INFECTION AND IMMUNITY, Mar. 2010, p. 939–953
Copyright © 2010, American Society for Microbiology. All Rights Reserved.
Vol. 78, No. 3
Pseudomonas aeruginosa-Mediated Damage Requires Distinct Receptors
at the Apical and Basolateral Surfaces of the Polarized Epithelium?†
Iwona Bucior,1,2Keith Mostov,2,3,4and Joanne N. Engel1,2,5*
Department of Medicine,1Microbial Pathogenesis and Host Defense Program,2Department of Anatomy,3Biochemistry and
Biophysics Program,4and Department of Microbiology and Immunology,5University of California,
San Francisco, California 94143
Received 26 October 2009/Returned for modification 18 November 2009/Accepted 7 December 2009
Pseudomonas aeruginosa, an important opportunistic pathogen of humans, exploits epithelial damage to
establish infection. We have rigorously explored the role of N-glycoproteins and heparan sulfate proteoglycans
(HSPGs) in P. aeruginosa-mediated attachment and subsequent downstream events at the apical (AP) and
basolateral (BL) surfaces of polarized epithelium. We demonstrate that the N-glycan chains at the AP surface
are necessary and sufficient for binding, invasion, and cytotoxicity to kidney (MDCK) and airway (Calu-3) cells
grown at various states of polarization on Transwell filters. Upregulation of N-glycosylation enhanced binding,
whereas pharmacologic inhibition of N-glycosylation or infection of MDCK cells defective in N-glycosylation
resulted in decreased binding. In contrast, at the BL surface, the HS moiety of HSPGs mediated P. aeruginosa
binding, cytotoxicity, and invasion. In incompletely polarized epithelium, HSPG abundance was increased at
the AP surface, explaining its increased susceptibility to P. aeruginosa colonization and damage. Using MDCK
cells grown as three-dimensional cysts as a model for epithelial organs, we show that P. aeruginosa specifically
colocalized with HS-rich areas at the BL membrane but with complex N-glycans at the AP surface. Finally, P.
aeruginosa bound to HS chains and N-glycans coated on plastic surfaces, showing the highest binding affinity
toward isolated HS chains. Together, these findings demonstrate that P. aeruginosa recognizes distinct recep-
tors on the AP and BL surfaces of polarized epithelium. Changes in the composition of N-glycan chains and/or
in the distribution of HSPGs may explain the enhanced susceptibility of damaged epithelium to P. aeruginosa.
Ninety-five percent of all infectious agents enter through
mucosal surfaces of the gastrointestinal, genitourinary, and
respiratory tracts (reviewed in reference 35). These mucosal
surfaces are usually lined by a single layer of epithelial cells,
which serves as the primary barrier against the entry of most
infectious agents and can be considered a primary component
of the innate immune system. Epithelial cells form highly po-
larized cell layers with apical (AP) and basolateral (BL) sur-
faces that exhibit distinct protein, lipid, and glycoconjugate
compositions. Pseudomonas aeruginosa is a ubiquitous oppor-
tunistic pathogen of humans that exploits injured mucosa to
cause acute and chronic infections with high morbidity and
mortality (reviewed in references 26 and 31). In the setting of
epithelial injury and immunocompromise, this Gram-negative
pathogen causes serious infections in patients with extensive
burns, corneal trauma, or catheter-related bladder injury or in
those on ventilators. In addition, P. aeruginosa chronically col-
onizes the lungs of patients with cystic fibrosis (CF) (4), leading
to severe pulmonary damage and death. Despite aggressive
antibiotic therapy, the fatality rate for many P. aeruginosa in-
fections is 40%, and new approaches to treatment are even
more critical now that antibiotic resistance is widespread
among P. aeruginosa isolates.
The first step in establishing P. aeruginosa infection is recep-
tor-mediated binding to the injured epithelium on the AP
and/or BL surface, leading to bacterial internalization and/or
direct host injury, as well as dissemination to distant tissues and
organs. Glycoconjugates, including glycolipids, glycosylated pro-
binding. Their long carbohydrate chains are prominently dis-
serve as receptors for many microorganisms (3). For P. aerugi-
nosa, however, conclusive in vitro or in vivo data are missing. For
example, the predilection of P. aeruginosa for injured epithelium
has been attributed to increased levels of asialo-GM1 on the AP
surface of regenerating cells (11, 23, 43, 44), though it remains
controversial whether asialo-GM1 and other glycosphingolipids
bind P. aeruginosa (13, 49). Furthermore, secreted O-glycopro-
teins, or mucins, have been associated with the binding of P.
aeruginosa to the AP surface (23, 37). N-glycosylated proteins,
in which mannose (Man), glucose (Glc), N-acetylglucosamine
(GlcN), and fucose are attached to core proteins to form
high-mannose, complex, and hybrid N-glycans, are also can-
didate receptors. For example, the N-glycoproteins CFTR
and CD95 have been shown to function as receptors for
bacterial binding and internalization (20). However, the role
of CFTR as a binding receptor for P. aeruginosa remains
In contrast to N-glycoproteins, which are present at the AP
and BL surfaces, heparan sulfate proteoglycans (HSPGs) are
preferentially expressed on the BL surface of the polarized
epithelium (3) and could serve as BL receptors for P. aerugi-
nosa. HSPGs are heterogeneous structures that are composed
of a core protein and one or more covalently attached heparan
sulfate (HS) chains. In addition to variability in the number of
* Corresponding author. Mailing address: Department of Medicine,
University of California, San Francisco, CA 94143. Phone: (415) 476-
7355. Fax: (415) 476-9364. E-mail: Jengel@medicine.ucsf.edu.
† Supplemental material for this article may be found at http://iai
?Published ahead of print on 14 December 2009.
HS repeating units and the identities of the core proteins, HS
chains are further modified by sulfation at the N, 2, 3, and/or
6 position, giving rise to enormous combinatorial diversity. The
primary HSPG families include syndecans, transmembrane
proteins located at the BL surface; perlecan and agrin, se-
creted HSPGs associated with the extracellular matrix; and
glycosylphosphatidylinositol-anchored glypicans, found at the
AP surface. HSPGs are known to mediate binding of various
bacterial and viral pathogens (2, 14, 21, 25). They have previ-
ously been postulated to modulate adhesion of P. aeruginosa to
incompletely polarized epithelial respiratory cells (41) and to
the exposed basement membrane of the mouse cornea (9), but
direct evidence for this function is lacking.
In this work, we have rigorously explored the role of N-
glycan chains of glycoproteins and HS chains of HSPGs in P.
aeruginosa infection at the AP and BL surfaces of polarized
kidney and airway epithelial cells and how changes in their
structure and/or expression affect bacterial binding and
downstream events, including internalization and cell dam-
age. Using two-dimensional (2D) and 3D cell cultures, we
show that N-glycans are necessary and sufficient for binding,
entry, and cytotoxicity at the AP surface of polarized epi-
thelium. Enhanced expression and/or expression of more
complex N-glycans, which can occur in damaged epithelium,
increases P. aeruginosa infection at the AP surface. We further
establish that HS chains of HSPGs are necessary and sufficient
to mediate binding, invasion, and cytotoxicity on the BL sur-
face in polarized cells and that sulfation is a critical determi-
nant. Finally, we show that in incompletely polarized cells, a
model of tissue injury, HSPGs are upregulated at the AP
surface, which leads to enhanced susceptibility to binding and
subsequent tissue damage by P. aeruginosa. Together, our re-
sults provide a basis for the increased susceptibility of acute or
chronically injured tissue to P. aeruginosa infections, and they
raise the possibility that well-studied molecules such as N-
glycans and HS might be useful therapeutic targets for the
treatment of P. aeruginosa infections.
MATERIALS AND METHODS
Bacterial strains and electroporation. P. aeruginosa strain K (PAK; obtained
from J. Mattick, University of Queensland, Brisbane, Australia) was routinely
grown with shaking overnight in Luria-Bertani broth (LB broth) at 37°C. To
create a plasmid which constitutively produces green fluorescent protein (GFP),
the pnpT2-GFP fragment from p519ngfg (33) was excised with HindIII and
ExoRI and cloned into the corresponding sites of pUCP20 to yield pnpT2-GFP-
pUCP20. This plasmid was introduced into PAK by electroporation with a
Bio-Rad Gene Pulser II, with settings of 1.6 V, 25 ?F, and 200 ?. The resulting
strain was named PAK-GFP.
2D and 3D cell culture. MDCK clone II and ConArMDCK cells obtained
from Keith Mostov (University of California, San Francisco, CA) were main-
tained in minimal essential medium (MEM) (51) supplemented with 5% fetal
bovine serum (FBS; Invitrogen) at 37°C with 5% CO2. Calu-3 cells were obtained
from the ATCC (Rockville, MD) and maintained in MEM supplemented with
10% FBS and L-glutamate at 37°C with 5% CO2. Cells were grown as 2D
monolayers on 12-mm Transwell filters (3-?m pore size; Corning Incorporated).
For all experiments, cells were plated under conditions such that they formed
confluent monolayers that exhibited basic features of early polarized cells, in-
cluding polarized distribution of some AP and BL membrane proteins and
functional tight junctions that were impermeable to small molecules such as
fluorescein isothiocyanate-inulin (FITC-inulin). Cells were grown for different
lengths of time to model different states of polarization. For incompletely po-
larized confluent monolayers, MDCK cells were seeded at 0.7 ? 106cells/well
and cultured for 24 h. For well-polarized confluent monolayers, MDCK cells
were seeded at 0.3 ? 106cells/well and cultured for 5 days. For incompletely
polarized confluent Calu-3 cell monolayers, Calu-3 cells were seeded at 1.5 ? 106
cells/well and cultured for 2 days. For well-polarized confluent Calu-3 cell mono-
layers, Calu-3 cells were seeded at 1 ? 106cells/well and cultured for 7 days.
For some experiments, MDCK or Calu-3 cells grown as 2D cultures on Trans-
well filters were treated with the following reagents. None of these treatments
inhibited bacterial growth (data not shown). To inhibit N-glycosylation, cells
were pretreated with 0.1 to 1 ?g/ml of tunicamycin (Sigma-Aldrich) for 16 h in
MEM supplemented with 5% FBS (MDCK and ConArcells) or 10% FBS
(Calu-3 cells). For competition blocking with glycosaminoglycans, cells were
pretreated with 0.1 to 10 ?g/ml of heparin or chondroitin sulfate (Sigma-Aldrich)
at 37°C for 1 h in serum-free MEM. To remove proteoglycans, cells were treated
with 1 to 200 mU of heparinase III (Sigma-Aldrich) or chondroitinase ABC
(Sigma-Aldrich) in Hank’s buffered salt solution (HBSS) containing 0.1% bovine
serum albumin (BSA) at 37°C for 2 h. Proteoglycan desulfation was performed
by overnight incubation of cells with 10 mM sodium chlorate in MEM containing
5% FBS (MDCK and ConArcells) or 10% FBS (Calu-3 cells). Resulfation was
accomplished by adding 1 mM sodium sulfate to sodium chlorate-containing
medium. Upregulation of N-glycosylation was performed as described previously
(46), with the following modifications. Cells were grown in the presence of 1 mM
Man, Glc, or Gal (Sigma-Aldrich) in MEM with 5% (MDCK and ConArcells)
or 10% (Calu-3 cells) FBS for 1 week (long upregulation of N-glycosylation) or
1 day (brief upregulation of N-glycosylation). Cells were stained with FITC-
concanavalin A (FITC-ConA; Sigma Aldrich) to assess cell surface N-glycosyla-
tion. Cell surface proteoglycans were visualized by immunofluorescence staining
with HS antibody (10E4; Seikagaku) or with FITC-WFA (a chondroitin sulfate-
specific lectin from Wisteria floribunda; Sigma Aldrich).
For 3D cell culture, MDCK or ConArMDCK cells were grown as cysts in
Matrigel-collagen as previously described (39, 52), with the following modifica-
tions. A single cell suspension of 4 ? 104cells/ml was added to a solution of
buffered, liquefied collagen (3 mg/ml). Two hundred fifty microliters of cells in
collagen I was plated in 8-well cover-glass chambers (Nalge Nunc International)
that had previously been covered with 100% Matrigel. The Matrigel served as a
solid base to which the cysts attached prior to collagen solidification. The colla-
gen was allowed to solidify into a gel by incubation at 37°C prior to the addition
of MEM containing 10% FBS. Over the 5-day growth period, individual cells in
the collagen matrix proliferated to form cysts, with the AP membrane facing the
lumen and the BL membrane facing the surrounding collagen (BL-side-out
cysts). For some experiments, the anti-beta-1 antibody AIIB2 (1:100 dilution in
the collagen I solution and/or in MEM; a kind gift of Caroline Damsky at the
University of California, San Francisco) was included for the entire culture
period. Under these conditions, polarized AP-side-out cysts were formed, al-
though the structures were less well organized than those in the BL-side-out cysts
(39). For bacterial infections, MDCK cysts grown in Matrigel-collagen were
treated with collagenase type VII (Sigma-Aldrich) at 100 U/ml in phosphate-
buffered saline (PBS) for 15 min at 37°C to digest the collagen gel. For some
experiments, cysts were treated with heparinase III in HBSS containing 0.1%
BSA at 37°C for 3 h, washed, and resuspended in serum-free MEM for bacterial
Bacterial adhesion and invasion assays on 2D monolayers. PAK cells grown
overnight in LB broth to stationary phase were diluted in 50 ?l of serum-free
MEM and added to cells at a multiplicity of infection (MOI) of 20. To infect the
AP side of 2D monolayers grown on Transwell filters, the bacteria were added to
the AP chamber. For BL infections, the Transwell insert was placed directly onto
50 ?l of serum-free MEM containing PAK. After 1 h of infection at 37°C,
adhesion and invasion assays were performed as described previously (27).
Briefly, for the adhesion assay, cells were washed in PBS to remove nonadherent
bacteria and were lysed in 1 ml Ca2?- and Mg2?-free PBS with 0.25% Triton
X-100 (Sigma-Aldrich) for 30 min. For invasion assays, cells were first incubated,
before lysis, in serum-free MEM containing 0.4 mg/ml amikacin (Fisher Scien-
tific) for 2 h. After lysis, cells were removed from the Transwell filters by gentle
scraping. Bacteria were enumerated by plating serial dilutions of cell lysates on
LB plates and counting the CFU. All assays were carried out on triplicate wells,
and results are reported as averages for three or four experiments. Because of
well-established variability in adhesion and invasion assays, data were normalized
to 100% of bacterial adhesion or internalization at the AP surface of cultured
epithelial cells to allow comparison between experiments performed on different
days. Consistent with previous reports, approximately 10% of the PAK inoculum
bound to cells, whereas 1 to 2% of the inoculum was internalized (29).
Bacterial infection of 3D cysts. Prior to bacterial infections, cysts were briefly
treated with type VII collagenase (100 U/ml in PBS for 15 min at 37°C), which
removed the thin layer of collagen that coated the cysts and allowed the bacteria
to access the surface of the cysts. GFP-expressing PAK cells (107) suspended in
serum-free MEM were incubated with the cysts for 2 h. Cells were washed with
940BUCIOR ET AL.INFECT. IMMUN.
PBS to remove nonbound bacteria and fixed in PBS containing 1% paraformal-
dehyde at 37°C for 0.5 h. Bacterial adherence and colocalization with specific
surface markers are described below.
Immunofluorescence microscopy and image analysis. HS chains were stained
with anti-heparan sulfate antibody (10E4), tight junctions were stained with
anti-ZO-1 (R40.76) antibody (a gift from B. Stevenson, University of Edmon-
ton, Alberta, Canada), actin filaments were stained with AlexaFluor594-
phalloidin (Invitrogen), and mannose residues were stained with FITC-ConA. Al-
exaFluor488- or 647-conjugated secondary antibodies were obtained from
Invitrogen. Monolayers grown on Transwell filters were fixed in PBS containing
1% paraformaldehyde at 37°C for 0.5 h. After being washed, cells were incubated
with primary antibodies overnight at 4°C and, afterwards, with fluorescent sec-
ondary antibodies for 2 h at room temperature. Filters were excised and mounted
on microscope slides (Fisher Scientific) in mounting medium (Vector Labora-
tories, Inc.). Cysts grown in eight-well cover-glass chambers were fixed and
stained with antibodies directly in chambers. Cysts were incubated with primary
antibodies overnight at 4°C and with secondary antibodies for 2 h at room
Samples were examined with a confocal microscope (LSM 510; Carl Zeiss
MicroImaging, Inc.). Images and 3D reconstructions were acquired by and pro-
cessed in Meta 510 software. Image J analysis was performed on TIFF files.
Bacterial binding to 3D cysts and colocalization with surface markers were
quantified using the Image J plug-in Voxel Counter on 3D reconstructions of
TIFF images acquired with Meta 510 software. Voxel Counter (ImageJ plug-in)
was used to quantify the volume of bound 3D bacterial aggregates, and a min-
imum volume was set as a threshold to enable automated cell counting using the
3D Object Counter (ImageJ plug-in). Any bacterial aggregate above the thresh-
old was counted as 1. The surface area of membrane regions either enriched with
or depleted of HS or N-glycans (determined by staining with an anti-HS antibody
or with FITC-ConA, respectively) was measured in pixels by ImageJ, and the
number of bacterial aggregates bound was normalized per pixel of each specific
surface area. The percentage (compared to the total) of bacterial aggregates
bound to each specific region was determined. For each treatment, 40 to 50 cysts
In vitro binding assay. Sugars, N-glycans, and glycosaminoglycans (0.1 to 10 ?g
in 0.2 ml double-distilled water [ddH2O]; Sigma Aldrich) were added to 96-well
plastic plates (Falcon, Becton Dickinson Labware) and incubated overnight at
37°C until evaporated. Wells were washed with ddH2O and blocked in 0.1% BSA
for 0.5 h at room temperature. Bound glycans were stained with 1% toluidine
blue (Sigma Aldrich), and absorbance was measured at 630 nm. The absorbance
values for known concentrations of glycans were used as the standard curve, and
the concentration of bound glycans (?g/well) was calculated. Stationary-phase
PAK-GFP cells (107in 0.1 ml ddH2O) were added to wells and incubated for 2 h
at room temperature. Nonadherent bacteria were removed by washing with
ddH2O. Bound PAK-GFP was quantified using a SpectraMax 340PC plate
reader using SOFTmaxPro software (Molecular Devices) at an excitation wave-
length (?ex) of 480 nm and an emission wavelength (?em) of 530 nm. PAK-GFP
bound to noncoated wells was used as a control and subtracted out as back-
ground. All assays were carried out on triplicate wells, and results are reported
as averages for four experiments.
Cytotoxicity assay. Epithelial cell cytotoxicity was quantified by colorimetric
quantification of lactate dehydrogenase (LDH) release, using a commercially
available kit (CytoTox 96 nonradioactive cytotoxicity assay; Promega) according
to the manufacturer’s instructions and as previously described (17), with the
following modifications. For both AP and BL infections, Transwell filters were
placed on 50-?l drops of medium only (for AP infections) or medium plus
bacteria (for BL infections). Transwell-grown cells were infected with PAK
(MOI of 20) for 5 h at the AP or BL surface, and 50 ?l of the supernatant was
collected from the AP chamber every 1 h. The absorbance of the sample was
measured at 490 nm on a SpectraMax 340PC plate reader using SOFTmaxPro
software. Results were normalized to the 100% LDH release in infected cells.
Assays were carried out on triplicate wells, and the results are reported as
averages for three or four experiments.
Transepithelial permeability. Transwell-grown cells were infected with PAK
for 5 h at the AP surface. Nonadherent bacteria were removed by washing with
PBS. FITC-inulin (Sigma-Aldrich; 100 ?g/ml) in PBS was added to the AP
chamber, and PBS alone was added to the BL chamber of Transwell plates. Cells
were incubated at 37°C, and 100-?l samples were collected from the BL chamber
every 0.5 h. Fluorescence was quantified using a fluorescence plate reader (?ex ?
480 nm and ?em ? 530 nm; SpectraMax 340PC plate reader using SOFTmaxPro
software). Known dilutions of FITC-inulin were used as a standard curve.
Statistical analysis. Data are expressed as means ? standard deviations (SD).
Statistical significance was estimated by paired Student’s t test. Differences were
considered to be significant at P values of ?0.05.
Modulations of specific enzymes in N-glycosylation pathway
affect the expression of N-glycan chains on the cell surface.
Previous studies have shown that P. aeruginosa cytotoxicity is
diminished in filter-grown confluent monolayers of concanava-
lin A-resistant Madin-Darby canine kidney (ConArMDCK)
cells compared to that for wild-type (wt) MDCK cells (1).
While the exact defect in the glycosylation pathway in ConAr
MDCK cells is not known, there are alterations in the Man
core of its N-linked carbohydrates, which impair the formation
of more complex N-linked carbohydrate structures (34). We
hypothesized that surface-exposed complex N-glycans are im-
portant determinants of P. aeruginosa adhesion to an injured
epithelium, thus explaining the reduced susceptibility of ConAr
MDCK cells to P. aeruginosa-mediated damage. Others have
suggested that P. aeruginosa binding and entry into ConAr
MDCK cells are diminished because these cells form hyper-
polarized monolayers (15).
To test our hypothesis, we utilized MDCK or human airway
epithelial (Calu-3) cells grown as confluent monolayers on
Transwell filters for various lengths of time. We have shown
that under these conditions, MDCK cells form functional tight
and adherens junctions within 24 h and show a polarized dis-
tribution of many AP and BL markers, such as gp135 and gp58
(28) (see Fig. S1 in the supplemental material). With increas-
ing time in culture, they exhibit an enhanced polarized distri-
bution of markers, such as the predominantly BL surface-
expressed epidermal growth factor (EGFR) (see Fig. S1 in the
supplemental material). We confirmed that cells grown for 1
day (MDCK cells) or 2 days (Calu-3 cells) formed confluent
monolayers with functional tight and adherens junctions, as
evidenced by impermeability to apically applied FITC-inulin
(see Fig. S1 in the supplemental material), a low-molecular-
weight molecule that is not able to diffuse through functional
tight junctions (1). Epithelial polarity continued to increase at
later times in culture (5 days for MDCK cells and 7 days for
Calu-3 cells), as evidenced by an increased polarized distribu-
tion of AP and BL markers, such as HSPGs and EGFR (see
Fig. 2 and Fig. S1 in the supplemental material). For ease of
nomenclature, we refer to cells cultured for 1 day (MDCK
cells) or 2 days (Calu-3 cells) as “incompletely polarized”
monolayers and those cultured for 5 days (MDCK cells) or 7
days (Calu-3 cells) as “well-polarized” monolayers. We empha-
size that under the conditions of our experiments, the term
“incompletely polarized monolayers” describes confluent
monolayers with functional tight and adherens junctions but
with an incompletely polarized distribution of some AP and BL
We qualitatively and quantitatively assessed the amount of
surface-exposed Man in N-glycan chains in wt MDCK, ConAr
MDCK, and Calu-3 cells at various states of polarization by
confocal microscopy and fluorescence plate assays using FITC-
conjugated ConA, a lectin that specifically recognizes nonre-
ducing terminal ?-D-glucosyl and ?-D-mannosyl groups. The
results obtained with incompletely polarized cells (see Fig. S2A
VOL. 78, 2010 DISTINCT RECEPTORS MEDIATE P. AERUGINOSA BINDING 941
in the supplemental material) were similar to those seen for
well-polarized cells (Fig. 1). FITC-ConA bound more effi-
ciently to well-polarized wt MDCK cells than to ConArMDCK
cells at both the AP and BL surfaces (Fig. 1A and B). FITC-
ConA binding to the AP and BL surfaces of Calu-3 cells was of
the same intensity as binding to wt MDCK cells. We used Man
or Glc (a Man precursor) supplementation to complement the
defect in ConArMDCK cells or to augment N-glycosylation in
wt MDCK and Calu-3 cells. Addition of these sugars is known
to upregulate the activities of phosphomannomutase (PMM)
and phosphomannose isomerase (PMI), enzymes that play a
critical role in maintaining the supply of D-Man derivatives
required for N-glycosylation (46). Man or Glc supplementation
restored the binding of FITC-ConA to ConArMDCK cells
(Fig. 1A and B). Increased binding of FITC-ConA to Man
residues on N-glycan chains was observed after only 5 to 6 days
of treatment with exogenous Man, eliminating the possibility
that the exogenous Man simply adhered to the cell surface and
facilitated the binding of the lectin. Addition of galactose
(Gal), which is not involved in Man synthesis, did not restore
binding of FITC-ConA. Moreover, binding of FITC-ConA was
enhanced for wt MDCK and Calu-3 cells cultured with Man or
Glc (but not Gal) compared to that with untreated cells. We
verified that MDCK and Calu-3 cells with normal or enhanced
levels of Man were sensitive to killing by ConA added to the
growth medium (see Fig. S3 in the supplemental material).
ConArMDCK cells grown in the presence of Man or Glc (but
not Gal) lost their normal resistance to the lectin and showed
enhanced sensitivity to ConA (see Fig. S3 in the supplemental
Several conclusions that are important for our subsequent
work can be drawn from these experiments. First, well-polar-
ized and incompletely polarized ConArMDCK cells have de-
creased levels of surface-exposed Man, and thus N-linked gly-
cans, on both the AP and BL surfaces. Second, Man or Glc
supplementation is able to restore the cell surface expression
of N-glycans in these cells. It is also sufficient to increase the
expression of more complex N-glycans on the AP and BL
surfaces of wt MDCK and Calu-3 cells. Third, and finally, the
surface presentation of N-glycans does not change as cells
become more polarized (compare Fig. 1B to Fig. S2A in the
The expression level and structure of N-glycan chains mod-
ulate P. aeruginosa binding at the AP surface of well-polarized
and incompletely polarized epithelia. To address the hypoth-
esis that modifications in the expression and structure of N-
glycan chains alter interactions of P. aeruginosa with the host
epithelium, we assayed the effects of various modifications in
N-glycosylation on bacterial adhesion to host epithelial cells.
MDCK, ConArMDCK, and Calu-3 cells were grown on Trans-
well filters as well-polarized or incompletely polarized mono-
layers, with or without sugar supplementation. Augmentation
of N-glycosylation by addition of Man or Glc (but not Gal) to
ConArMDCK cells increased bacterial binding nearly three-
fold, to at least wt levels, at the AP surface of well-polarized
cells (Fig. 1C). Likewise, addition of Man or Glc (but not Gal)
to wt MDCK cells (Fig. 1C) or Calu-3 cells (Fig. 1D) was
sufficient to increase bacterial binding at the AP surface. Sim-
ilar results were observed in incompletely polarized cells (see
Fig. S2B and C in the supplemental material). None of these
treatments affected bacterial binding at the BL surface of
ConAror wt MDCK cells (Fig. 1C) or Calu-3 cells (Fig. 1D),
eliminating the possibility that these treatments nonspecifically
affected bacterial binding.
We next pretreated cells with tunicamycin, a well-character-
ized inhibitor of GlcN phosphotransferase, the enzyme that
catalyzes the first step of N-glycoprotein synthesis. In control
experiments, we determined that tunicamycin treatment re-
duced the amounts of N-glycans on the AP and BL cell sur-
faces of well-polarized and incompletely polarized wt MDCK
and Calu-3 cells, to levels roughly similar to those for un-
treated ConArMDCK cells (see Fig. S4 in the supplemental
material). As expected, tunicamycin treatment had minimal
effect on FITC-ConA binding to ConArMDCK cells, which
already express fewer N-glycans on their surfaces. Tunicamycin
treatment of well-polarized wt MDCK or Calu-3 cells de-
creased P. aeruginosa adhesion to the AP surface up to twofold
(Fig. 1E and F), in a dose-dependent manner, indicating that
N-glycans may function as AP binding receptors. Drug treat-
ment did not further decrease bacterial binding to the AP
surface of ConArMDCK cells (Fig. 1E), which was already
decreased approximately twofold compared to that for wt cells,
strengthening our conclusion that N-glycosylation is defective
in these cells. P. aeruginosa adhesion to the BL surface was not
affected in tunicamycin-treated wt or ConArMDCK cells (Fig.
1E) or in tunicamycin-treated Calu-3 cells (Fig. 1F). Similar
results were observed in incompletely polarized cells (see Fig.
S2D and E in the supplemental material).
Consistent with previously published results (15, 28), we
found that P. aeruginosa bound more efficiently to the AP
surface of incompletely polarized cells than to the AP surface
of well-polarized cells (see Fig. S5 in the supplemental mate-
rial). We determined if alteration in the expression of N-gly-
cans in incompletely polarized cells is responsible for the in-
creased binding of P. aeruginosa to the AP surface. Bacterial
binding to the AP surface of incompletely polarized cells was
twofold higher than binding to well-polarized kidney and lung
cells (see Fig. S6A in the supplemental material). Treatment
with tunicamycin did not change the binding ratio. These re-
sults establish that N-glycans are important determinants of P.
aeruginosa binding to the AP surface of polarized epithelium.
However, enhanced bacterial binding to the AP surface of
incompletely polarized cells is N-glycan independent; other-
wise, tunicamycin treatment would have abrogated the in-
creased binding. Together, these results firmly establish that an
increase in the abundance and/or length and complexity of
N-glycan chains enhances P. aeruginosa binding at the AP
surface independent of the state of epithelial polarization.
HSPGs are expressed preferentially at the BL surface of
well-polarized epithelium but on both surfaces of incompletely
polarized epithelium. In contrast to N-glycoproteins, which are
present at the AP and BL surfaces, HSPGs are preferentially
expressed on the BL surface of polarized epithelium (3). We
considered the possibilities that (i) HS chains of HSPGs mod-
ulate P. aeruginosa attachment to the BL surface of mucosal
surfaces and/or (ii) enhanced AP expression of HS explains the
increased binding of P. aeruginosa to the AP surface of incom-
pletely polarized epithelium, since the increase is independent
We first assessed expression of HS qualitatively and quanti-
942BUCIOR ET AL.INFECT. IMMUN.
FIG. 1. P. aeruginosa adhesion to the AP surface is N-glycan dependent. (A and B) Addition of Man or Glc, but not Gal, to the growth medium
increases the level of Man on the cell surface. MDCK, ConAr, and Calu-3 cells were cultured in the presence or absence of the indicated sugar.
Shown are well-polarized monolayers grown on Transwells that were fixed without permeabilization and stained at either the AP or BL surface
with FITC-ConA, which binds to Man in N-glycan chains. ConArcells have less Man on the AP and BL surfaces than do wt MDCK cells, but the
addition of Man or Glc restores FITC-ConA binding to wt levels. The addition of Man or Glc enhances FITC-ConA binding to MDCK and Calu-3
cells at both the AP and BL surfaces. (A) 3D reconstructions of z-stack images acquired by confocal microscopy. Shown are AP and BL projections
of well-polarized monolayers. Man is stained with FITC-ConA (green), and actin is stained with phalloidin (red). (B) FITC-ConA fluorescence
(in arbitrary units) of well-polarized cells treated with the indicated sugars, as measured in a fluorescence plate reader. Shown are the means ?
SD for three separate experiments.*, P ? 0.05 compared to AP-side-untreated MDCK cells; ?, P ? 0.05 compared to AP-side-untreated ConAr
cells; ?, P ? 0.05 compared to AP-side-untreated Calu-3 cells;**, P ? 0.05 compared to BL-side-untreated MDCK cells; ??, P ? 0.05 compared
to BL-side-untreated ConArcells; ??, P ? 0.05 compared to BL-side-untreated Calu-3 cells. (C and D) Addition of Man or Glc is sufficient to
enhance P. aeruginosa binding. Cells were grown in the presence or absence of the indicated sugar, bacteria were added for 1 h to the AP or BL
surface of well-polarized cells grown on Transwell filters, and standard adhesion assays were performed. Shown are the means ? SD for three
separate experiments. (C) P. aeruginosa binding to well-polarized MDCK and ConArcells, normalized to AP-side-infected, untreated MDCK cells.
(D) P. aeruginosa binding to well-polarized Calu-3 cells, normalized to AP-side-infected, untreated Calu-3 cells. (E and F) Tunicamycin inhibits
bacterial binding to the AP surface of wt MDCK and Calu-3 cells in a dose-dependent manner. Cells were pretreated with the indicated amounts
of tunicamycin to inhibit N-glycosylation. Bacterial binding was measured as described above. (E) P. aeruginosa binding to well-polarized MDCK
and ConArcells, normalized to AP-side-infected, untreated MDCK cells (point “0”). (F) P. aeruginosa binding to well-polarized Calu-3 cells,
normalized to AP-side-infected, untreated Calu-3 cells (point “0”).*, P ? 0.05 compared to AP-side-infected, untreated MDCK cells; ?, P ? 0.05
compared to AP-side-infected, untreated ConArcells; ?, P ? 0.05 compared to AP-side-infected, untreated Calu-3 cells.
VOL. 78, 2010 DISTINCT RECEPTORS MEDIATE P. AERUGINOSA BINDING 943
tatively at the AP and BL surfaces of MDCK cells and Calu-3
cells at various stages of polarization, using a commercially
available antibody that recognizes the HS moiety of HSPGs
independent of the identity of the core protein. Confocal mi-
croscopy revealed that the HS antibody preferentially bound to
the BL surface of well-polarized Calu-3 cells (Fig. 2A) as well
as to the BL surface of well-polarized wt MDCK and ConAr
MDCK cells (see Fig. S1C and D in supplemental material). In
contrast, the HS antibody bound approximately equally to the
AP and BL surfaces of incompletely polarized cells (Fig. 2A).
Both polarization models had functional tight junctions, as evi-
denced by ZO-1 staining (Fig. 2A) and by the lack of permeability
to apically applied FITC-inulin (data not shown). Quantification
of bound FITC-conjugated antibody confirmed that HSPGs were
largely restricted to the BL surface of well-polarized wt MDCK,
ConArMDCK, and Calu-3 cells (Fig. 2B), whereas in incom-
pletely polarized cells, there were approximately equivalent levels
at both the AP and BL surfaces (Fig. 2C). Together, the experi-
ments demonstrate that HS chains of HSPGs fulfill the charac-
teristics predicted for a BL receptor in multiple cell lines. There
is preferential presentation of HSPGs at the BL surface of fully
polarized cells, while there is increased presentation of HSPGs at
the AP surface of incompletely polarized cells.
HS chains of HSPGs contribute to P. aeruginosa binding at
the BL surface of well-polarized epithelium and at both sur-
faces of incompletely polarized epithelium. We utilized com-
prehensive approaches to determine the role of HSPGs in
bacterial adherence to the AP and BL surfaces of well-polar-
ized and incompletely polarized epithelial monolayers. First,
we added excess heparin to block the interaction between P.
aeruginosa and HS chains on the cell surface. In well-polarized
epithelium, the addition of heparin inhibited bacterial binding
in a dose-dependent manner at the BL surface of wt MDCK,
ConArMDCK, and Calu3 cells (Fig. 3A and C). At 10 ?g/ml
heparin, binding was decreased ?50% compared to that in
cells without the addition of heparin. As expected, exogenous
addition of heparin had minimal or no effect on P. aeruginosa
binding at the AP surface of well-polarized cells (Fig. 3A and
C). The results were distinctly different for incompletely po-
larized cells. Addition of heparin reduced bacterial adhesion
50% at both the AP and BL surfaces of incompletely polarized
wt MDCK (Fig. 3B) and Calu-3 (Fig. 3D) cells. Notably, hep-
arin also decreased bacterial attachment to the AP surface of
incompletely polarized ConArMDCK cells (Fig. 3B). To rule
out nonspecific charge effects, we demonstrated that the addi-
tion of another highly negatively charged glycosaminoglycan
chain, chondroitin sulfate (CS), had no effect on bacterial
binding to either surface in any of these cell types (see Fig. S7A
to D in the supplemental material).
We further examined the role of HS chains in mediating the
interaction between P. aeruginosa and HSPGs in well-polarized
and incompletely polarized cells. Pretreatment of cells with
heparinase III, an enzyme that cleaves HS chains, reduced
bacterial adhesion to the BL surface of well-polarized wt
MDCK, ConArMDCK, and Calu3 cells in a dose-dependent
manner, with reductions of up to 50% at 200 mU (Fig. 3E and
G). A minimal effect was observed on bacterial binding at the
AP surface. In incompletely polarized cells, heparinase III
treatment decreased bacterial attachment to both the AP and
BL surfaces of all three cell lines (Fig. 3F and H). Enzymatic
removal of CS by chondroitinase ABC did not affect bacterial
attachment, confirming the specific role of HS in mediating in-
teractions with P. aeruginosa (see Fig. S7E to H in the supple-
mental material). We qualitatively and quantitatively confirmed
FIG. 2. HSPGs are expressed on the AP surface in incompletely
polarized cells. In well-polarized monolayers grown on Transwell fil-
ters, there was a greater amount of HSPGs on the BL surface than on
the AP surface. In incompletely polarized monolayers, there was in-
creased expression of HSPGs on the AP surface. (A) 3D reconstruc-
tion of z-stack images acquired by confocal microscopy. Shown are AP
and BL projections of well-polarized and incompletely polarized
Calu-3 cells stained with an antibody against the HS chain of HSPGs
(FITC-HS; purple), with phalloidin (red) to visualize actin, and with
ZO-1 (green) to visualize tight junctions. (B and C) FITC-HS fluores-
cence (in arbitrary units) measured in a fluorescence plate reader.
Well-polarized (B) or incompletely polarized (C) MDCK, ConAr, and
Calu-3 cells were stained with FITC-HS added to the AP or BL
surface. Shown are the means ? SD for three separate experiments.*,
P ? 0.05 compared to AP surface-stained MDCK cells; ?, P ? 0.05
compared to AP surface-stained ConArcells; ?, P ? 0.05 compared to
AP surface-stained Calu-3 cells.
944BUCIOR ET AL.INFECT. IMMUN.
the specificity of enzymatic treatment with heparinase III by
showing decreased HS antibody staining at the BL surface of
well-polarized wt MDCK, ConArMDCK, and Calu-3 cells and at
the AP and BL surfaces of incompletely polarized cells (see Fig.
S8 in the supplemental material). Likewise, the efficacy of chon-
droitinase ABC treatment was confirmed by staining with FITC-
conjugated Wisteria floribunda agglutinin (WFA), a lectin that
binds to the N-acetylgalactosamine moiety in CS (data not
shown). Treatment with chondroitinase ABC did not attenuate
the staining of the HS antibody (data not shown).
The sulfate moieties on HS chains are major contributors to
their net negative charge and may provide the basis for ionic
FIG. 3. HS chains mediate P. aeruginosa adhesion to the BL surface and contribute to binding to the AP surface in incompletely polarized cells.
Competitive inhibition with heparin (A to D) or enzymatic removal of HS chains with heparinase III (E to H) inhibits P. aeruginosa binding to
the BL surface of well-polarized and incompletely polarized cells and to the AP surface of incompletely polarized cells. Cells were pretreated with
the indicated amount of heparin or heparinase III, bacteria were added for 1 h, and standard adhesion assays were performed. Shown are the
means ? SD for three separate experiments. P. aeruginosa binding to well-polarized (A) and incompletely polarized (B) MDCK and ConArcells
pretreated with increasing amounts of heparin was determined and normalized to that of AP-side-infected, untreated MDCK cells. P. aeruginosa
binding to well-polarized (C) and incompletely polarized (D) Calu-3 cells pretreated with increasing amounts of heparin was determined and
normalized to that of AP-side-infected, untreated Calu-3 cells. P. aeruginosa binding to well-polarized (E) and incompletely polarized (F) MDCK
and ConArcells pretreated with increasing doses of heparinase III was determined and normalized to that of AP-side-infected, untreated MDCK
cells. P. aeruginosa binding to well-polarized (G) and incompletely polarized (H) Calu-3 cells pretreated with increasing doses of heparinase III
was determined and normalized to that of AP-side-infected, untreated Calu-3 cells.*, P ? 0.05 compared to AP-side-infected, untreated MDCK
cells; ?, P ? 0.05 compared to AP-side-infected, untreated ConArcells; ?, P ? 0.05 compared to AP-side-infected, untreated Calu-3 cells;**,
P ? 0.05 compared to BL-side-infected, untreated MDCK cells; ??, P ? 0.05 compared to BL-side-infected, untreated ConArcells; ??, P ?
0.05 compared to BL-side-infected, untreated Calu-3 cells.
VOL. 78, 2010DISTINCT RECEPTORS MEDIATE P. AERUGINOSA BINDING 945
forces that underlie noncovalent interactions between HSPGs
and P. aeruginosa. To study the role of sulfation in bacterial
binding, we pretreated wt MDCK, ConArMDCK, and Calu-3
cells with sodium chlorate, an inhibitor of sulfate adenyltrans-
ferase and HS sulfation. As shown in Fig. 4, chemical desulfa-
tion of HS reduced bacterial binding over twofold at the BL
surface of well-polarized cells and at both surfaces in incom-
pletely polarized cells. Resulfation by addition of sodium sul-
fate to chlorate-treated cells (53) restored bacterial binding to
well-polarized and incompletely polarized epithelia. The effi-
ciencies of desulfation and resulfation were monitored by
staining with the anti-HS antibody (data not shown).
In summary, our results suggest that HS chains with intact
sulfate groups play an important role in binding of P. aerugi-
nosa to the BL surface of the polarized epithelium. Impor-
tantly, all three treatments that affected HSPGs (addition of
excess exogenous heparin, removal of HS chains by digestion
with heparinase III, and removal of sulfate residues by chem-
ical desulfation) reduced bacterial binding to the AP surface of
incompletely polarized cells approximately two-fold for all
three cell lines compared to that for well-polarized cells (see
Fig. S6B in the supplemental material). We concluded that the
increased presence of HSPGs on the AP surface of incom-
pletely polarized cells accounts at least in part for their in-
creased susceptibility to P. aeruginosa infection.
P. aeruginosa binds directly to isolated HS and N-glycan
chains in vitro. If N-glycans and HS chains serve as receptors
for P. aeruginosa binding, then it should be possible to dem-
onstrate direct binding in vitro. We established an in vitro
binding affinity assay where informative glycosylated molecules
were used to coat 96-well plastic plates at different concen-
trations, GFP-conjugated bacteria were then added to
coated plates, and fluorescence was measured after 1 h. As
shown in Fig. 5, P. aeruginosa bound in a dose-dependent
manner to HS or to a complex hybrid N-glycan chain [(Gal-
GlcN)4Man3(GlcN)2], with the strongest binding to HS.
Minimal or no binding was observed with other glycosamino-
glycans, such as alternatively sulfated CS chains or nonsul-
fated hyaluronic acid (HA). These results suggest that (i) P.
aeruginosa binds to specific sulfated sequences in HS chains
FIG. 4. P. aeruginosa binding to HS is dependent on sulfation of HS chains. Chemical inhibition of sulfation inhibits P. aeruginosa binding to
the BL surface of well-polarized and incompletely polarized cells and to the AP surface of incompletely polarized cells. Cells were pretreated with
sodium chlorate for desulfation (desulf) of HS chains, and sulfation was restored (resulf) by treatment with sodium sulfate. Bacteria were added
for 1 h, and standard adhesion assays were performed. Shown are the means ? SD for three separate experiments. P. aeruginosa binding to
well-polarized (A) and incompletely polarized (B) MDCK and ConArcells was determined and normalized to that of AP-side-infected, untreated
MDCK cells. P. aeruginosa binding to well-polarized (C) and incompletely polarized (D) Calu-3 cells was determined and normalized to
AP-side-infected, untreated Calu-3 cells.*, P ? 0.05 compared to AP-side-infected, untreated MDCK cells; ?, P ? 0.05 compared to AP-side-
infected, untreated ConArcells; ?, P ? 0.05 compared to AP-side-infected, untreated Calu-3 cells;**, P ? 0.05 compared to BL-side-infected,
untreated MDCK cells; ??, P ? 0.05 compared to BL-side-infected, untreated ConArcells; ??, P ? 0.05 compared to BL-side-infected,
untreated Calu-3 cells.
FIG. 5. P. aeruginosa directly binds in vitro to isolated HS and
N-glycan chains in a dose-dependent manner. Ninety-six-well plastic
plates were coated overnight with increasing concentrations of the
indicated molecules. GFP-conjugated bacteria were added for 1 h, and
fluorescence was quantified in a fluorescence plate reader. Control
bacterial binding to noncoated wells was set as a background, and the
percentage of binding above that of the control is indicated. Shown are
means ? SD for four separate experiments. HS, heparan sulfate; CS-4,
4-0-sulfated chondroitin sulfate; CS-6, 6-0-sulfated chondroitin sulfate;
HA, hyaluronic acid; GlcN, N-acetylglucosamine.
946BUCIOR ET AL.INFECT. IMMUN.
and (ii) the anionic charge provided by sulfate groups is
necessary for the interaction. Moreover, no binding was
observed for wells coated with GlcN, which is one of the
sugar residues present in N-glycan chains. Our findings sug-
gest that single sugars are not sufficient and that recognition
of specific ordered combinations of sugar sequences along
the N-glycan chain are required for bacterial adhesion. To-
gether, these results strongly indicate that HS and N-glycans
can function as binding receptors for P. aeruginosa.
P. aeruginosa-induced host cell injury or internalization into
host cells is mediated by binding to N-glycans at the AP sur-
face or to HS at the BL surface. Following binding to epithelial
cells, P. aeruginosa is able to enter into cells and/or, at later
time points, cause type III secretion-dependent cytotoxicity. In
order to study the extent of host cell damage after bacterial
attachment to N-glycans or HS, variously treated cells were
incubated with bacteria for 5 h, and standard LDH release
assays (which measure cell damage) and FITC-inulin mono-
layer permeability assays (which measure the integrity of tight
junctions) were performed. We first studied the role of N-
glycans by pretreating cells with tunicamycin to inhibit N-gly-
cosylation or by exogenous addition of Man to increase N-
glycosylation. At up to 5 h postinfection, after AP-side addition
of bacteria to tunicamycin-treated cells, LDH release was re-
duced 50 to 70% in both well-polarized and incompletely po-
larized wt MDCK and Calu-3 cells (Fig. 6; see Fig. S9A to H
in the supplemental material). In contrast, Man supplementa-
tion augmented bacterium-induced cytotoxicity at the AP sur-
face of well-polarized and incompletely polarized monolayers,
as evidenced by increased LDH release. Bacterium-induced
cell death correlated well with the loss of the epithelial barrier,
as measured by FITC-inulin diffusion through the epithelial
monolayer (data not shown; see Fig. S10 in the supplemental
material). As expected, ConArMDCK cells were resistant to
bacterial killing at the AP surface, and tunicamycin treatment
did not further decrease LDH release (Fig. 6; see Fig. S9I to L
in the supplemental material) or affect FITC-inulin diffusion
(data not shown). However, incubation with Man increased the
sensitivity of ConArMDCK cells to bacterium-induced injury
at the AP surface (Fig. 6). Consistent with our binding data
(Fig. 1), cytotoxicity induced by bacteria added to the BL
surface was unaffected by tunicamycin treatment or Man sup-
plementation (Fig. 6; see Fig. S9 in the supplemental material).
Next, we investigated the role of HS chains of HSPGs in
promoting host cell injury upon P. aeruginosa binding. Hepa-
rinase III digestion or HSPG desulfation decreased LDH re-
lease upon BL-side addition of bacteria to well-polarized wt
MDCK, ConArMDCK, and Calu-3 cells (Fig. 6A and C; see
Fig. S9 in the supplemental material). Importantly, these treat-
ments also decreased cytotoxicity at the AP surface of incom-
pletely polarized cells (Fig. 6B and D; see Fig. S9 in the sup-
plemental material). Resulfation of chemically desulfated HS
chains restored cytotoxicity when bacteria were added to the
BL surface of well-polarized cells or to both surfaces of incom-
FIG. 6. Bacterial cytotoxicity is mediated upon P. aeruginosa binding to N-glycans on the AP surface of polarized cells, to HSPGs on the BL
surface of polarized cells, and to both molecules on the AP surface of incompletely polarized cells. Cells were pretreated with mannose (man) to
upregulate N-glycosylation, with tunicamycin (tun) (32) to inhibit N-glycosylation, with heparinase III (hepIII) to remove HS chains, with sodium
chlorate for desulfation (desulf), or with sodium sulfate for resulfation (resulf). Host cell treatments are color coded as follows: modifications of
HSPGs are shown in shades of red and modifications of N-glycans are shown in shades of blue. Bacteria were added for 5 h, and standard LDH
release assays were performed. Shown are the means ? SD for four separate experiments. Percentages of LDH release in well-polarized (A) and
incompletely polarized (B) MDCK and ConArcells were determined and normalized to that for AP-side-infected, untreated MDCK cells.
Percentages of LDH release in well-polarized (C) and incompletely polarized (D) Calu-3 cells were determined and normalized to AP-side-
infected, untreated Calu-3 cells.*, P ? 0.05 compared to AP-side-infected, untreated MDCK cells; ?, P ? 0.05 compared to AP-side-infected,
untreated ConArcells; ?, P ? 0.05 compared to AP-side-infected, untreated Calu-3 cells;**, P ? 0.05 compared to BL-side-infected, untreated
MDCK cells; ??, P ? 0.05 compared to BL-side-infected, untreated ConArcells; ??, P ? 0.05 compared to BL-side-infected, untreated Calu-3
VOL. 78, 2010DISTINCT RECEPTORS MEDIATE P. AERUGINOSA BINDING947
pletely polarized cells (Fig. 6A to D). As expected, decreased
cytotoxicity correlated with decreased FITC-inulin diffusion
(data not shown; see Fig. S10 in the supplemental material).
In order to examine the roles of N-glycans and HS chains in
P. aeruginosa entry into host cells, variously pretreated cells
were infected for 1 h and standard bacterial internalization
assays were performed. Bacterial internalization was reduced
at the AP membrane of well-polarized ConArMDCK cells
compared to that with wt MDCK cells, but internalization at
the BL surface was equivalent in the two cell lines (Fig. 7A and
C). It was restored to wt levels in ConArMDCK cells supple-
mented with Man or Glc (Fig. 7A). An increase in bacterial
internalization at the AP surface was observed in well-polar-
ized wt MDCK or Calu-3 cells expressing more complex N-
glycans after supplementation with Man or Glc (Fig. 7A and
B). Addition of Man or Glc did not enhance internalization at
the BL surface of wt MDCK, ConArMDCK, or Calu-3 cells
(Fig. 7A and B). Inhibition of N-glycosylation with tunicamycin
reduced bacterial entry at the AP surface of well-polarized
MDCK and Calu-3 cells but did not further decrease bacterial
internalization into ConArMDCK cells (Fig. 7C and D).
Tunicamycin had no effect on P. aeruginosa internalization from
the BL surface of any of these cell types. Similar results were
observed in incompletely polarized monolayers (data not
shown). Finally, enzymatic cleavage of HS by heparinase III
reduced bacterial invasion in a dose-dependent manner on
both the AP and BL surfaces of incompletely polarized cells
but only at the BL surface of well-polarized cells (Fig. 7E to
H). Chondroitinase ABC treatment had no effect on invasion
under any of these conditions (data not shown).
Importantly, bacterial internalization at the AP surface of
incompletely polarized cells was twofold higher than that in
well-polarized cells (see Fig. S6C in the supplemental mate-
rial). This twofold difference was still observed after tunicamy-
cin treatment, again indicating that the enhanced internaliza-
tion observed at the AP surface of incompletely polarized cells
is not due to upregulation of N-glycans at the AP surface.
However, pretreatment of incompletely polarized cells with
heparinase III reduced bacterial internalization at the AP sur-
face to the levels observed in well-polarized cells. Together,
these data confirm that P. aeruginosa-induced binding and sub-
sequent host injury and entry into well-polarized epithelium
are mediated by bacterial binding to HS chains at the BL
surface and to N-glycans at the AP surface. In contrast, these
events are mediated by HS at the BL surface and by both HS
and N-glycans at the AP surface in incompletely polarized
epithelium. An increase in the abundance and complexity of
N-glycan chains enhances P. aeruginosa binding at the AP
surface of both well-polarized and incompletely polarized ep-
ithelia. However, the enhancement of bacterial internalization
on the AP surface of incompletely polarized cells compared to
that of well-polarized cells is HS dependent.
P. aeruginosa colocalizes with N-glycans at the AP mem-
brane or with HS chains at the BL membrane of 3D cysts.
MDCK cells grown in collagen or Matrigel form highly polar-
ized 3D clonal cysts comprised of a layer of well-polarized cells
surrounding a simple lumen, with the AP side facing the lumen
and the BL side facing the surrounding collagen (BL-side-out
cysts). This system recapitulates the organization of simple
epithelial tissues (5). It also affords the possibility of studying
microbe interactions with the BL surface of the mucosal bar-
rier in the absence of a Transwell filter. If an antibody to the
extracellular domain of integrin is present during growth, cysts
with opposite polarity are formed, in which the AP surface
faces outward (AP-side-out cysts), allowing direct comparison
with BL-side-out cysts (52). These cysts, while less well orga-
nized, are as well polarized as BL-side-out cysts. We used this
model to test whether P. aeruginosa binding to the outside
surface of highly polarized BL-side-out and AP-side-out cysts
occurs at HSPG-rich or N-glycan-rich regions. Without addi-
tional manipulations, the 3D cysts are not representative of
incompletely polarized epithelium.
We first examined the distributions of HS chains and N-
glycans in cysts. An antibody to HS exhibited uniform staining
of the outer membrane of BL-side-out cysts, consistent with
the known polarized distribution of HSPGs in the BL mem-
brane (Fig. 8A, left panel). In AP-side-out cysts, it showed a
chicken-wire-like pattern of staining of the inner BL mem-
brane (Fig. 8A, right panel). Heparinase III treatment of BL-
side-out cysts resulted in patchy staining of HS compared to
that in untreated cysts, affording us the possibility of correlat-
ing bacterial binding to HS-rich patches (Fig. 8C and D).
N-glycans, revealed by staining with FITC-ConA, were found
on the BL surface of BL-side-out cysts and on both surfaces of
AP-side-out cysts (Fig. 8B). Staining of the luminal surface of
BL-side-out cysts with FITC-ConA was not detectable, since
the lectin could not penetrate through well-polarized cells. As
expected, ConArMDCK BL-side-out (Fig. 8E and F) and
AP-side-out (Fig. 8I) cysts exhibited decreased staining with
FITC-ConA compared to wt MDCK cells, with a patchy dis-
tribution of N-glycans. Brief Man supplementation of ConAr
MDCK cysts resulted in increased surface FITC-ConA stain-
ing (Fig. 8J). However, the uniform staining seen in wt MDCK
cysts was not achieved because the sugar was added for a
shorter time (1 day) than that for complete supplementation (1
week). Together, these studies suggest that the distributions of
N-glycans and HSPGs are similar in MDCK cells grown as 3D
cysts or as well-polarized 2D monolayers.
We used heparinase III-treated BL-side-out MDCK cell
cysts and Man-supplemented AP-side-out ConArMDCK cysts
to ask whether P. aeruginosa binding to cysts correlated with
HS- or N-glycan-rich regions at the BL or AP surface. Since
BL-side-out cysts show a uniform distribution of HSPGs on the
BL surface, we briefly exposed these cysts to heparinase III and
then correlated the binding of GFP-expressing P. aeruginosa
with HS-rich or HS-poor patches. For comparison, we deter-
mined the association of binding to Man-rich regions by using
cysts formed from ConArMDCK cells or ConArMDCK cells
briefly supplemented with Man (1 day), where N-glycosylation
is upregulated but not restored to wt levels. Under these con-
ditions, a nonuniform distribution of N-glycans was observed,
as determined by binding of FITC-ConA. As shown in Fig. 8G,
significantly more GFP-expressing bacteria colocalized to HS-
rich patches (70%) than to HS-poor patches (30%) in BL-side-
out wt MDCK cysts. Only 25% of P. aeruginosa organisms
bound to Man-rich patches expressed on the BL membrane of
BL-side-out ConArMDCK cysts (data not shown) or ConAr
MDCK cysts grown briefly in the presence of Man (Fig. 8H).
We also tested whether bacteria bound preferentially to
Man-rich regions on the AP surface of AP-side-out cysts by
948 BUCIOR ET AL.INFECT. IMMUN.
FIG. 7. P. aeruginosa internalization is dependent on bacterial binding to N-glycans on the AP surface of well-polarized cells, to HSPGs on the
BL surface of well-polarized cells, and to both molecules at the AP surface of incompletely polarized cells. Cells were pretreated with the indicated
sugars to upregulate N-glycosylation (A and B), with the indicated concentrations of tunicamycin to inhibit N-glycosylation (C and D), or with the
indicated amounts of heparinase III to remove HS chains (E to H). Bacteria were added for 1 h, and standard invasion assays were performed.
Shown are the means ? SD for three separate experiments. (A and B) Man and Glc are sufficient to enhance bacterial invasion of MDCK, ConAr,
and Calu-3 cells from the AP surface. (A) P. aeruginosa internalization in well-polarized MDCK and ConArcells, normalized to AP-side-infected,
untreated MDCK cells. (B) P. aeruginosa internalization in well-polarized Calu-3 cells, normalized to AP-side-infected, untreated cells. (C and D)
Tunicamycin inhibits invasion from the AP surface of well-polarized MDCK and Calu-3 cells in a dose-dependent manner. (C) P. aeruginosa
internalization in well-polarized MDCK and ConArcells, normalized to AP-side-infected, untreated MDCK cells. (D) P. aeruginosa internalization
in well-polarized Calu-3 cells, normalized to AP-side-infected, untreated cells. (E to H) Heparinase III treatment inhibits invasion from the BL
surface of well-polarized and incompletely polarized MDCK, ConAr, and Calu-3 cells and from the AP surface of incompletely polarized cells. P.
aeruginosa binding to well-polarized (E) and incompletely polarized (F) MDCK and ConArcells was determined and normalized to that of
AP-side-infected, untreated MDCK cells. P. aeruginosa binding to well-polarized (G) and incompletely polarized (H) Calu-3 cells was determined
and normalized to that of AP-side-infected, untreated cells.*, P ? 0.05 compared to AP-side-infected, untreated MDCK cells; ?, P ? 0.05
compared to AP-side-infected, untreated ConArcells; ?, P ? 0.05 compared to AP-side-infected, untreated Calu-3 cells;**, P ? 0.05 compared
to BL-side-infected, untreated MDCK cells; ??, P ? 0.05 compared to BL-side-infected, untreated ConArcells; ??, P ? 0.05 compared to
BL-side-infected, untreated Calu-3 cells.
VOL. 78, 2010 DISTINCT RECEPTORS MEDIATE P. AERUGINOSA BINDING 949
comparing binding to AP-side-out ConArMDCK cysts grown
in the absence or presence of supplemental Man. As shown in
Fig. 8K, only ?30% of PAK-GFP colocalized with Man-rich
regions in AP-side-out ConArMDCK cysts. When the cysts
were supplemented briefly with Man, the fraction of P. aerugi-
nosa bacteria colocalizing with more complex N-glycans in-
creased to 70% (compare panels K and L). In summary, in 3D
cysts, P. aeruginosa binds preferentially to more complex N-
glycans on the AP surface or to HS chains of HSPGs on the BL
surface, supporting our hypothesis that these molecules are
specific binding partners for P. aeruginosa.
Successful opportunistic pathogens, of which P. aeruginosa is
a prime example, exploit specific niches in the host in order to
facilitate attachment, colonization, damage, and dissemina-
tion. Our work was inspired by the fact that long carbohydrate
chains of various glycoconjugates, including HSPGs and N-
glycoproteins, could potentially serve as bacterial receptors
since they are prominent cell surface-exposed structures at the
mucosal epithelium. We extensively characterized the distribu-
tion of HSPGs and the structure of N-glycan chains in cultured
epithelial cells grown at various states of polarization. Since P.
aeruginosa requires preexisting epithelial damage and the loss
of at least some degree of polarity in order to cause disease,
these in vitro epithelial cell culture systems recapitulate impor-
tant aspects of human infections and serve as a useful model to
further dissect mechanisms of disease. Using comprehensive
and multifaceted approaches, we demonstrated that complex
N-glycan chains are necessary and sufficient to mediate P.
aeruginosa binding at the AP surface, whereas the HS moieties
of HSPGs mediate binding at the BL surface of the polarized
FIG. 8. P. aeruginosa colocalizes with HS-rich patches on the exposed BL membrane and with N-glycan-rich patches on the exposed AP membrane
of highly polarized 3D cysts. (A and B) Representative x-y confocal sections of BL-side-out or AP-side-out MDCK cysts cultured for 5 days and stained
with anti-HS antibody (HS; purple) or ConA (Man; purple) and with phalloidin (actin; red). (A) BL-side-out (left) and AP-side-out (right) MDCK cysts
show preferential localization of HSPGs to the BL surface of the cysts. (B) BL-side-out (left) and AP-side-out (right) MDCK cysts show localization of
Man to both the AP and BL surfaces of the cysts. (C to F) 3D reconstructions of z-stack images acquired by confocal microscopy. HS is stained with an
anti-HS antibody (HS; purple), Man is stained with ConA (Man; purple), and actin is stained with phalloidin (actin; red). (C) BL-side-out MDCK cysts
show uniform staining of HS at the exposed BL membrane. (D) MDCK cysts treated with heparinase III and infected with GFP-PAK cells for 2 h (green)
show nonuniform patchy HS staining, and the bacteria bind preferentially to HS-rich areas (purple). (E) BL-side-out wt MDCK cysts show uniform
staining of Man at the exposed BL membrane. (F) BL-side-out ConArMDCK cysts briefly treated with Man (to induce expression of more complex
N-glycans) and infected with GFP-PAK cells for 2 h (green) show nonuniform patchy Man staining, and the bacteria do not bind preferentially to
Man-rich areas (purple). (G) Quantification of GFP-PAK bound to HS-rich versus HS-poor patches in BL-side-out MDCK cysts pretreated with
heparinase III. (H) Quantification of bacteria bound to Man-rich versus Man-poor patches in BL-side-out ConArcysts briefly treated with Man. (I and
J) 3D reconstructions of z-stack images of ConArMDCK cysts acquired by confocal microscopy. Man is stained with ConA (purple), and actin is stained
with phalloidin (red). (I) AP-side-out ConArcysts infected with PAK-GFP cells show nonuniform patchy staining of Man at the exposed AP membrane,
and the bacteria do not bind preferentially to Man-rich areas. (J) ConArcysts briefly treated with Man and infected with GFP-PAK cells (green) show
nonuniform patchy Man staining, and the bacteria bind preferentially to Man-rich areas (purple). (K) Quantification of GFP-PAK bound to Man-rich
versus Man-poor patches in AP-side-out ConArcysts. (L) Quantification of GFP-PAK bound to Man-rich versus Man-poor patches in AP-side-out
ConArcysts briefly treated with Man.*, P ? 0.01.
950BUCIOR ET AL.INFECT. IMMUN.
epithelium. During epithelial injury and dedifferentiation,
HSPG presentation at the AP surface is increased, explaining
at least in part the predilection of this important pathogen for
such injured tissues. Changes in the composition of N-glycan
chains and/or in the polarized segregation of HSPGs could
contribute to the pathogenesis of acute and chronic diseases.
We first focused on N-glycosylation, as increased or altered
expression of N-glycans could result in enhanced susceptibility
to P. aeruginosa infections in the setting of acute or chronic
injury. Using chemical and enzymatic inhibitors, we showed
that N-glycans are important contributors to P. aeruginosa
binding, entry, and damage at the AP surface of airway and
kidney cells grown at various states of polarity as confluent 2D
monolayers. ConArMDCK cells, which are defective in N-
glycosylation, were particularly informative in identifying the
role of N-glycans in P. aeruginosa binding and subsequent
internalization and host injury. Although the specific defect in
these cells is not known, the fact that N-glycosylation could be
restored by growing these cells in the presence of excess Man
or Glc suggested that they are defective in the activities of
PMM and PMI enzymes (46). When P. aeruginosa was added
to the AP surface of ConArMDCK cells, decreased binding,
entry, and cytotoxicity were observed compared to those in wt
cells, whereas no difference was observed at the BL surface.
While it has previously been suggested that ConArMDCK
cells form more highly polarized monolayers, which leads to
resistance to P. aeruginosa infection (15), we found no evidence
for altered polarity in these cells under our experimental con-
ditions. Importantly, when ConArMDCK cells were grown in
the presence of Man or Glc, P. aeruginosa binding, entry, and
cytotoxicity were restored to wild-type levels at the AP surface.
We could also enhance the binding and subsequent damage at
the AP surface of wt MDCK and airway epithelial cells grown
in the presence of excess Man or Glc. We demonstrated in-
creased colocalization of bacteria with more complex N-glycan
patches on the AP membrane of highly organized 3D cysts
grown in the presence of Man to upregulate N-glycosylation.
Finally, P. aeruginosa preferentially bound in vitro to a mixture
of complex N-glycans over an individual sugar. Together, these
results suggest that N-glycan chains of one or more N-glyco-
sylated proteins at the AP surface serve as important receptors
for AP binding of P. aeruginosa. The N-glycosylated molecule
is unlikely to be the previously identified receptors CD95 (Fas
receptor), integrin, and fibronectin, as these molecules are
preferentially expressed at the BL surface (50). Likewise, it is
unlikely to be CFTR, since similar results were obtained with
Calu-3 cells, which express high levels of CFTR at the AP
surface, and wt MDCK cells, which express very little CFTR
(18). Future studies will be aimed at identifying this important
molecule. In summary, N-glycans and particular enzymes in
the N-glycosylation pathway could be potential targets for
novel therapeutic approaches in treatment of P. aeruginosa
infections. Most importantly, simple sugars could be used to
either modify the course of the disease or competitively inhibit
bacterial binding and subsequent infection, a line of treatment
currently being investigated.
While these results identified N-glycan chains as AP recep-
tors, they did not reveal the identity of the BL receptor or the
receptor that is upregulated in incompletely polarized cells.
We therefore examined whether HSPGs, which are abundantly
expressed on the BL surface (3), can serve as specific BL
receptors for P. aeruginosa. We demonstrated that HSPGs are
upregulated in incompletely polarized cells and lose their
polarized distribution. Competitive inhibition, enzymatic re-
moval, or desulfation of HS chains decreased P. aeruginosa bind-
ing, entry, and cytotoxicity at the BL surface of well-polarized
cells and at both surfaces in incompletely polarized cells. We
corroborated these results by showing that P. aeruginosa binds
to HS in vitro, with the highest affinity among all tested com-
pounds, and that it preferentially binds to HS-rich patches on
the BL surface of 3D cysts.
If N-glycans can function as binding receptors and are found
on both the AP and BL surfaces, why do they not contribute to
P. aeruginosa binding to the BL surface? First, the identity of
the protein core that is N-glycosylated may also contribute to
binding specificity and the BL N-glycoproteins may not func-
tion as binding receptors or may not be readily accessible. In
addition, or alternatively, the binding affinity for HSPGs may
be higher than that for N-glycan chains (as suggested by our in
vitro data). While our results demonstrate that AP N-glycans
can function as receptors, in healthy hosts with well-polarized
intact mucosal barriers P. aeruginosa does not cause disease.
This observation likely reflects the importance of local defense
mechanisms, such as a functional innate immune system. How-
ever, upregulation of AP HSPGs, particularly in the context of
altered local innate immune responses, may offer an explana-
tion for the increased binding and subsequent damage seen
when P. aeruginosa infects incompletely polarized but intact
monolayers, such as might be seen during regenerating epithe-
lium. In more severe injury, when the monolayer is disrupted,
increased access to HSPGs on the BL surface may also con-
tribute to enhanced susceptibility to infection. Interestingly, it
has been shown that inhibition of shedding of the HSPG syn-
decan-1 or degradation of the shed HS chains attenuates
mouse lung infection (40). These results suggest an additional
possible role for secreted HSPGs in mediating microbial viru-
lence. They also indirectly suggest that P. aeruginosa binds to
HS chains on syndecan-1, thus supporting the work presented
Flagella and type IV pili are the predominant P. aeruginosa
adhesins. Studies are under way to determine their binding
specificities toward N-glycans and HSPGs and whether they
play different roles in adhesion at the AP versus BL surface.
Previous studies have suggested that glycosphingolipids may
serve as AP receptors for type IV pili (10, 47), although these
findings remain controversial (13). Flagellar components have
been shown to bind to Lewis X derivatives that are found on
secreted mucins (48). Moreover, Toll-like receptor 5, predom-
inantly found on the AP surface, has been found to bind
flagellin (24). Finally, there can be flagellum- and pilus-inde-
pendent bacterial binding to the cell surface, since two differ-
ent lectins have been described for P. aeruginosa, specifically
binding to either Fuc or Gal, sugars present in both N- and
O-glycan chains (8, 22).
There is a great deal of potential to use heparin or heparin-
like structures as drugs to treat a wide range of disorders,
including respiratory diseases (12, 30). Although heparin has
been in use as an anticoagulant for over 70 years, heparan
sulfate-like structures are attracting considerable interest as a
source of new therapeutics, since it has been proposed that the
VOL. 78, 2010DISTINCT RECEPTORS MEDIATE P. AERUGINOSA BINDING951
main purpose of heparin is in a defensive mechanism at sites of
tissue injury against invading bacteria and other foreign mate-
rials (36). HSPGs are attractive as therapeutic targets since a
wide range of viral and bacterial pathogens are known to bind
to HS chains (2, 14, 21, 25), and our work further shows that
the sulfate moieties of HSPGs function as P. aeruginosa-bind-
ing receptors. Drugs that disrupt HSPG-pathogen interactions
may be an effective strategy for preventing or treating acute P.
aeruginosa infections, particularly as inhaled therapy for pneu-
An important recent advance in understanding epithelial
cell biology is the ability to grow epithelial cells as 3D cysts in
a collagen-Matrigel matrix, which recapitulates the develop-
ment and structural organization of tubular structures such as
the lung alveolus. (5). While there are a few reports of studies
of the interactions of pathogens with epithelial cells grown as
aggregates (7, 38), to the best of our knowledge our study is the
first that examines the interaction of bacteria with epithelial
cells grown as organized 3D cysts. This approach required that
we overcome several technical challenges. First, the cysts had
to be treated briefly with collagenase in order to expose their
surface. Second, bacterial binding had to be quantified by di-
rect confocal microscopy, as bacteria also bind avidly to the
collagen-Matrigel matrix. Third, during the 7 days it takes for
mature cysts to form, the cells become highly polarized, and
bacterial binding is limited. In addition, N-glycans and HSPGs
are expressed at high levels in a highly polarized manner, with
uniform distributions on the AP and BL sides, respectively.
Therefore, we either exposed the BL surface of BL-side-out
cysts to heparinase III (to correlate binding to HS-rich do-
mains) or utilized ConArMDCK cells (to correlate binding to
Man-rich domains). Altogether, cysts provide a promising
avenue for further study of host-pathogen interactions in vitro
in a biologically relevant context, particularly as they relate to
microbes that infect tubular or luminal structures. Future di-
rections will include observing these events by time-lapse con-
focal microscopy to delineate the temporal and spatial events
that occur during bacterial attachment, internalization, and
While our work examined epithelial cells in various states of
polarization as a model for acute or chronic epithelial injury,
upregulation of N-glycosylation and/or altered expression or
localization of HSPGs may be specifically relevant to the
chronic P. aeruginosa infections that result in end-stage lung
disease in patients with CF. CFTR itself is a highly glycosylated
protein, and there are published data that suggest that the
sugar composition of membrane glycoproteins and secreted
mucins is altered in CF, though the exact mechanism remains
unknown (45). We are currently testing whether N-glycosyla-
tion or HSPGs are upregulated in lung tissues from patients
Another possible way in which upregulation of HSPG ex-
pression could affect both chronic and acute P. aeruginosa
infections is through enhancing the binding and dimerization
of growth factors and their receptors as well as by stabilizing
these receptors. Of particular relevance are the epidermal
growth factor (EGF) and its receptor, EGFR. EGFR activa-
tion is important in wound repair. In addition, EGFR has been
shown to be hyperphosphorylated in CF patients (6). HSPGs
also bind and stabilize the activity of interleukin-8 (IL-8),
which may play a role in IL-8-driven hyperinflammatory re-
sponses in CF (16, 19). Thus, upregulation of HSPGs during
acute and chronic epithelial injury may amplify P. aeruginosa-
induced changes in growth factor signaling. In preliminary
studies, we have found that P. aeruginosa activates EGFR in an
The studies presented here on the pathogenesis of P. aerugi-
nosa infections provide key insights into how changes in the
presentation of AP and BL molecules contribute to acute and
chronic P. aeruginosa infections. The use of cultured epithelial
cells grown as 3D cysts provides a new and powerful in vitro
tool that has the potential to offer better insights into under-
standing the complex mechanism(s) involved in the establish-
ment of the infection and in subsequent organ damage. Our
data show an important role for HS chains and N-glycans in
mediating microbial adherence, entry, and cytotoxicity and
provide a basis for designing therapeutic strategies based on
these interactions. The use of combined therapeutic strategies
that target N-glycosylation, HSPG synthesis, or polarized seg-
regation of these molecules may provide powerful new thera-
peutic approaches to correct the abnormal epithelial architec-
ture that occurs in the setting of chronic lung disease, including
CF, in order to improve clinical outcomes. In summary, our
work establishes fertile ground for better understanding the
connection between specific physiological alterations in vari-
ous acute and chronic disorders and the predilection to infec-
tions by P. aeruginosa.
This work was supported by the Elizabeth Nash Memorial Fellow-
ship of the Cystic Fibrosis Research Inc. (I.B.) and by the National
Institutes of Health (P01 AI053194 [J.N.E. and K.M.], R01 AI065902
[J.N.E.], and R01 DK067153 [K.M.]).
We thank Armando Lemus for his kind gift of pnpT2-GFP-pUCP20.
We thank members of our laboratories for experimental suggestions
and for advice and comments on the paper.
1. Apodaca, G., M. Bomsel, R. Lindstedt, J. Engel, D. Frank, K. Mostov, and
J. Wiener-Kronish. 1995. Characterization of Pseudomonas aeruginosa-in-
duced MDCK cell injury: glycosylation defective host cells are resistant to
bacterial killing. Infect. Immun. 63:1541–1551.
2. Avirutnan, P., L. Zhang, N. Punyadee, A. Manuyakorn, C. Puttikhunt, W.
Kasinrerk, P. Malasit, J. P. Atkinson, and M. S. Diamond. 2007. Secreted
NS1 of dengue virus attaches to the surface of cells via interactions with
heparan sulfate and chondroitin sulfate E. PLoS Pathog. 3:e183.
3. Bishop, J. R., M. Schuksz, and J. D. Esko. 2007. Heparan sulphate proteo-
glycans fine-tune mammalian physiology. Nature 446:1030–1037.
4. Borkowski, T., B. J. Van Dyke, K. Schwarzenberger, V. MacFarland, A. Farr,
and M. Udey. 1994. Expression of E-cadherin by murine dendritic cells:
E-cadherin as a dendritic cell-differentiation antigen characteristic of epi-
dermal Langerhans cells and related cells. Eur. J. Immunol. 24:2767–2774.
5. Bryant, D. M., and K. E. Mostov. 2008. From cells to organs: building
polarized tissue. Nat. Rev. Mol. Cell. Biol. 9:887–901.
6. Burgel, P. R., D. Montani, C. Danel, D. J. Dusser, and J. A. Nadel. 2007. A
morphometric study of mucins and small airway plugging in cystic fibrosis.
7. Carterson, A. J., K. Honer zu Bentrup, C. M. Ott, M. S. Clarke, D. L.
Pierson, C. R. Vanderburg, K. L. Buchanan, C. A. Nickerson, and M. J.
Schurr. 2005. A549 lung epithelial cells grown as three-dimensional aggre-
gates: alternative tissue culture model for Pseudomonas aeruginosa patho-
genesis. Infect. Immun. 73:1129–1140.
8. Chemani, C., A. Imberty, S. de Bentzmann, M. Pierre, M. Wimmerova, B. P.
Guery, and K. Faure. 2009. Role of LecA and LecB lectins in Pseudomonas
aeruginosa-induced lung injury and effect of carbohydrate ligands. Infect.
9. Chen, L. D., and L. D. Hazlett. 2000. Perlecan in the basement membrane of
corneal epithelium serves as a site for P. aeruginosa binding. Curr. Eye Res.
952BUCIOR ET AL.INFECT. IMMUN.
10. Comolli, J. C., A. R. Hauser, L. Waite, C. B. Whitchurch, J. S. Mattick, and
J. N. Engel. 1999. Pseudomonas aeruginosa gene products PilT and PilU are
required for cytotoxicity in vitro and virulence in a mouse model of acute
pneumonia. Infect. Immun. 67:3625–3630.
11. Comolli, J. C., L. L. Waite, K. E. Mostov, and J. N. Engel. 1999. Pili binding
to asialo-GM1 on epithelial cells can mediate cytotoxicity or bacterial inter-
nalization by Pseudomonas aeruginosa. Infect. Immun. 67:3207–3214.
12. Coombe, D. R., and W. C. Kett. 2005. Heparan sulfate-protein interactions:
therapeutic potential through structure-function insights. Cell Mol. Life Sci.
13. Emam, A., A. R. Yu, H. J. Park, R. Mahfoud, J. Kus, L. L. Burrows, and C. A.
Lingwood. 2006. Laboratory and clinical Pseudomonas aeruginosa strains do
not bind glycosphingolipids in vitro or during type IV pili-mediated initial
host cell attachment. Microbiology 152:2789–2799.
14. Fleckenstein, J. M., J. T. Holland, and D. L. Hasty. 2002. Interaction of an
outer membrane protein of enterotoxigenic Escherichia coli with cell surface
heparan sulfate proteoglycans. Infect. Immun. 70:1530–1537.
15. Fleiszig, S. M., D. J. Evans, N. Do, V. Vallas, S. Shin, and K. E. Mostov. 1997.
Epithelial cell polarity affects susceptibility to Pseudomonas aeruginosa inva-
sion and cytotoxicity. Infect. Immun. 65:2861–2867.
16. Frevert, C. W., M. G. Kinsella, C. Vathanaprida, R. B. Goodman, D. G.
Baskin, A. Proudfoot, T. N. Wells, T. N. Wight, and T. R. Martin. 2003.
Binding of interleukin-8 to heparan sulfate and chondroitin sulfate in lung
tissue. Am. J. Respir. Cell Mol. Biol. 28:464–472.
17. Garrity-Ryan, L., S. Shafikhani, P. Balachandran, L. Nguyen, J. Oza, T.
Jakobsen, J. Sargent, X. Fang, S. Cordwell, M. A. Matthay, and J. N. Engel.
2004. The ADP ribosyltransferase domain of Pseudomonas aeruginosa ExoT
contributes to its biological activities. Infect. Immun. 72:546–558.
18. Gerc ¸eker, A. A., T. Zaidi, P. Marks, D. E. Golan, and G. B. Pier. 2000.
Impact of heterogeneity within cultured cells on bacterial invasion: analysis
of Pseudomonas aeruginosa and Salmonella enterica serovar Typhi entry into
MDCK cells by using a green fluorescent protein-labeled cystic fibrosis
transmembrane conductance regulator receptor. Infect. Immun. 68:861–870.
19. Goger, B., Y. Halden, A. Rek, R. Mosl, D. Pye, J. Gallagher, and A. J. Kungl.
2002. Different affinities of glycosaminoglycan oligosaccharides for mono-
meric and dimeric interleukin-8: a model for chemokine regulation at in-
flammatory sites. Biochemistry 41:1640–1646.
20. Grassme ´, H., S. Kirschnek, J. Riethmueller, Andrea Riehle, G. von Ku ¨rthy,
F. Lang, M. Weller, and E. Gulbins. 2000. CD95/CD95 ligand interactions on
epithelial cells in host defense to Pseudomonas aeruginosa. Science 290:527–
21. Hess, D. J., M. J. Henry-Stanley, S. L. Erlandsen, and C. L. Wells. 2006.
Heparan sulfate proteoglycans mediate Staphylococcus aureus interactions
with intestinal epithelium. Med. Microbiol. Immunol. 195:133–141.
22. Imberty, A., M. Wimmerova, E. P. Mitchell, and N. Gilboa-Garber. 2004.
Structures of the lectins from Pseudomonas aeruginosa: insight into the
molecular basis for host glycan recognition. Microbes Infect. 6:221–228.
23. Imundo, L., J. Barasch, A. Prince, and Q. Al-Awqati. 1995. Cystic fibrosis
epithelial cells have a receptor for pathogenic bacteria on their apical sur-
face. Proc. Natl. Acad. Sci. USA 92:3019–3023.
24. Jacchieri, S. G., R. Torquato, and R. R. Brentani. 2003. Structural study of
binding of flagellin by Toll-like receptor 5. J. Bacteriol. 185:4243–4247.
25. Johnson, K. M., R. C. Kines, J. N. Roberts, D. R. Lowy, J. T. Schiller, and
P. M. Day. 2009. Role of heparan sulfate in attachment to and infection of
the murine female genital tract by human papillomavirus. J. Virol. 83:2067–
26. Kazmierczak, B., K. Mostov, and J. Engel. 2001. Interaction of bacterial
pathogens with polarized epithelium. Annu. Rev. Microbiol. 55:407–435.
27. Kazmierczak, B. I., T. S. Jou, K. Mostov, and J. N. Engel. 2001. Rho GTPase
activity modulates Pseudomonas aeruginosa internalization by epithelial cells.
Cell. Microbiol. 3:85–98.
28. Kazmierczak, B. I., K. Mostov, and J. N. Engel. 2004. Epithelial cell polarity
alters Rho-GTPase responses to Pseudomonas aeruginosa. Mol. Biol. Cell
29. Kierbel, A., A. Gassam, K. Mostov, and J. Engel. 2005. The phosphoinositol-
3-kinase-protein kinase B/Akt pathway is critical for Pseudomonas aerugi-
nosa strain PAK internalization. Mol. Biol. Cell 16:2577.
30. Lever, R., and C. P. Page. 2002. Novel drug development opportunities for
heparin. Nat. Rev. Drug Discov. 1:140–148.
31. Mandell, G. L., J. E. Bennett, and R. Dolin. 2005. Principles and practice of
infectious diseases, 6th ed. Churchill Livingstone Inc., New York, NY.
32. Marjomaki, V., V. Pietiainen, H. Matilainen, P. Upla, J. Ivaska, L. Nissinen,
H. Reunanen, P. Huttunen, T. Hyypia, and J. Heino. 2002. Internalization of
echovirus 1 in caveolae. J. Virol. 76:1856–1865.
33. Matthysse, A. G., S. Stretton, C. Dandie, N. C. McClure, and A. E. Goodman.
1996. Construction of GFP vectors for use in gram-negative bacteria other
than Escherichia coli. FEMS Microbiol. Lett. 145:87–94.
34. Meiss, H. K., R. F. Green, and E. J. Rodriguez-Boulan. 1982. Lectin-resistant
mutants of polarized epithelial cells. Mol. Cell. Biol. 2:1287–1294.
35. Mostov, K., T. Su, and M. ter Beest. 2003. Polarized epithelial membrane
traffic: conservation and plasticity. Nat. Cell Biol. 5:287–293.
36. Nader, H. B., S. F. Chavante, E. A. dos-Santos, T. W. Oliveira, J. F. de-Paiva,
S. M. Jeronimo, G. F. Medeiros, L. R. de-Abreu, E. L. Leite, J. F. de-Sousa-
Filho, R. A. Castro, L. Toma, I. L. Tersariol, M. A. Porcionatto, and C. P.
Dietrich. 1999. Heparan sulfates and heparins: similar compounds perform-
ing the same functions in vertebrates and invertebrates? Braz. J. Med. Biol.
37. Nichols, G. E., T. Shiraishi, and W. W. J. Young. 1988. Polarity of neutral
glycolipids, gangliosides, and sulfated lipids in MDCK epithelial cells. J.
Lipid Res. 29:1205–1213.
38. Nickerson, C. A., T. J. Goodwin, J. Terlonge, C. M. Ott, K. L. Buchanan,
W. C. Uicker, K. Emami, C. L. LeBlanc, R. Ramamurthy, M. S. Clarke, C. R.
Vanderburg, T. Hammond, and D. L. Pierson. 2001. Three-dimensional
tissue assemblies: novel models for the study of Salmonella enterica serovar
Typhimurium pathogenesis. Infect. Immun. 69:7106–7120.
39. O’Brien, L. E., T. S. Jou, A. L. Pollack, Q. Zhang, S. H. Hansen, P. Yurch-
enco, and K. E. Mostov. 2001. Rac1 orientates epithelial apical polarity
through effects on basolateral laminin assembly. Nat. Cell Biol. 3:831–838.
40. Park, P. W., G. B. Pier, M. T. Hinkes, and M. Bernfield. 2001. Exploitation
of syndecan-1 shedding by Pseudomonas aeruginosa enhances virulence. Na-
41. Plotkowski, M. C., A. O. Costa, V. Morandi, H. S. Barbosa, H. B. Nader, S.
de Bentzmann, and E. Puchelle. 2001. Role of heparan sulphate proteogly-
cans as potential receptors for non-piliated Pseudomonas aeruginosa adher-
ence to non-polarised airway epithelial cells. J. Med. Microbiol. 50:183–190.
42. Plotkowski, M. C., S. de Bentzmann, S. H. Pereira, J. M. Zahm, O. Bajolet-
Laudinat, P. Roger, and E. Puchelle. 1999. Pseudomonas aeruginosa inter-
nalization by human epithelial respiratory cells depends on cell differentia-
tion, polarity, and junctional complex integrity. Am. J. Respir. Cell Mol. Biol.
43. Prince, A. 1992. Adhesins and receptors of Pseudomonas aeruginosa associ-
ated with infection of the respiratory tract. Microb. Pathog. 13:251–260.
44. Ramphal, R., C. Carnoy, S. Fievre, J. C. Michalski, and N. Houdret. 1991.
Pseudomonas aeruginosa recognizes carbohydrate chains containing type 1
(Gal ?1–3GlcNac) or type 2 (Gal ?1-4GlcNac) disaccharide units. Infect.
45. Rhim, A. D., L. I. Stoykova, A. J. Trindade, M. C. Glick, and T. F. Scanlin.
2004. Altered terminal glycosylation and the pathophysiology of CF lung
disease. J. Cyst. Fibros. 3(Suppl. 2):95–96.
46. Rush, J. S., K. Panneerselvam, C. J. Waechter, and H. H. Freeze. 2000.
Mannose supplementation corrects GDP-mannose deficiency in cultured
fibroblasts from some patients with congenital disorders of glycosylation
(CDG). Glycobiology 10:829–835.
47. Saiman, L., and A. Prince. 1993. Pseudomonas aeruginosa pili bind to
asialoGM1 which is increased on the surface of cystic fibrosis epithelial
cells. J. Clin. Invest. 92:1875–1880.
48. Scharfman, A., S. K. Arora, P. Delmotte, E. Van Brussel, J. Mazurier, R.
Ramphal, and P. Roussel. 2001. Recognition of Lewis X derivatives present
on mucins by flagellar components of Pseudomonas aeruginosa. Infect. Im-
49. Schroeder, T. H., T. Zaidi, and G. B. Pier. 2001. Lack of adherence of clinical
isolates of Pseudomonas aeruginosa to asialo-GM(1) on epithelial cells. In-
fect. Immun. 69:719–729.
50. Tan, K. H., and W. Hunziker. 2003. Compartmentalization of Fas and Fas
ligand may prevent auto- or paracrine apoptosis in epithelial cells. Exp. Cell
51. Viala, J., C. Chaput, I. G. Boneca, A. Cardona, S. E. Girardin, A. P. Moran,
R. Athman, S. Memet, M. R. Huerre, A. J. Coyle, P. S. DiStefano, P. J.
Sansonetti, A. Labigne, J. Bertin, D. J. Philpott, and R. L. Ferrero. 2004.
Nod1 responds to peptidoglycan delivered by the Helicobacter pylori cag
pathogenicity island. Nat. Immunol. 5:1166–1174.
52. Yu, W., A. Datta, P. Leroy, L. E. O’Brien, G. Mak, T. S. Jou, K. S. Matlin,
K. E. Mostov, and M. M. Zegers. 2005. Beta1-integrin orients epithelial
polarity via Rac1 and laminin. Mol. Biol. Cell 16:433–445.
53. Zweegman, S., J. Van Den Born, A. M. Mus, F. L. Kessler, J. J. Janssen, T.
Netelenbos, P. C. Huijgens, and A. M. Drager. 2004. Bone marrow stromal
proteoglycans regulate megakaryocytic differentiation of human progenitor
cells. Exp. Cell Res. 299:383–392.
Editor: B. A. McCormick
VOL. 78, 2010DISTINCT RECEPTORS MEDIATE P. AERUGINOSA BINDING953