washed once with Hank's balanced salt solution (HBSS; GIBCO, Santa Clara, Calif.) supplemented with 1 mM CaCl2, 2 mM MgCl2, 20 mM HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, pH 7.3) (this solution will be referred to as HBSS with supplements in this report), and resuspended in HBSS with supplements to the desired bacterial concentration before
Corresponding author. 822
VOL. 59, 1991
being added to A549 cells. The bacterial concentration was estimated by measuring the A60. An OD6. of 1.0 corresponds to 6 x 108 viable bacteria, as established from the number of CFU resulting from plating dilutions of bacterial suspension onto L-agar plates and overnight incubation. Cell culture methods. The A549 cell line ATCC CCL 165, passage 77 (13), was obtained from the American Type Culture Collection (Rockville, Md.). The cells were maintained in 10% fetal calf serum in Waymouth MB752/1 medium, supplemented with 2 mM glutamine, 1 mM sodium pyruvate, and a standard antibiotic mixture (100 U of penicillin, 100 ,ug of streptomycin, and 0.5 ,ug of amphotericin B per ml) and incubated at 37°C in 5% CO2. Passage 91 to 99 was used for all experiments in this report. The cells were fed every 3 to 4 days and passaged at 7- to 10-day intervals. Briefly, cells were rinsed with 0.53 mM Na-EDTA in PBS (20 mM sodium phosphate, 150 mM NaCl [pH 7.4]) and treated with 0.25% (wt/vol) trypsin in PBS, followed by addition of 50% fetal calf serum to stop the protease activity. The cells were then plated at 0.5 x 106 per 25-cm2 flask (Corning 25100) or 35-mm-diameter petri dish (Falcon 3001). All tissue culture reagents were obtained from GIBCO. Quantitation of lactate dehydrogenase release, cell recovery, and viability. Lactate dehydrogenase released into the culture fluid from A549 cells incubated with bacteria was determined by the method of Amador and Wacker (1) and compared with the total enzyme activity recovered from monolayers and A549 cells following lysis of cells with 0.1% Triton X-100 in PBS. Bacteria and cellular debris were removed from the lysates by centrifugation at 5,000 x g for 5 min before assaying for the enzyme. Bacterial adherence assay. Approximately 1 x 105 to 2 x 105 A549 cells were seeded into 35-mm dishes and incubated overnight prior to binding assays. To determine the kinetics of bacterial association, the monolayers were first washed three times with warm (37°C) serum-free HBSS with supplements, and the cells were overlaid with 1 ml of this solution. A 0.2-ml aliquot of bacterial suspension was then added to give a final ratio of 50 bacteria per A549 cell. The plates were gently swirled and incubated for 1 to 4 h at 37°C. When constructing binding curves with multiple ratios of bacteria to cultured cells, twofold dilutions of the bacteria in 1-ml aliquots were added directly to washed A549 monolayers and incubated for 1 h at 37°C. At the conclusion of the adherence experiments, the unbound bacteria were removed by washing the monolayers three times with 1 ml of HBSS with supplements. The monolayers were then fixed for 2 h with 2.5% (vol/vol) glutaraldehyde in PBS (pH 7.4) or a fixative composed of 5% (vol/vol) formaldehyde and 5% (vol/vol) glacial acetic acid in 70% (vol/vol) methanol. The monolayers were washed once with water and stained with 10% Giemsa stain for 10 min. The stain was removed by washing three times with water and air-drying the plates. The extent of bacterial adherance to A549 cells was determined by examining the monolayers under a light microscope at 400 x magnification and enumerating the bacterial cells residing over 100 A549 cells. Because this technique does not distinguish internalized from surface-bound bacteria, binding refers to bacteria associated with A549 cells and includes those bacteria located on the surface as well as those internalized by the A549 cells. Determination of uptake of P. aeruginosa. A549 cells grown in 35-mm dishes to 75 to 100% confluency were washed three times with 1 ml of HBSS with supplements, warmed to 37°C. A 1.5-ml suspension of bacteria in HBSS with supplements was added to each monolayer, and the dishes were incubated
P. AERUGINOSA ADHERENCE
823
at 37°C in an air incubator for the indicated periods. The nonadherent bacteria were removed from the monolayer by
washing the monolayers three times with 1 ml of HBSS maintained at 37°C. Fresh HBSS containing gentamicin sulfate (Sigma Chemical Co., St. Louis, Mo.) at 50 jig/ml was added to kill extracellular bacteria. The MIC of gentamicin for P. aeruginosa is 2 to 4 ,ug/ml. The antibiotic was removed at the conclusion of the experiment by washing the monolayers four times with 1 ml of HBSS. The monolayers containing the internalized bacteria were rinsed with 1 ml of 0.53 mM Na-EDTA in PBS, followed by 1 ml of trypsin (0.25%, wt/vol) for 5 min. The trypsinized cells were scraped off the plates with a rubber policeman and
transferred to a sterile centrifuge tube, and 1 ml of soybean trypsin inhibitor (0.005%, wt/vol) was added. The total number of viable A549 cells was determined by hemacytometer counting after addition of 5 RI of 0.4% trypan blue (GIBCO) to 50 RI of cell suspension. To release intracellular bacteria, the A549 cells were sedimented in a Beckman microfuge for 5 min, and the pellet was resuspended in 20 p1 of 1% Triton X-100. After 10 min of incubation at room temperature, the lysate was diluted with 1 ml of Luria broth, serial 10-fold dilutions were made, and aliquots were plated on L-agar plates. After overnight incubation at 37°C, the number of CFU was determined. Electron microscopy. The cells for electron microscopy were fixed with 2% glutaraldehyde in PBS for 2 h, washed with 0.1 M cacodylate buffer (pH 7.4), and postfixed in 1% OS04 in cacodylate buffer, as described previously (2). Following dehydration of samples through a graded alcohol series, the cells were embedded in Epon. Thin sections were cut with a diamond knife, stained with uranyl acetate and lead citrate, and then examined with a JEOL-1OOB electron microscope at 60 kV. Scanning electron microscopy was performed with a JEOL-35C microscope, with the methods of sample preparation described previously (2). RESULTS Adhesion of P. aeruginosa to A549 cells. The interaction of P. aeruginosa PAK and a nonpiliated isogenic mutant, PAK-NP, with A549 cells was studied by incubating a suspension of bacteria with monolayer-grown A549 cells for 1 to 4 h at a ratio of 50 bacteria per A549 cell. Following removal of nonadherent bacteria by extensive washing, the number of P. aeruginosa associated with 100 A549 cells was determined. As shown in Fig. 1, the number of bound PAK cells increased linearly with time during the course of the experiment. The rate of association of PAK-NP cells was significantly slower and showed indications of leveling off after 2 h. The observed binding was dependent on the presence of cells, because no adhesion of either PAK or PAK-NP was observed when bacteria were incubated in tissue culture flasks under identical experimental conditions
(data not shown). Figure 2 shows the dose dependence of bacterial association with A549 cells. At an input ratio of less than 100 bacteria per A549 cell, the binding of wild-type P. aeruginosa was linear in relation to the number of bacteria present in the assay. When higher concentrations of bacteria were added to A549 monolayers, a decreasing fraction of these bacteria bound, indicating an approaching saturation of the sites on the A549 cells available for bacterial adhesion. A Scatchard plot (5, 24) derived from these data (Fig. 2B) was used to calculate the association constant, Ka, as 24 pl per
bacterium.
INFECT. IMMUN.
CHI ET AL.
824
We compared the adherence of two different isogenic, nonpiliated mutants of P. aeruginosa with that of their wild-type parent. Strain PAK-NP, carrying a mutation in the pilin structural gene, and PAK-Nl, containing a mutation in the pilin regulatory gene rpoN, showed binding properties significantly different from those of the parental strain PAK. PAK-NP bound to A549 cells in a dose-dependent manner over a wide range of bacterial concentrations. However, the number of PAK-NP cells associated with A549 cells ranged from 10 to 20% of that observed for the wild-type strain PAK. No significant association of the mutant strain PAK-Ni with the A549 cells was observed even at very high input ratios of bacteria to A549 cells. An association constant of 47 pl per bacterium for the binding of PAK-NP, based on the Scatchard plot shown in Fig. 2C, was similar to that calculated for the wild-type PAK strain. A fraction of the A549 cells, as examined by light microscopy, underwent pronounced rounding following their interaction with P. aeruginosa. At an input ratio of 50 bacteria (PAK or PAK-NP) per A549 cell, approximately 10% of the cells were noticeably rounded in 1 h. After 3 h, 40% of the A549 cells were rounded when associated with PAK, while the fraction of rounded A549 cells remained at 10% following their interaction with the same number of nonpiliated PAKNP. These findings suggest that the observed morphological changes are due to an association of piliated bacteria with the A549 cells or a release of cellular components from these bacteria. No difference in the distribution of adherent bac-
18 --
16-
*
PAK PAK-NP
14ci 01
0
12-
X) 10Q, L.
8-
g
64-
20
0.5
1
2.5
2
1.5
Time (htours)
3
FIG. 1. Kinetics of binding of P. aeruginosa PAK and PAK-NP to A549 cells. The association of P. aeruginosa strains with A549 cells was estimated at bacteria-to-cell ratios of 50:1 as described Materials and Methods. The binding data are expressed as the mean ± standard error of the mean of four experiments (number of bacteria on 100 A549 cells was counted at each time point for each experiment).
PAK
*
-+-. PAK-NP PAK-N1
--
A) 2.5
2
Bacteria (x 108) 80,
200-
180-
,, 60
'J 140:
01
-v.
L* 120-
oz
so-
60:
.t
v
10
20
0
30
_ 20
A
`4 40
B)
50
40
_100-
,;,
PAK-NP
70
PAK
A
;D160-
0
10
20
30
40
6 50 ... 60
B (bacteria per A549 cell)
70
70
80
C)
v
o
v
0
1
2
3
4
5
v
6
7
B (bacteria per A549 cell)
FIG. 2. (A) Dose-response curves of bacterial association with A549 cells. P. aeruginosa PAK (3.6 x 106 to 2.13 x 108 bacteria per ml) and PAK-NP (4.8 x 106 to 3.0 x 108 bacteria per ml) were incubated with 4 x 105 A549 cells per ml for 1 h. After monolayers were washed, 100 stained cells were located and associated bacteria were counted. Each point represents the mean + standard error of the mean for 100 cells counted. Scatchard plots were constructed for PAK (B) and PAK-NP (C) from the binding data; B is the number of bacteria per A549 cell, F is the concentration of bacteria in the assay, Kc, (association constant) is the slope of the least-square regression line, and N is the number of binding sites per cell at saturation.
VOL. 59, 1991
teria on flat versus rounded cells was noticed. The rounded cells were not lysed, since no significant release of a cytoplasmic marker (lactate dehydrogenase) from A549 cells was detected during the course of the experiments (data not shown). Furthermore, the level of viability of the A549 cells, as measured by the exclusion of trypan blue dye, was greater than 90% in cultures used in adherence assays with PAK or PAK-NP as well as in control cultures that were not exposed to bacteria. The observed association of P. aeruginosa PAK was temperature dependent. Following a 1-h incubation of bacteria with A549 cells, only 0.13 adherent bacteria per A549 cell were detected at 4°C, compared with an average of 3.44 bacteria bound in the same time period at 37°C. Heat treatment of bacteria (50°C for 30 min) also resulted in a 10-fold reduction in the number of adherent bacteria, while treatment of P. aeruginosa PAK with gentamicin (100 ,ug/ml for 1 h) prior to the assay resulted in an eightfold reduction in adherence. These findings suggest the involvement of a bacterial adhesive component that is thermolabile and is rapidly turned over in the bacterial cell. Internalization of bacteria following their association with A549 cells. Thin-section transmission electron microscopy was used to examine the interaction of P. aeruginosa PAK with A549 cells. Monolayers were incubated with bacteria for 2 or 3 h at 37°C at a ratio of 50 bacteria per A549 cell, and after extensive washing, the monolayers were fixed and processed for electron microscopy. While many P. aeruginosa PAK cells were seen to be attached to the membranes of A549 cells, frequent intracellular localization of bacteria was also observed (Fig. 3). The intracellular bacteria were always enclosed within a vesicle and in the vicinity of the Golgi apparatus. A peribacillary clear space separated the microorganisms from the surrounding membrane of the enclosing vesicle. Many vesicles were lined with the spherical projections on their luminal side. Occasionally, multiple bacteria were seen in the same vesicle, suggesting that cell division may have occurred while they were internalized (Fig. 3C). Lamellar bodies, the organelles that typify type II alveolar cells such as A549, were also seen closely associated with the vesicles containing the bacteria (Fig. 3B). Both the bacteria and the A549 cells were well preserved, even after a 3-h incubation prior to fixation and sectioning. Survival of bacteria in A549 cells. The significant number of relatively intact bacteria internalized by the A549 cells suggested that these bacteria somehow escaped the normal bactericidal mechanisms of the A549 cell. Monolayers of A549 cells were exposed to PAK and PAK-NP at a ratio of 50 bacteria per host cell for 2 h. Nonadherent bacteria were removed by washing, and those cells that were adherent but not internalized were killed by exposure to gentamicin (50 ,ug/ml). At intervals thereafter, internalized bacteria were released by detergent lysis of A549 cells and viable P. aeruginosa were enumerated as CFU by plating dilutions from cell lysates on L-agar plates and incubating overnight. Figure 4 shows the kinetics of survival of the fraction of bacteria that were internalized by A549 cells during the experiment. While a noticeable decrease in the number of viable bacteria over time was observed for P. aeruginosa PAK, a significant fraction (30%) of the viable bacteria were recovered from A549 cells after 4 h. In contrast, only a small fraction of nonpiliated PAK-NP bacteria were initially internalized (ca. 0.05 per A549 cell), and the viability of these bacteria decreased 20-fold in 4 h. To estimate the efficiency of internalization and survival, we determined the fraction of adherent bacteria that were
P. AERUGINOSA ADHERENCE
825
subsequently internalized. The number of A549-associated bacteria was first estimated following a 2.5-h incubation. In replicate experiments, the fraction of viable bacteria was estimated following exposure to gentamicin during the last half hour of the experiment. While approximately 12% of adherent PAK bacteria were surviving intracellularly, only 1.6% of adherent nonpiliated PAK-NP bacteria were determined to be viable, i.e., not killed by external gentamicin (data not shown). DISCUSSION We have examined the interaction of P. aeruginosa with A549 cells, an established pulmonary epithelial cell line, in order to develop an in vitro assay system for studying the role of bacterial adhesins during the critical early phase of human respiratory tract infection by this organism. The data presented in this article strongly suggest that P. aeruginosa utilizes pili or a component of pili for attachment, confirming the observations from several laboratories with different cell lines. Two nonpiliated mutants of P. aeruginosa PAK showed less adherence to A549 cells than the wild-type parental strain. While the pilin-deficient strain PAK-NP adhered poorly to A549 cells, the few bacteria that adhered showed an affinity for the receptors on the A549 membrane similar to that of the wild-type PAK strain. These findings suggest that P. aeruginosa possesses one major adhesin that is associated with the ability of bacteria to form functional pili, while a different class of adhesin molecules may be found in the bacterial cell envelope. Alternatively, only one pilus-associated adhesin is made by P. aeruginosa, and the observed reduced binding of the pilin-deificient PAK-NP strain might be due to the localization of the adhesin in the outer membrane, where adhesin interaction with the host cell surface receptors is less efficient. Several of the previously described accessory proteins of type 1 pili (8, 11) and pili of uropathogenic Escherichia coli (25) have been implicated in binding of bacteria to cellular receptors. In the absence of pilin subunits, these proteins were membrane localized but still allowed the bacteria to adhere to cell surfaces. Similarly, Paruchuri et al. (17) have demonstrated the presence of a pilus-independent adhesin on the surface of Neisseria gonorrhoeae. We have attempted to determine the kinetic constants for the interaction of P. aeruginosa with the cultured A549 cells under the conditions of the assay. The similar association constants calculated for the interaction of the bacteria with A549 cells (24 pl for PAK and 83 pl for PAK-NP) confirm that the adhesin molecules responsible for binding of the wild-type PAK strain and the nonpiliated mutant PAK-NP are similar or perhaps identical. The use of in vitro adhesion assays for determination of the kinetic parameters of the bacterium-A549 cell association may be influenced by several additional factors, such as modification of receptors on A549 cells by bacterial products or, alternatively, degradation of bacterial adhesins by enzymes released from the epithelial cells. The complete lack of adhesion by P. aeruginosa PAK-N1, carrying a mutation in the pleiotropic regulatory gene rpoN, suggests that the expression of the gene(s) encoding the putative adhesin molecules on the bacterial surface is dependent on the presence of this regulatory polypeptide. RpoN is a sigma factor of RNA polymerase and is responsible for the transcription of a number of genes involved in a variety of metabolic functions, including pilin expression. The complete repertoire of genes in P. aeruginosa that are under
A.
FIG. 3. Morphology of P. aeruginosa within A549 cells. Transmission electron micrographs showing internalized P. aeruginosa PAK in A549 cells after a 3-h incubation. (A) Portion of a cell showing at least 11 bacteria in membrane-bound vacuoles. The bacteria shown are located in the vicinity of the nucleus (N) and the Golgi apparatus (G). The characteristic lamellar bodies (LB) of this cell line are seen. A few dividing bacteria are also seen in the vacuoles (arrows). (B) Micrograph showing lamellar bodies (LB) and a mitochondrion (M) closely associated with the bacteria-containing vacuoles. (C) Cytoplasmic vacuole containing four bacteria. The particles, possibly glycogen, are clearly seen attached to the vacuolar (V) membrane (arrows). 826
P. AERUGINOSA ADHERENCE
VOL. 59, 1991
-+-
0.1
HeLa cells require expression of a number of virulence factors by B. pertussis that are under control of the vir regulatory element (6). Our study has also shown that the association of P. aeruginosa with cultured cells may require the expression of several genes under the control of a common regulatory element, RpoN. The successful colonization of a host, including attachment to and invasion of eukaryotic cells by P. aeruginosa, may also require the coordinate expression of a number of different virulence factors. Environmental modulation of the signals received by such controlling elements may be an important component of the natural host defense mechanism and may present new and effective approaches to treatment of a variety of infections caused by P. aeruginosa.
A549 Cells + PAK
-a- A549 Cells + PAK-NP
I.4 .
0.01
.0b
*;je
0.001
0.5
1
1.5
2
2.5
827
3.5
Time (hiours)
FIG. 4. Kinetics of survival of P. aeruginosa in A549 cells. Monolayers of A549 cells were incubated with P. aeruginosa PAK or PAK-NP. Gentamicin was added at the times indicated to kill adherent or extracellular bacteria, and intracellular survival was determined as described in Materials and Methods. Each point represents the mean standard error of the mean of four different experiments. ±
RpoN control is not known; however, coregulation of adhesion with pilin expression suggests that the adhesin may be responding to the same regulatory stimuli as pilin. It is known that related functions are often coregulated in bacteria. Therefore, it is possible that the synthesis of pilin and the adhesin molecule is coordinated in the bacterium to allow their interaction during pilus assembly. The entry and survival of P. aeruginosa within the A549 cells was a surprising finding, since P. aeruginosa is usually considered an extracellular parasite which generates tissue damage during colonization by producing extracellular toxic substances. There have not been any reports to date of intracellular bacteria in tissues of heavily colonized patients, such as the lungs of cystic fibrosis patients. Since only a minority of adherent bacteria are internalized, the lack of histological evidence may reflect the relatively few epithelial cells containing bacteria at any one time. Our observation that piliated P. aeruginosa is capable of surviving inside A549 cells with minimal loss of viability while the nonpiliated mutant is rapidly killed by the same cells suggests that pili may interfere with killing mechanisms following endocytosis by epithelial cells. Alternatively, the enhanced killing of the nonpiliated mutant may simply reflect the lower numbers of bacteria attached to each A549 cells. Similarly, enhanced survival of piliated PA but not of nonpiliated PAK-NP was observed when a primary cell line derived from the trachea of a fetal monkey was used in the assay (data not shown). This suggests that the ability to harbor viable bacteria intracellularly may be a property of most epithelial cells. The availability of genetically engineered isogenic mutants of P. aeruginosa with mutations in a number of extracellular and cell-associated virulence factors may allow identification of the bacterial components that play a role in invasion. Recently, Ewanowich et al. demonstrated invasion of HeLa cells by Bordetella pertussis (6) and Bordetella parapertussis (7), pathogens normally considered noninvasive. These workers also showed that optimal invasion of and survival in
ACKNOWLEDGMENTS We thank Jean Reding for technical assistance, Arnie Smith and Mark Strom for helpful discussions, and Karyn Ishimoto for construction of strains used in this study. This work was supported by an RDP grant from the Cystic Fibrosis Foundation and grant HL 30542 from NIH. D.N. is a postdoctoral fellow of the Cystic Fibrosis Foundation. S.L. holds a Research Scholar Award from the Cystic Fibrosis Foundation.
REFERENCES 1. Amador, E., and W. E. C. Wacker. 1965. Enzymatic methods used for diagnosis. Methods Biochem. Anal. 13:265-356. 2. Chi, E. Y., and W. R. Henderson. 1984. Ultrastructure of mast cell degranulation induced by eosinophil peroxidase: use of diaminobenzidine cytochemistry by scanning electron microscopy. J. Histochem. Cytochem. 32:332-341. 3. Doig, P., P. A. Sastry, R. S. Hedges, K. K. Lee, W. Paranchych, and R. T. Irvin. 1990. Inhibition of pilus-mediated adhesion of Pseudomonas aeruginosa to buccal epithelial cells by monoclonal antibodies directed against pili. Infect. Immun. 58:124-130. 4. Doig, P., T. Todd, P. A. Sastry, K. K. Lee, R. S. Hodges, W. Paranchych, and R. T. Irvin. 1988. Role of pili in adhesion of Pseudomonas aeruginosa to human respiratory epithelial cells. Infect. Immun. 56:1641-1646. 5. Doyle, R. J., J. D. Oakley, K. R. Murphy, D. McAlister, and K. G. Taylor. 1985. Graphical analyses of adherence data, p. 109-113. In S. E. Mergenhagen and B. Rosen (ed.), Molecular basis of oral microbial adhesion. American Society for Microbiology, Washington, D.C. 6. Ewanowich, C. A., A. R. Melton, A. A. Weiss, R. K. Sherburne, and M. S. Peppler. 1989. Invasion of HeLa 229 cells by virulent Bordetella pertussis. Infect. Immun. 57:2698-2704. 7. Ewanowich, C. A., R. K. Sherburne, S. F. Man, and M. S. Peppler. 1989. Bordetella parapertussis invasion of HeLa 229 cells and human respiratory epithelial cells in primary culture. Infect. Immun. 57:1240-1247. 8. Hanson, M. S., and C. C. Brinton. 1988. Identification and characterisation of the Escherichia coli type 1 pilus tip adhesin protein. Nature (London) 322:265-268. 9. Hazlett, L. D., M. Moon, M. Strejc, and R. S. Berk. 1987. Evidence for N-acetylmannosamine as an ocular receptor for Pseudomonas aeruginosa adherence to scarified cornea. Invest. Ophthalmol. Visual Sci. 28:1978-1985. 10. Ishimoto, K., and S. Lory. 1989. Formation of pilin in Pseudomonas aeruginosa requires the alternative sigma factor (RpoN) of RNA polymerase. Proc. Natl. Acad. Sci. USA 86:1954-1957. 11. Klemm, P., K. A. Krogfelt, L. Hedegaard, and G. Christiansen. 1990. The major subunit of Escherichia coli type 1 fimbriae is not required for D-mannose-specific adhesion. Mol. Microbiol. 4:553-559. 12. Krivan, H. C., V. Ginsburg, and D. D. Roberts. 1988. Pseudomonas aeruginosa and Pseudomonas cepacia isolated from cystic fibrosis patients binds specifically to gangliotetraosylceramide (asialo GM1) and gangliotriaosylceramide (asialo GM2). Arch. Biochem. Biophys. 260:493-496. 13. Lee, K. K., P. Doig, R. T. Irvin, W. Paranchych, and R. D.
828
14.
15.
16. 17.
18. 19.
CHI ET AL.
Hodges. 1989. Mapping the surface region of Pseudomonas aeruginosa PAK pilin: the importance of the C-terminal region for adherence to human buccal epithelial cells. Mol. Microbiol. 3:1493-1499. Lieber, M., B. Smith, A. Szakal, W. Nelson-Rees, and G. Tadaro. 1976. A continuous tumor cell line from human lung carcinoma with properties of type II alveolar epithelial cells. Int. J. Cancer 17:62-70. Lund, B., F. Lindberg, B.-I. Marklund, and S. Normark. 1987. The PapG protein is the alpha-D-galactopyranosyl-(1-4)-J3-Dgalactopyranose-binding adhesin of uropathogenic Escherichia coli. Proc. Natl. Acad. Sci. USA 84:5898-5902. Marcus, H., A. Austria, and N. R. Baker. 1989. Adherence of Pseudomonas aeruginosa to tracheal epithelium. Infect. Immun. 57:1050-1053. Paruchuri, D. K., H. S. Seifert, R. S. Ajioka, K.-A. Karisson, and M. So. 1990. Identification and characterization of a Neisseria gonorrhoeae gene encoding a glycolipid-binding adhesin. Proc. Natl. Acad. Sci. USA 87:333-337. Pier, G. B. 1985. Pulmonary disease associated with Pseudomonas aeruginosa in cystic fibrosis: current status of the hostbacterium interaction. J. Infect. Dis. 151:575-580. Ramphal, R., C. Guay, and G. B. Pier. 1987. Pseudomonas aeruginosa adhesins for tracheobronchial mucins. Infect. Immun. 55:600-603.
INFECT. IMMUN.
20. Ramphal, R., J. C. Sadoff, M. Pyle, and J. D. Silipingi. 1984. Role of pili in adherence of Pseudomonas aeruginosa to injured tracheal epithelium. Infect. Immun. 44:38-40. 21. Saiman, L., K. Ishimoto, S. Lory, and A. Prince. 1990. The effect of piliation and exoproduct expression on the adherence of Pseudomonas aeruginosa to respiratory epithelial monolayers. J. Infect. Dis. 161:541-548. 22. Saiman, L., J. Sadoff, and A. Prince. 1989. Cross-reactivity of Pseudomonas aeruginosa antipilin monoclonal antibodies with heterogeneous strains of P. aeruginosa and Pseudomonas cepacia. Infect. Immun. 57:2764-2770. 23. Sato, H., and K. Okinaga. 1987. Role of pili in the adherence of Pseudomonas aeruginosa to mouse epidermal cells. Infect. Immun. 55:1774-1778. 24. Scatchard, G. 1949. The attraction of proteins for small molecules and ions. Ann. N.Y. Acad. Sci. 51:660-672. 25. Uhlin, B. E., M. Norgren, M. Baga, and S. Normark. 1985. Adhesion to human cells lacking the major subunit of a digalactoside-specific pilus adhesin. Proc. Natl. Acad. Sci. USA 82: 1800-1804. 26. Woods, D. E., D. C. Strauss, W. G. Johanson, Jr., V. K. Berry, and J. A. Bass. 1980. Role of pili in adherence of Pseudomonas aeruginosa to buccal epithelial cells. Infect. Immun. 29:11461151.
