From the Veterans Affairs Medical Center, New
York, New York 10010, the Departments of § Urology and
** Microbiology, Kaplan Comprehensive Cancer Center, New York University
School of Medicine, New York, New York 10016, the ¶ Department of
Anatomy and Neurobiology, University of Tennessee, Memphis, Tennessee
38163, and the
Veterans Affairs Medical Center,
Memphis, Tennessee 38104
Received for publication, September 20, 2000, and in revised form, December 19, 2000
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ABSTRACT |
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The adherence of uropathogenic Escherichia
coli to the urothelial surface, a critical first step in the
pathogenesis of urinary tract infection (UTI), is controlled by three
key elements: E. coli adhesins, host receptors, and host
defense mechanisms. Although much has been learned about E. coli adhesins and their urothelial receptors, little is known
about the role of host defense in the adherence process. Here we show
that Tamm-Horsfall protein (THP) is the principal urinary protein that
binds specifically to type 1 fimbriated E. coli, the main
cause of UTI. The binding was highly specific and saturable and could
be inhibited by D-mannose and abolished by endoglycosidase
H treatment of THP, suggesting that the binding is mediated by the
high-mannose moieties of THP. It is species-conserved, occurring in
both human and mouse THPs. In addition, the binding to THP was much
greater with an E. coli strain bearing a phenotypic variant
of the type 1 fimbrial FimH adhesin characteristic of those prevalent
in UTI isolates compared with the one prevalent in isolates from the
large intestine of healthy individuals. Finally, a physiological
concentration of THP completely abolished the binding of type 1 fimbriated E. coli to uroplakins Ia and Ib, two putative
urothelial receptors for type 1 fimbriae. These results establish, on a
functional level, that THP contains conserved high-mannose moieties
capable of specific interaction with type 1 fimbriae and strongly
suggest that this major urinary glycoprotein is a key urinary
anti-adherence factor serving to prevent type 1 fimbriated E. coli from binding to the urothelial receptors.
The adhesion of Escherichia coli, the most common cause
of urinary tract infection
(UTI),1 to urothelial cells
is a crucially important first step in UTI pathogenesis (1-5). This
adhesion process frequently requires filamentous surface appendages of
uropathogenic E. coli that are called fimbriae, or pili.
Epidemiological studies have shown that >90% of all E. coli isolates from UTI patients elaborate type 1 fimbriae
(also named mannose-sensitive fimbriae) (4, 6, 7). Although
controversies existed for several years, recent investigations have
unequivocally documented the importance of type 1 fimbriae as a major
urovirulence factor. For instance, of the nine most common E. coli virulence factors, the genes for type 1 fimbriae emerged as
the only trait common in all 203 UTI isolates examined (8). In
addition, 26% of the 203 strains were positive only for type 1 fimbrial genes and were negative for the eight other urovirulence
factors tested, including P, S, and Dr fimbriae. In experimental
mouse models, type 1 fimbriae were shown to be indispensable for
bladder colonization and infection (9). Conversely, systemic
immunization of mice with the FimH tip adhesin of type 1 fimbriae
reduced bladder colonization of E. coli by 99% even in
neutropenic mice, suggesting that blocking type 1 fimbriae could
completely abolish E. coli adhesion (10). Further evidence
supporting an important role of type 1 fimbriae in UTIs has come from
the identification of phenotypic variants of the type 1 fimbrial FimH
adhesin. On the basis of the receptor binding specificity for
monomannose residues, type 1 fimbriae can be divided into the low
monomannose-binding variant (ML) and the high monomannose-binding
variant (MH) (4, 11-13). Interestingly, the ML phenotype predominates
in the large intestine, whereas the MH phenotype predominates in the
UTI isolates, suggesting a selective advantage for certain subtypes of
type 1 fimbriated E. coli in the urinary tract (14). These
recent results, along with numerous previous studies demonstrating the
direct binding between type 1 fimbriae and the urothelium (15-17),
clearly established the functional importance of type 1 fimbriae in
UTI.
The established importance of type 1 fimbriae in urovirulence prompted
us to search for their urothelial receptors. The apical surface of
mammalian urothelia is covered by numerous rigid-looking plaques whose
luminal leaflets are twice as thick as the cytoplasmic ones, hence the
name asymmetric unit membrane (AUM) (18-20). Since AUM constitutes
>90% of the luminal surface of the urinary tract, including the
proximal urethra, bladder, ureters, and renal pelvis, we explored the
role of AUM in E. coli adherence. Using sucrose gradient
followed by detergent washing, we isolated milligram quantities of
highly purified AUMs (21, 22). Such isolated AUMs contain four major
integral membrane proteins that were designated as uroplakins Ia (27 kDa), Ib (28 kDa), II (15 kDa), and III (47 kDa) (21, 23, 24).
Together, these four proteins form natural two-dimensional crystals
arranged in hexagonal arrays (25-28). By generating antibodies to each
of these proteins, we have demonstrated that all these proteins are
urothelium-specific, are confined to the apical surface of the
urothelium (21, 29), and are highly conserved morphologically and
biochemically in all these mammalian species (22). Using an in
vitro adherence system, we recently showed that type 1 fimbriated
E. coli can bind to uroplakins Ia and Ib, two major high
mannose-type glycoproteins of apical urothelial plaques (30). The
binding is highly specific, saturable, and species-conserved and can be
inhibited by D-mannose. We further showed that the allelic
variants of type 1 fimbriated E. coli that are prevalent in
UTIs, but not those prevalent in feces, bind to the uroplakins
(14).2 These results strongly
suggest that the uroplakins can serve as the urothelial receptors for
type 1 fimbriae and that these receptors can provide a selective
advantage for certain E. coli strains to survive in the
urinary niche. Our in vitro data showing the functional
interaction between type 1 fimbriated E. coli and uroplakins
have been recently confirmed morphologically by an in vivo
infection model. Using quick-freeze and deep-etch electron microscopy
of infected mouse bladders, Mulvey et al. (31) demonstrated that the tip of type 1 fimbriae interacted directly with the central depression (3.7 nm in diameter) of hexagonal uroplakin particles. This
finding parallels our previously proposed structural model of uroplakin
particles in which either uroplakin Ia or Ib, the two in
vitro urothelial receptors for type 1 fimbriae, occupies the inner
ring surrounding the central depression of hexagonal uroplakin
particles (32). Together, these in vitro and in
vivo data strongly indicate that uroplakins Ia and Ib can serve as the major urothelial receptors for type 1 fimbriae.
Despite these developments, little attention has been paid to the host
defense factors that may interfere with E. coli adhesion to
the urothelial receptors. Previous studies suggested that Tamm-Horsfall protein (THP; also named uromodulin), the most abundant protein in
mammalian urine, can bind to type 1 fimbriated E. coli, thus implying a role for THP in urinary defense (33-36). These studies were, however, not performed in the context of well defined E. coli strains or in the presence of the cognate urothelial
receptors. It was also unclear whether THP possesses a single, well
conserved, high-mannose chain that is necessary for type 1 fimbrial
binding (37, 38) and whether soluble urinary THP is capable of
interacting at all with the fimbriae (35). There have also been
questions regarding the specificity of the THP-E. coli
interaction as non-mannose-specific P fimbriae were found to bind THP
(39). In addition, it remains uncertain what the relative contribution
of THP would be to urinary defense compared with other urinary
proteins. Finally, it is unclear how THP interacts with the two
recently identified major phenotypic FimH variants (14). We have
undertaken this study to address some of these questions and to examine
the potential role of THP in host urinary tract defense.
Bacterial Strains, Culture, and Metabolic Labeling--
The
fimbrial expression of clinical and recombinant E. coli
strains was determined by yeast aggregation and hemagglutination as
previously described (14, 30). Thus, J96 (O4, K6), a human pyelonephritis isolate, expresses both type 1 (MH variant of FimH) and
P (PapG1 and PapG3) fimbriae (34, 40). P678-54, a minicell-producing E. coli K12 derivative, expresses no fimbriae. SH48 and
HU849, two recombinant strains derived by transfecting the
non-fimbriated P678-54 strain with J96 genomic DNA fragments, express
type 1 and P fimbriae (PapG1), respectively (41). IA2 (O6,
H
All strains were cultured in Luria-Bertani medium at 37 °C for
16 h in methionine- and cysteine-free Dulbecco's modified
Eagle's medium (glucose-free; Life Technologies, Inc.) for 2 h
and then in Dulbecco's modified Eagle's medium containing
[35S]methionine and [35S]cysteine
(PerkinElmer Life Sciences) for 2 h. The labeled E. coli cells were washed four times in phosphate-buffered saline (PBS) and stored in 30% glycerol in PBS at Purification of THP and Asymmetric Unit Membrane--
Human THP
was purified from pooled fresh urine samples collected from three
healthy male donors by three rounds of precipitation in 0.58 M NaCl and solubilization and dialysis in distilled water (43). Mouse THP was purified from pooled fresh urine of BALB/c and 129/svj strains using the same method. The yield and purity of THP
were assessed by SDS-PAGE followed by silver nitrate staining (see
below), and both human and mouse THPs migrated as a single band.
AUM was purified from bovine urinary bladders by first isolating total
urothelial membranes with gradient centrifugation and then treating
them with 2% Sarkosyl followed by 25 mM NaOH (21, 22). AUM
was quantified using bicinchoninic acid reagent (Pierce) in the
presence of 1% SDS. On SDS gel, purified AUM contained four major
proteins: 27-kDa uroplakin Ia, 28-kDa uroplakin Ib, 15-kDa uroplakin
II, and 47-kDa uroplakin III (21, 24, 29, 44).
E. coli Binding Assays--
For the bacterial overlay assay,
total urinary proteins or purified THP was resolved by SDS-PAGE (15%
acrylamide, 120:1 acrylamide/bisacrylamide ratio), electrophoretically
transferred onto nitrocellulose membrane, and reacted with
[35S]methionine-labeled E. coli strains
reconstituted in 3% bovine serum albumin and 0.1% NaN3 in
PBS. After washing in PBS, the binding was visualized by autoradiography.
For the microtiter well binding assay, purified THP was dissolved in
distilled water and incubated in 96-well polystyrene microtiter plates
at room temperature for 30 min and at 4 °C overnight. After washing,
the microtiter wells were blocked with 3% bovine serum albumin in PBS
for 2 h, and immobilized THP was then incubated with
[35S]methionine-labeled E. coli strains
reconstituted in 3% bovine serum albumin and 0.1% NaN3 in
PBS at room temperature for 2 h. After washing four times in PBS,
the bound bacteria were dissolved in 1% SDS and quantified using
scintillation counting. All binding was performed in triplicate.
Deglycosylation of THP--
Purified human THP was reduced and
S-carboxymethylated according to van Rooijen et
al. (45). THP was dissolved in 1 M Tris-HCl (pH 8.25)
containing a final concentration of 50 mM dithiothreitol (Sigma), 6 M guanidine chloride, and 1 mM EDTA.
After incubation at 37 °C for 2 h, the mixture was supplemented
with iodoacetic acid (Sigma) to a final concentration of 100 mM. The reaction proceeded in the dark for 30 min and was
stopped by the addition of 200 mM Silver Nitrate Staining and Western Blotting--
After
electrophoresis, the polyacrylamide gel was prefixed with 50% methanol
and 7% acetic acid and then incubated with 10% glutaraldehyde for 30 min. After extensive washing with distilled water, the gel was exposed
to a solution containing 20% silver nitrate, 0.4% NaOH, 0.1%
NH4OH, and 2% ethanol for 6 min. The gel was washed with
distilled water for 1 h and developed in a solution containing
0.005% citric acid, 0.02% formaldehyde, and 10% ethanol. The
reaction was stopped by incubating the gel with 10% acetic acid. For
Western blotting, proteins resolved by SDS-PAGE were
electrophoretically transferred onto nitrocellulose membrane and
incubated first with an anti-human THP polyclonal antibody (BIODESIGN
International) and then with a secondary antibody conjugated with
peroxidase. The membrane was developed in a
diaminobenzidine/H2O2 solution.
Identification of Urinary Proteins That Bind to Type 1 Fimbriated
E. coli--
To identify urinary proteins that can potentially serve
as defense factors against E. coli adherence, we examined
the interaction between type 1 fimbriated E. coli and total
urinary proteins using the gel overlay assay. Pooled fresh urine
samples from healthy male donors were immediately denatured and reduced
in SDS/ Binding of Two Major Phenotypic Variants of Type 1 Fimbriae to
Purified THP--
It has been recently documented that two major
phenotypic variants exist for type 1 fimbriae based on their binding
specificity for the monomannose residues: the low monomannose-binding
(ML) and high monomannose-binding (MH) variants (14). They predominate in different niches, with 80% of fecal E. coli expressing
the ML phenotype and >70% of UTI isolates expressing the MH
phenotype. A previous in vitro adherence assay showed that
the MH (UTI) variant bound to the purified urothelial plaques
containing uroplakins Ia and Ib, two putative urothelial receptors for
type 1 fimbriae, in significantly greater numbers than the ML (fecal)
variant (14). This provides an explanation for the selective advantage
of the urothelium for the MH variant and raises the interesting
possibility that the two variants might bind differentially to THP
(47).
To further study this possibility, we performed an in vitro
adherence assay to test the binding between a panel of E. coli strains and purified human THP. THP, shown to be a single
species by silver nitrate staining (see Figs. 4A and
6A), was immobilized on microtiter wells and incubated with
equivalent numbers of each [35S]methionine-labeled
E. coli strain (Fig. 2). The
first experiment tested isogenic strains representing the two major
phenotypic variants of type 1 fimbriae. Although KB91 and KB54, which
express the ML and MH adhesins, respectively, aggregated
Saccharomyces cerevisiae equally well (data not shown), KB54
bound to THP four times better compared with KB91. This result suggests
that it is the unmodified terminal mannose, most likely a moiety within the high-mannose residues, but not the bulky complex-type sugars, of
THP that is responsible for the binding. Not surprisingly, the negative
control strain, KB18, which expresses a nonfunctional mutant adhesin,
showed little binding (Fig. 2A). The second experiment examined strains generated in the E. coli P678-54
background, with SH48 expressing type 1 fimbriae (MH type), HU849
expressing PapG1-type P fimbriae, IA2 expressing PapG2-type
P fimbriae, and P678 expressing no fimbriae. High-level binding to THP
was observed only with the type 1 fimbriated SH48 strain, with no
significant binding of the P or non-fimbriated E. coli
strains (Fig. 2B). With both sets of strains, the binding to
bovine serum albumin was consistently at a background level. These
results strongly suggest that the binding of E. coli to THP
is specific for type 1 fimbriated E. coli. Since THP
preferentially binds to the MH variant of type 1 fimbriae, which is
prevalent in UTIs, this lends further support that THP may play an
important role in urinary tract defense.
Characteristics of Type 1 Fimbrial Binding to THP--
To further
examine the binding specificity between type 1 fimbriated E. coli and THP, we performed two saturation binding assays (Fig.
3). In the first, we incubated increasing
numbers of [35S]methionine-labeled E. coli
cells with immobilized THP. Again, of five strains tested, only type 1 fimbriated E. coli (SH48, MH type) bound strongly to
immobilized THP. The binding of J96, which expresses both type 1 and P
fimbriae, was most likely due to its type 1 fimbriae, as isogenic
strains expressing P fimbriae (HU849 or IA2) alone failed
to bind. With both type 1 fimbria-expressing strains, the binding to
THP was linearly proportional to the E. coli input and
eventually reached a plateau. P fimbriated and non-fimbriated E. coli cells did not exhibit any appreciable binding even at very
large bacterial input. The saturation binding of type 1 fimbriated
E. coli was also true when a fixed amount of E. coli was incubated with increasing amounts of immobilized THP. Together, these experiments suggest that the binding between type 1 fimbriae and THP possesses characteristics of a typical ligand-receptor interaction.
To directly establish that the binding between type 1 fimbriated
E. coli and THP was mannose-mediated, we carried out a gel overlay assay in the presence of various synthetic carbohydrates (Fig.
4, A-C). Binding could be
inhibited in a concentration-dependent manner by
D-mannose (Fig. 4B), but not by
D-galactose (Fig. 4C). In addition,
deglycosylation of THP using endoglycosidase H (Fig. 4,
D-F), which specifically cleaves the high-mannose residues, completely abolished the binding of type 1 fimbriated E. coli to THP (Fig. 4E). Moreover, type 1 fimbriated
E. coli was found to bind equally well to equal moles of
immobilized THP and bovine RNase B, the latter of which is known to
possess a single high-mannose chain (48). These results establish that
human THP contains the high-mannose moiety responsible for the binding
of type 1 fimbriated E. coli (Figs. 4 and
5).
To determine whether the high-mannose moiety of THP is conserved in
different species, we isolated THP from mouse urine using a protocol
designed for isolating human THP. A total of 30 ml of pooled mouse
urine yielded ~3 mg of THP (0.1 mg/ml), a concentration comparable to
that of human urinary THP. THP from two different mouse strains (BALB/c
and 129/svj) had a similar yield and purity, and both samples showed a
slightly higher molecular mass compared with human THP (Fig.
6A). This might be related to
the fact that the mouse THP sequence contains two more Asn-linked
glycosylation consensus sites and therefore is more heavily
glycosylated by complex-type moieties. Microtiter well assays showed
that type 1 fimbriated E. coli bound equally well to both
human and mouse THPs (Fig. 6B), indicating that the
high-mannose glycosylation of THP is evolutionarily conserved.
Competitive Inhibition of Type 1 Fimbrial Binding to Uroplakins by
Soluble THP--
To determine whether THP can inhibit the binding of
type 1 fimbriated E. coli to the uroplakin Ia and Ib
urothelial receptors, we performed an in vitro adherence
competition assay. Type 1 fimbriated E. coli cells (SH48)
were incubated with immobilized AUM containing uroplakins Ia and Ib in
the presence of increasing amounts of THP. Fig.
7 shows that THP effectively blocked the
binding of type 1 fimbriated E. coli to AUMs in a
concentration-dependent manner. At a concentration of 0.01 mg/ml, 50% inhibition was achieved; and at 0.1 mg/ml, almost complete
inhibition was achieved (Fig. 7). Since the physiological concentration
of THP is ~0.1 mg/ml (46) and since the number of E. coli
cells invading the urinary tract under physiological conditions must be
several orders of magnitude lower than that used in our experiments
(2 × 108 colony-forming units/ml) (2), our
data indicate that the physiological concentration of THP is in great
excess for saturating type 1 fimbriated E. coli and can
therefore provide a powerful and physiologically relevant defense
against E. coli adhesion.
Tamm-Horsfall Protein as a Major Urinary Defense Factor: A Working
Model--
Although direct interaction between bacterial adhesins and
host receptors is critical for bacterial adherence to host epithelial cells, almost all mucosal epithelial cells are naturally resistant to
bacterial adhesion due to potent host defense mechanisms (49-52). Much
has been learned about such antibacterial defense mechanisms in the
secretory epithelia of the respiratory and intestinal tracts. In these
tissues, a powerful mucus secretion covers the epithelium, thus
shielding the underlying receptors from invading bacteria (50). Less is
known, however, about the biochemical basis of the anti-adherence
mechanisms of the urinary tract. Unlike other epithelia, the urothelium
is not known to have a major secretory function because it lacks
secretory goblet cells and glands and because the apical urothelial
cells do not contain secretory granules, but are covered by
rigid-looking plaques (AUM) that are seemingly incompatible with a
secretory function (53, 54). Furthermore, quick-freeze and deep-etch
microscopic studies of unperturbed mouse bladder surfaces showed that
the uroplakins, the putative receptors for type 1 fimbriae, are not
covered by a detectable glycocalyx or mucus layer, but appear to be
directly exposed to the urinary space (28). Similar observations were
recently made using a murine infection model in which type 1 fimbriae
appeared to bind directly to the naturally exposed uroplakin particles of the bladder luminal surface (31). The absence of detectable protective layers covering the uroplakin E. coli receptors
of the mouse urinary bladder suggests that the urinary tract defense must rely largely on the physical forces imparted by micturition and,
perhaps as important, by the soluble factors present in urine.
In this study, we presented several lines of evidence strongly
implicating THP as the major urinary defense factor. These include the
following. 1) THP is the major urinary protein that binds to type 1 fimbriated E. coli. 2) A strain expressing a UTI-prevalent phenotypic variant of type 1 fimbriae, but not one expressing a
feces-prevalent variant, binds avidly to THP. 3) Type 1 fimbriated E. coli, but not P fimbriated E. coli, binds to
THP in a saturable and mannose-dependent manner. 4) Type 1 fimbriated E. coli binds equally well to THP and to bovine
RNase B, the latter of which is known to possess a single high-mannose
chain. 5) The high-mannose moiety of THP is evolutionarily conserved.
6) At physiological levels, THP completely abolishes the binding of
type 1 fimbriae to the urothelial receptors. These results strongly
suggest that THP, the most abundant glycoprotein in mammalian urine,
may serve as a soluble receptor for type 1 fimbriated E. coli, coating the adhesins and preventing them from binding to
urothelial receptors, thereby facilitating their elimination from the
urinary tract.
High-mannose Glycosylation, an Important Function of THP--
Our
results documented, on a functional level, that THP contains
high-mannose residues capable of interacting with type 1 fimbriated
E. coli. THP is known to be a heavily glycosylated protein,
with carbohydrates accounting for 30% of the total molecular mass
(55). About 80% of the carbohydrate moieties expressed on human THP
are of the complex type, with sialylated tri- and tetraantennary
structures (37). However, proteolytic digestion of human urinary THP
consistently yielded high mannose-bearing glycopeptides. Serafini-Cessi
et al. (56) separated a high-mannose glycopeptide from the
THP digests and analyzed its oligosaccharides by endoglycosidase
digestion and thin-layer chromatography. They showed that native
urinary THP bears one unprocessed high-mannose chain of predominantly
the Man6-GlcNAc2 type (73% versus
19% Man7-GlcNAc2 and 8%
Man5-GlcNAc2). They extended this finding by
isolating chemical (50 mg) amounts of high-mannose chains and by
establishing the primary structure of the glycan by 1H NMR
spectroscopy (57). The relative proportion of high-mannose to
polyantennary complex moieties was ~1:5 (~20% of the total neutral
sugars), suggesting that at least one of the eight potential glycosylation sites in human THP carries high-mannose chains. Other
investigators have identified high-mannose glycans not only from human
THP, but also from bovine THP, indicating the highly conserved nature
of this glycosylation (58, 59). Moreover, the high-mannose
glycosylation in THP does not occur only in vivo; it also
occurs in culture. Serafini-Cessi et al. (60) showed that
when THP cDNA is expressed in HeLa cells, recombinant THP retains
the high-mannose chains in a proportion similar to urinary THP. These
results strongly suggest that the high-mannose glycosylation of THP is
host cell-independent, being imposed by the primary sequence of THP and
folding of specific peptides near the site(s) bearing the high-mannose
glycan. The folding results in the inaccessibility of the glycosylation
site(s) to the endoplasmic reticulum/Golgi enzymes responsible for
converting the high-mannose glycans to the complex type (60). Taken
together, the high-mannose glycosylation of THP has been established by
different glyco-analytical methods, in vivo, in
vitro, in multiple animal species, structurally, and functionally.
Finally, van Rooijen et al. (45) have recently mapped the
high-mannose moiety to a single site at Asn251 of human
THP.
The constant presence of the high-mannose moiety of THP, the ability of
THP to bind to type 1 fimbriae in a mannose-sensitive fashion, and the
strong inhibition of type 1 fimbrial binding to uroplakins by THP raise
the intriguing possibility that THP can serve as a soluble urinary
inhibitor for type 1 fimbriated E. coli (Fig.
8). By saturating the FimH adhesin of
type 1 fimbriae, THP can prevent type 1 fimbriated E. coli
from binding to the urothelial surface receptors. This hypothesis has
three components: bacterial adhesin, urothelial receptors, and THP
(Fig. 8). Our recent studies showed that both uroplakins Ia and Ib bear
exclusively high mannose-type oligosaccharides (30). Therefore, these
two uroplakins can serve as the "immobilized" receptors, allowing type 1 fimbriated E. coli to adhere to the urothelium (30,
31). However, this binding can be effectively blocked by THP, a major high mannose-containing urinary protein. This hypothesis is attractive because it addresses three key elements in UTI pathogenesis,
i.e. the type 1 fimbrial adhesins, their host receptors, and
a potential host urinary tract defense factor. The hypothesis also
implies that quantitative or qualitative defects in THP may predispose certain individuals to bacterial infections, leading to recurrent UTIs.
Additional in vitro and in vivo studies are
required to further verify this hypothesis.
Differential Binding of FimH Variants to Uroplakins and to
THP--
Sokurenko et al. (14, 47) previously found
that the MH variant of type 1 fimbriae binds to bovine AUMs containing
the uroplakin Ia and Ib urothelial receptors much more effectively than
the ML variant. This provides an explanation as to why the MH variant
is a better colonizer in the bladder and why this variant predominates
in UTI isolates. It is therefore of interest to note that the binding
of the two FimH variants to THP is also different, favoring more the MH
variant. Although this seems to be paradoxical, the phenomenon can be
readily explained from an evolutionary standpoint, as the selective
advantage of the urinary tract for the MH variant will likely be
countered by an effective defense mechanism, specifically the
modification of THP by the high-mannose residues. The intricate balance
among E. coli adhesins, urothelial receptors, and urinary tract defense factors, both on quantitative and qualitative levels, may
well determine whether an infection occurs. For example, when urothelial receptor expression and host defense factors are within normal range, E. coli inoculum will be a critical factor.
Similarly, if host urinary defense is compromised, even a small
E. coli inoculum could cause infection. By the same token,
overexpression of urothelial receptors, which could conceivably occur
in certain disease states, could increase the chance of E. coli bladder colonization. Further studies to better define each
of these conditions in humans should no doubt enhance our understanding
of how multiple factors interact in contributing to the pathogenesis of UTIs.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
), a clinical isolate from a patient with acute UTI,
expresses the PapG2-type P fimbriae (42). KB54 and KB91, two
recombinant strains obtained by transfecting a
fimH-null E. coli AAEC191A strain with
fimH genes isolated from UTI and intestine, respectively, express a high monomannose-binding variant (MH) and a low
monomannose-binding variant (ML), respectively (14). KB18, a negative
control strain, expresses a nonfunctional FimH mutant.
70 °C until use.
-mercaptoethanol
followed by dialysis against distilled water. For deglycosylation,
reduced and carboxymethylated THP was digested with endoglycosidase H
(0.05 units) or with N-glycosidase F (2500 units) in 50 mM phosphate buffer containing 0.5% SDS, 1% Nonidet P-40,
1%
-mercaptoethanol, 10 mM EDTA, and 0.05%
NaN3 at 37 °C for 16 h.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-mercaptoethanol solution to minimize protein degradation and
aggregation and subsequently analyzed by SDS-PAGE. Silver nitrate
staining revealed two major protein species at 90 and 65 kDa, along
with many minor proteins at a lower molecular mass range (Fig.
1A). When duplicate samples resolved by SDS-PAGE were electrotransferred onto nitrocellulose membrane and reacted with [35S]methionine-labeled type 1 fimbriated E. coli (strain SH48, MH type), the bacteria
reacted specifically with the 90-kDa protein band, with very little
binding to any other urinary proteins (Fig. 1B). On the
basis of the predominance and the molecular mass range, we speculated
that the 90-kDa protein was THP (46). This was proven to be the case,
as immunoblotting using a polyclonal antibody raised against human THP
specifically reacted with the 90-kDa band (Fig. 1C). The
fact that as little as 20 µl of the unconcentrated urine contained
sufficient amounts of THP to bind a detectable number of type 1 fimbriated E. coli cells suggested that this protein is the
major urinary protein that can potentially block E. coli
adherence to urothelial receptors (see below).
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Fig. 1.
Identification of THP as the major urinary
protein that binds to type 1 fimbriated E. coli.
A, SDS-PAGE of the total human urinary proteins visualized
by silver nitrate staining. Lanes 1-3 contains 10, 20, and
40 µl of pooled fresh human urine, respectively. Lane M
contains molecular mass marker (from top to bottom: 116, 97, 84, 66, 49, 29, 18, 14, and 7 kDa). Note the 90-kDa protein as one of the two
major urinary proteins. B, gel overlay. Total urinary
proteins as shown in A were incubated with
35S-labeled type 1 fimbriated E. coli (SH48, MH
phenotype). Note that THP is the major protein that binds to E. coli. C, immunoblotting with anti-THP antibody
confirming the identity of the 90-kDa band as THP.
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Fig. 2.
In vitro adherence of E. coli strains to purified human THP. A, three
isogenic E. coli strains expressing a mutant FimH adhesin
(KB18), an ML FimH adhesin (KB91), and an MH FimH adhesin (KB54) were
metabolically labeled with [35S]methionine/cysteine.
Equal amounts of the labeled bacteria (2 × 105 cpm)
were incubated with the same amount of immobilized THP (1 µg/well).
Note the high-level binding with the MH strain. The open
bars indicate bovine serum albumin (BSA; 1 µg/microtiter well) as a negative control. B, four
isogenic strains generated in a different genetic background and
expressing MH-type FimH (SH48), PapG1 (HU849), PapG2, and no fimbriae
(P678) were incubated with THP as described for A. Note that
only the FimH-expressing strain bound to THP.
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Fig. 3.
Saturation binding of E. coli
to purified human THP. A, a fixed amount (1 µg)
of immobilized THP was incubated with increasing amounts of
35S-labeled E. coli as indicated. Note that only
the FimH-expressing type 1 fimbriated E. coli strains (SH48
and J96) bound to THP. Also note that the binding was linearly
proportional to the E. coli input and was saturable.
H denotes FimH of type 1 fimbriae, and G1,
G2, and G3 denote the three PapG adhesins of P
fimbriae. B, a fixed amount of 35S-labeled type
1 fimbriated E. coli (5 × 105 cpm) was
incubated with increasing amounts of immobilized THP. Note the
saturable binding of E. coli to THP.
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Fig. 4.
Binding of type 1 fimbriated E. coli (SH48, MH type) to THP is mediated by high-mannose
moieties of THP. A, purified THP
shown as a single band by SDS-PAGE and silver nitrate staining
(lane 1) reacted with anti-THP antibody upon immunoblotting
(lane 2). B, gel-resolved THP was
electrotransferred onto nitrocellulose and incubated with
35S-labeled type 1 fimbriated E. coli in the
absence (lane 1) or presence of increasing concentration of
D-mannose (lanes 2-7, 0.0001, 0.001, 0.01, 0.1, 1, and 10%, respectively). C, shown are the results of
E. coli binding to THP in the presence of
D-galactose (lanes 1-3, 0.1, 1, and 10%,
respectively). Note that E. coli binding to THP was
inhibited by D-mannose, but not by D-galactose.
D, purified human THP was treated with buffer only
(lane 1), endoglycosidase H (lane 2), or
N-glycosidase F (lane 3); resolved by SDS-PAGE;
and immunoblotted with anti-THP antibody. Note the slight decrease in
molecular mass with endoglycosidase H treatment and the larger decrease
in molecular mass with N-glycosidase F treatment.
E and F, duplicated nitrocellulose blots were
reacted with the type 1 fimbriated SH48 strain and the non-fimbriated
P678-54 control strain, respectively . Note the complete loss of
reactivity of deglycosylated THP with type 1 fimbriated E. coli (E, lanes 2 and 3).
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Fig. 5.
Comparative binding of type 1 fimbriated
E. coli to immobilized THP and bovine RNase B. Note that 35S-labeled type 1 fimbriated E. coli
bound equally well to equal moles (10 pmol) of THP and bovine RNase B
(bRNase B), the latter of which contains a single
high-mannose chain, indicating the high mannose-mediated binding of
THP. The p value indicates the significance level of the
binding to SH48 between the two proteins.
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Fig. 6.
Type 1 fimbriated E. coli
binds to both human and mouse THPs. A, SDS-PAGE
analysis of purified human and mouse THPs. THP was isolated from BALB/c
(lane 2) and 129/svj (lane 3) mouse urine using
the NaCl precipitation method; 1 µg of each sample was analyzed by
SDS-PAGE and silver staining. Lane 1 is a human THP sample
(control). Note that the molecular mass of mouse THP is slightly higher
than that of human THP. B, comparative binding of E. coli strains to purified human and mouse THPs.
35S-Labeled isogenic E. coli strains expressing
no fimbriae (None), type 1 fimbriae (Type 1:
SH48, MH type), PapG1-type P fimbriae (P (G-1)), or
PapG2-type P fimbriae (P (G-2)) (2 × 105
cpm/strain) were incubated with 1 µg of immobilized human or mouse
THP. The number of E. coli cells bound to THP is indicated
as (counts/min) × 10 4. Note that type 1 fimbriated E. coli bound to THP isolated from both species
equally efficiently, suggesting that the high-mannose residues in THP
are highly conserved.
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Fig. 7.
THP blocks the binding of type 1 fimbriated
E. coli (SH48, MH type) to uroplakins. A fixed
amount of E. coli (2 × 105 cpm = 2 × 108 colony-forming units/ml) was incubated, in
the presence of increasing amounts of THP, with immobilized AUMs (2 µg/well). Note that the binding of type 1 fimbriated E. coli to AUMs was greatly inhibited by THP.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 8.
Schematic model showing that THP can bind,
via its highly conserved high-mannose residues, to type 1 fimbriated
E. coli. This binding can effectively block the
attachment of the E. coli cells to the uroplakin Ia and Ib
urothelial receptors. Thus, THP can act as a soluble urinary receptor
analog in defending the urothelium from E. coli
attachment.
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FOOTNOTES |
---|
* This work was supported in part by a merit review grant from the Veterans Affairs Medical Research Service (to X.-R. W.) and by National Institutes of Health Grants 1R01DK56903-01A1 (to X.-R. W.) and 1R01AI42886-01A1 (to D. L. H.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Urology,
New York University School of Medicine, 550 First Ave., Rm. Skirball
10U, New York, NY 10016. Tel.: 212-951-5429; Fax: 212-951-5424; E-mail:
xue-ru.wu@med.nyu.edu.
Published, JBC Papers in Press, December 27, 2000, DOI 10.1074/jbc.M008610200
2 J. Pak, D. L. Hasty, and X.-R. Wu, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: UTI, urinary tract infection; AUM, asymmetric unit membrane; THP, Tamm-Horsfall protein; PBS, phosphate-buffered saline; PAGE, polyacrylamide gel electrophoresis.
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REFERENCES |
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