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

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 37C 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

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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.

VOL. 59, 1991

823

at 37C 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 37C. 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 37C, 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.

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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 37C 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 (37C) 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 37C. 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 37C. 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 37C.
A 1.5-ml suspension of bacteria in HBSS with supplements
was added to each monolayer, and the dishes were incubated

P. AERUGINOSA ADHERENCE

INFECT. IMMUN.

CHI ET AL.

824

--

16-

PAK
PAK-NP

14ci
01

12-

X) 10Q,
L.

8-

64-

20

0.5

2.5

1.5

Time (htours)

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

Bacteria (x 108)
80,

200-

180-

,, 60

'J 140:

01

-v.

L* 120-

oz

so-

60:

.t

10

20

30

_ 20

`4 40

B)

50

40

_100-

,;,

PAK-NP

70

PAK

;D160-

10

20

30

40

6
50 ... 60

B (bacteria per A549 cell)

70

70

80

C)

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.

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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

VOL. 59, 1991

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

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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 4C, compared with an average of 3.44
bacteria bound in the same time period at 37C. Heat
treatment of bacteria (50C 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 37C 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

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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

1.5

2.5

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.

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