| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
* Departments of Medical Microbiology and Virology, University of Kiel, Kiel, Germany;
Department of Human Microbiology, Tel Aviv University, Tel Aviv, Israel;
Division of Medical and Biochemical Microbiology, Center for Medicine and Biosciences, Research Center Borstel, Borstel, Germany; and
Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO 63110
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Most secreted collectins, including SP-A and SP-D, are assembled as multimers of trimeric subunits. Each subunit consists of an amino-terminal cross-linking domain, a collagen domain, a linking peptide, and a mannose-type, C-type lectin carbohydrate recognition domain (CRD) (3, 4). Each of the trimeric subunits appears "functionally univalent" with respect to binding to particulate, multivalent ligands. Multimerization of the trimeric subunits of SP-D to form dodecamers permits bridging interactions that can lead to efficient microbial agglutination and enhance the interactions of various microorganisms with phagocytic cells (1).
Klebsiella pneumoniae is an important cause of pneumonia, particularly among hospitalized patients. Pattern recognition molecules of the innate immune system are especially important for the clearance of such opportunistic pathogens. In this regard, there is growing evidence that SP-D and SP-A, and the macrophage mannose receptor (MR), another C-type lectin, participate in the host defense against this Gram-negative microorganism through interactions with cell wall glycoconjugates (5, 6, 7, 8). For example, we have previously shown that SP-A and MR preferentially interact with encapsulated strains of K. pneumoniae containing dimannose or dirhamnose sequences in their capsular polysaccharides (6).
Although the expression of capsular polysaccharides is an important mechanism for the evasion of opsonic host defenses, nonencapsulated organisms probably play particularly important roles in the initial stages of lung epithelial attachment and colonization. Nonencapsulated variants of K. pneumoniae spontaneously emerge from the colonies of encapsulated strains as sectors typical of the phase variation phenomenon (9). These nonencapsulated variants bind to and invade epithelial cells more efficiently than the parental encapsulated strains, presumably through enhanced interactions of the epithelial cells with bacterial adhesins (10, 11). It is likely that colonization of the respiratory tract is selective for these nonencapsulated variants because the nonadherent, encapsulated bacteria are expected to be cleared more efficiently by the mechanical defense systems of the lung (12). These and other observations suggest that phase variation to a nonencapsulated form is required to initiate infection in a susceptible host.
In previous studies, we found that SP-D interacts with nonencapsulated strains of K. pneumoniae, but not the corresponding encapsulated strains (8). The major surface glycoconjugate of nonencapsulated Klebsiella is the LPS. In Klebsiella, nine different LPS O-serotypes have been described based on reactivity of the antigenic O-polysaccharides with specific Abs (13, 14). Of these, the O1 serotype is the most common O-Ag found among clinical isolates (15). The molecular basis for the predominance of the O1 serotype is not clear. However, it has been suggested that O-Ags of the LPSs of pathogenic bacteria play a major role in conferring resistance of the bacteria to the bactericidal activity of human serum (16, 17).
LPS is the major SP-D ligand associated with the cell wall of strains of Escherichia coli and Salmonella minnesota (18). In addition, SP-D shows CRD-dependent binding to the LPS isolated from a number of other Gram-negative bacteria, including K. pneumoniae (19). The characterization of mutant forms of E. coli and S. minnesota LPS suggested that the glucose-containing core oligosaccharide of the LPS is necessary and sufficient for CRD-dependent binding (18). In addition, SP-D was found to selectively aggregate strains of E. coli expressing immature, "rough" forms of LPS, which lack the O-polysaccharide (O-Ag) domain (18, 20).
More recently, we demonstrated that the binding of SP-D to the LPS of unencapsulated Klebsiella results in bacterial agglutination and opsonization, with enhanced internalization and killing of the nonencapsulated variants by alveolar macrophages (8). Consistent with earlier studies of E. coli, SP-D was observed to potently agglutinate Klebsiella that express a high proportion of rough LPS (R-LPS) (21). However, in preliminary experiments, we also observed the agglutination of organisms expressing "smooth" LPS (S-LPS), suggesting previously unrecognized modes of interaction with the LPS molecule.
In the present study, we investigated the role of the O-polysaccharide (O-Ag) domain on the interaction of SP-D with LPS purified from 16 well-characterized clinical strains of K. pneumoniae. We found that the structure of the oligosaccharide-repeating units of the O-Ag determines the ability of SP-D to efficiently react with the bacterium, and with its ability to bind to the mature smooth forms of purified LPS. These observations expand the range of potential recognition sites for SP-D on Gram-negative bacteria and bacterial LPS. We also found that SP-D dodecamers decrease the adhesion of Klebsiella expressing "reactive" O-polysaccharides to lung epithelial cells in vitro. Thus, in addition to its opsonic activity, SP-D may act as an anti-adhesion molecule to provide innate immunity against certain O-serotypes of Klebsiella. Notably, Klebsiella isolates expressing "nonreactive" O-polysaccharides were more frequently associated with pneumonia among hospitalized patients.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Clinical isolates included in the O-Ag seroepidemiology (see
Table II
) were from patients hospitalized at the University of Kiel
Hospital (Kiel, Germany), three other affiliated teaching
hospitals, and two cooperating clinical centers. All patients
(n = 138) fulfilled the CDC criteria of nosocomial
pneumonia, urinary tract infections (UTIs), and primary bloodstream
infections (22, 23). In particular, the diagnosis of
Klebsiella pneumonia was based on clinical and radiographic
presentation, and bacterial cultures. In addition to the isolation of
K. pneumoniae, the patients had one or more of the
following: 1) rales or dullness on chest percussion and new onset of
purulent sputum; or 2) new lung infiltrates, consolidation, pulmonary
cavitation, or pleural effusion. Bacterial isolates were from sputum,
transtracheal aspirates, bronchoalveolar lavage fluid that contained a
significant number of bacteria (
104
organisms/ml), or open-lung biopsies. The origin of each strain was
noted and only one strain per patient was included. In most cases,
other clinical information was not available.
|
The bacteria were grown either in Luria broth or on nutrient agar (Difco, Detroit, MI) for 24 h at 37°C. The bacteria were harvested from broth by centrifugation 3000 x g or by scraping the confluent growth from agar, respectively. The bacteria were washed three times with endotoxin-free HEPES-buffered saline (HBS) (5 mM HEPES, 150 mM NaCl, pH 7.5), and then resuspended at the desired density in the same buffer. The O-serotyping of the strains was performed by Dr. J. V. Benedí (Universidad de las Islas Baleares, Palma de Mallorca, Spain) (13), and confirmed by using rabbit anti-O Ag Abs and a competitive ELISA as previously described previously (26).
Cell line and culture conditions
Human A549 adenocarcinoma cells (CCL-185; American Type Culture Collection, Manassas, VA), which are of probable alveolar type II cell derivation, were cultivated as described (27). The cells were grown and maintained in F-12K medium with 10% (v/v) FCS and 2 mM glutamine.
Biological reagents
Rabbit polyclonal Abs specific for the O-Ags of LPS from O1, O3, O4, and O5 reference strains (26) were kindly supplied by Prof. M. Trautmann (Ulm University Hospital, Ulm, Germany). The preparation of mouse monoclonal anti-lipid A and anti-core Abs are described elsewhere (28). LPS was isolated and purified from the reference O-serotypes and from the clinical isolates using conventional methods (8, 29).
Recombinant human SP-D dodecamers, recombinant wild-type rat SP-D dodecamers (RrSP-D), recombinant rat SP-D trimers (RrSP-Dser15,20), and recombinant rat SP-D dodecamers lacking the consensus for asparagine-linked glycosylation at residue 70 of the mature protein (recombinant rat SP-D dodecamers lacking N-linked sugars; RrSP-Dala72), were isolated by sequential saccharide affinity chromatography and gel filtration chromatography (30, 31, 32, 33). All showed minimal endotoxin contamination as confirmed using a chromogenic assay. Purified proteins were stored at -80°C in HBS containing 10 mM EDTA.
Gel electrophoresis and immunoblotting of LPS
Purified LPS (1 µg/lane) was resolved on SDS-PAGE slab gels without urea as described previously (8). One of the gels was stained with silver according to the method of Tsai and Frasch (34), while the LPS bands from the other gels were transferred to nitrocellulose sheets using a wet transfer apparatus as described earlier (8). In some experiments, strips of the blotted membranes were incubated at 100°C in 0.1 M HCl for 1 h to remove the LPS O-side chains and the core oligosaccharide (35). All strips were overlaid with either purified SP-D at indicated concentrations; or with 1/1000 dilutions of anti-lipid A, anti-core, or anti-O-sera. BSA (Sigma-Aldrich, St. Louis, MO) was used as a blocking agent. Bound SP-D was detected using rabbit polyclonal Abs to SP-D, as described previously (8). The detection of bound anti-LPS-core or anti-lipid A or anti-O-Ag Abs was performed as described elsewhere (28, 36).
Epithelial adhesion assay
Recalcified SP-D in HBS, or control buffer, was added to
suspensions of Klebsiella at 0.5 OD
(108 CFU/ml) to obtain the final indicated
concentrations. After 30 min at room temperature, the bacteria were
added to wells of 96-microtiter plates flat-bottom containing confluent
monolayers of A549 cells, which had been briefly washed with HBS
containing 20 mM CaCl2. After 30 min at room
temperature, the nonadherent bacteria were removed by repeated washing
and the cell monolayer was fixed with 2.5% glutaraldehyde in HBS for 5
min. The plates were then washed and incubated in blocking buffer
overnight at 4°C. The bound bacteria were quantified using
anti-Klebsiella serum at 1/1000 in ELISA as described
previously (37). Each adhesion experiment shown in
Figs. 4–6 was performed at least three times; statistical significance was
calculated using the Student’s t test.
|
|
|
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
In our previous study, we observed that human SP-D was more
effective in the agglutination of Klebsiella K50-3OF (a
nonencapsulated phase variant of the O3 serotype) than K21a/3 (a
nonencapsulated phase variant of the O1 serotype) (8).
Determination of the minimal SP-D concentration needed to induce
agglutination of the O1-serotype was 
10-fold higher than that needed
for the O3 serotype (K50-3OF) (4.2 and 0.5 µg/ml, respectively; Table I). The encapsulated parent strains K21a
(O1) and K50 (O3) were not agglutinated at concentrations as high as 10
µg/ml (data not shown).
|
LPS extracted from the two Klebsiella strains differed
markedly in their ability to inhibit SP-D-induced agglutination of
Klebsiella (Table I
, column 2). In particular, much lower
concentrations of the O3-LPS were required to inhibit agglutination.
Capsular polysaccharides isolated from the parent K21a/3 (O1) and
K50-OF (O3) strains did not inhibit agglutination at concentrations as
high as 150 µg/ml (data not shown).
Binding of SP-D to Klebsiella LPS is influenced by the presence of O-Ag
As shown in Table I, an O1 Klebsiella mutant lacking
the O1-polysaccharide in its LPS (but retaining the core region) was
agglutinated at much lower concentrations of SP-D than the wild-type O1
strain. Additional experiments were performed to determine whether the
differences in SP-D binding to the O-serotypes are due to the structure
of their O-Ags.
For this purpose, the LPS isolated from K50-3OF (O3) and K21a/3 (O1)
were subjected to SDS-PAGE to resolve subpopulations of LPS molecules
containing the lipid A plus core region (R-LPS) from populations
containing various numbers of repeating units of O-Ag linked to their
core oligosaccharides (S-LPS). Silver staining of the gels showed that
LPS isolated from both serotypes has a typical ladder-like banding
pattern with a major polydisperse component that migrates slightly
slower than the buffer front (Fig. 1, lanes 1 and 10).
|
, the
slowly and rapidly migrating components of the ladder both reacted with
the anti-core Ab. However, reactivity was lost from both
populations following removal of the carbohydrate with HCl treatment.
As expected, the slowly migrating components of the ladder, but not the
rapidly migrating components, reacted with the Ab to O-Ag; this
reactivity was also lost following treatment with HCl. In contrast, the
Abs to lipid A reacted with all components, but only following removal
of the carbohydrate, which masks epitopes associated with the lipid A
(38). Thus, the rapidly migrating components in our
preparations are definitively shown to consist of LPS molecules
consisting of lipid A conjugated to core oligosaccharide but lacking
O-Ag (R-LPS), while the more slowly migrating components of the ladder
consist of mature LPS molecules containing O-Ag linked to their lipid A
and core domains (S-LPS).
Binding of human SP-D was then examined by incubating parallel blots
with SP-D, followed by detection of the bound SP-D using the indirect
immunoassay described in Materials and Methods. As shown in
Fig. 1 (lanes 8–9 and 17–18), SP-D
strongly and specifically reacted with the rapidly migrating components
(i.e., R-LPS) of the K21a/3 (O1 serotype), but showed no reaction with
the slowly migrating, O-Ag containing species (S-LPS). By contrast,
SP-D reacted strongly with both populations of LPS molecules derived
from the K50-3OF (O3 serotype). Thus, SP-D can bind to the smooth or
O-Ag-containing LPS of an O3 serotype, but not the corresponding
species from an O1 serotype. These SP-D binding data are representative
of several independent experiments, and similar results were obtained
with RrSP-D (data not shown).
Limited acid hydrolysis eliminated all reactivity of the LPS with the
SP-D (Fig. 1, lane 18), despite retention of epitopes for
the anti-lipid A Ab, as described above. This finding is consistent
with previous studies showing that the CRD of SP-D specifically
interacts with saccharides associated with the LPS (18).
Binding was prevented by the presence of EDTA. As expected, binding was
also prevented by 10 mM maltose, a competing sugar of SP-D lectin
activity, but not 10 mM lactose, a noncompeting sugar (data not shown).
Thus, the observed interactions involve the divalent cation-dependent
binding of the CRD of SP-D to carbohydrates associated with the
LPS.
SP-D shows a higher apparent affinity for mature LPS of the O3 serotype
In other experiments, we examined the dose-dependence of SP-D
binding to the LPS. As shown in Fig. 2, there was no obvious difference in the SP-D concentration needed to
visualize the R-LPS components of the two serotypes, consistent with
previous studies that demonstrated binding to the core oligosaccharide.
Although the dose response for SP-D binding to the R-LPS and S-LPS
components was similar for the O3 serotype, a higher concentration of
SP-D was needed to visualize binding of SP-D to the S-LPS forms. In
fact, binding to the O1-Ag containing species was only barely
discernible on the original blot at the highest concentrations.
|
Because the chemical structure of the O-Ags, which also determines
the serotype of Klebsiella, has been characterized
(39), we sought to determine whether the differential
binding of SP-D to the examined O1 and O3 strains is dependent on
specific structure. For this purpose, we performed blotting experiments
of LPS isolated from each of four major Klebsiella serotypes
(O1, O3, O4, and O5) that were characterized as part of recent
epidemiological studies (15). All isolates were smooth
strains and showed similar ladders of S-LPS on silver-stained gels
(data not shown). As shown in Fig. 3, LPS
from all isolates of a given serotype showed comparable profiles of
reactivity with SP-D, suggesting that the reactivity is related to the
structural differences of the O-Ag. Although SP-D (1 µg/ml) bound to
the R-LPS bands of all strains tested, binding to the S-LPS bands was
restricted to the O3 and O5 serotypes, which express O-Ags containing
mannose, rather than galactose, repeating units (39).
|
We next sought to determine whether the serotype-dependent
differences in SP-D reactivity correlate with differences in the
propensity of these organisms to cause infection. For this purpose, we
statistically analyzed the relative frequency of isolation of strains
belonging to the O1, O3, O4, and O5 LPS serotypes among
Klebsiella strains isolated from patients with nosocomial
pneumonia, UTI, and primary bacteremia. Nonreactive strains were
operationally defined as those that express S-LPS that does not react
with low concentrations of SP-D (as observed for the 16 strains
studied,
Figs. 1–3). Reactive strains were defined as those that
express S-LPS that binds to SP-D under the same conditions. Pearson’s
2 test showed that the frequency of isolation
of the SP-D reactive O-serotypes was significantly lower (28%) than
that of the nonreactive serotypes (72%) (Table II; p < 0.001). Although
the difference between the pneumonia and nonpneumonia groups was
significant (p < 0.019), there was no
significant difference among isolates from blood and urine (Table II;
p0.2). These frequencies of isolation are consistent with the results
of our recent European multicenter study (15) and with
other studies of a number of European countries (13, 26).
SP-D preferentially inhibits the adhesion of the O3-serotype of Klebsiella to epithelial cells
Epithelial adhesion is believed to constitute a critical event in
the process of infection. Given our experimental findings and the
epidemiological data, we examined the effects of SP-D on the adhesion
of the O1 and O3 Klebsiella serotypes to A549 lung
epithelial cells as a model of epithelial colonization. Recent studies
have shown that nonencapsulated phase variants are required for
efficient epithelial attachment and invasion, presumably through the
enhanced exposure of bacterial adhesins. For this purpose, we used a
well-characterized immunological method that employs specific
Klebsiella Ab to quantify adherent bacteria
(37). Previous studies have shown that under these
conditions of assay the number of bound bacteria correlates directly
with the ELISA signal. As shown in Fig. 4, human SP-D showed dose-dependent
inhibition of bacterial adhesion. Notably, the minimal
concentration of SP-D needed to significantly inhibit the adhesion of
nonencapsulated Klebsiella to the epithelial cells was 3
µg/ml for the O1 serotype, as compared with 0.75 µg/ml for the O3
serotype. Thus, SP-D decreases bacterial adhesion in vitro, and the
potency of this inhibitory effect is greater for the SP-D reactive than
for the nonreactive O-serotype.
The effects of SP-D on bacterial adhesion require the multimerization of trimeric subunits
SP-D is preferentially, but not exclusively, assembled as dodecamers consisting of four trimeric subunits. Some of the known activities of SP-D, such as particle aggregation, are dependent on multimers (2). However, trimeric subunits or recombinant trimeric lectin domains can mediate other activities, including the neutralization of certain respiratory viruses in vitro and in vivo.
To further define mechanisms of the inhibitory effect of SP-D on
epithelial adhesion, we next compared the activity of RrSP-D dodecamers
(four arms) with single arm, trimeric subunits (RrSP-Dser15,20). We
have previously shown that the mutant protein is fully active as a
lectin, but is defective as a bacterial agglutinin (40).
In contrast with wild-type RrSP-D dodecamers, RrSP-Dser15,20 did not
significantly inhibit the adhesion of the K50 (O3) serotype to
epithelial cells, even at concentrations as high as 5 µg/ml (Fig. 5). Interestingly, 5 µg/ml of
RrSP-Dser15,20 was able to significantly decrease the inhibitory effect
of 3.5 µg/ml SP-D dodecamers. In microscopic slide agglutination
assays, a 5-fold weight excess of RrSP-Dser15,20 markedly inhibited the
agglutination by RrSP-D (data not shown).
Asparagine-linked glycosylation of SP-D is not required for inhibition of epithelial adhesion
Gram-negative organisms, including Klebsiella, can
express a variety of adhesins, some of which can interact with complex
oligosaccharides expressed on host glycoproteins (12). A
single site of N-linked is near the amino-terminal end of the collagen
domain (Asn70) of SP-D. Thus, each dodecamer
contains up to 12 complex oligosaccharides near the amino-terminal hub
of the dodecamer. To further exclude the effects of bacterial
attachment to these oligosaccharides, we examined the inhibitory effect
of a mutant SP-D (RrSP-Dala72) that lacks the sole consensus for
N-linked glycosylation. Previous studies have shown that the molecule,
which is assembled as dodecamers, is fully functional as a lectin and
bacterial agglutinin (40). As shown in Fig. 6, RrSP-Dala72 showed dose-dependent
inhibition of binding of K50-3OF (O3), with reproducible inhibition of
adhesion at concentrations down to 0.75 µg/ml SP-D.
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Interactions with smooth forms of LPS
When populations of LPS expressed by 16 Klebsiella
isolates were separated by SDS-PAGE, the rough forms (R-LPS), which
lack O-Ag, uniformly reacted with SP-D. In contrast, the reaction of
SP-D with smooth forms (S-LPS) was restricted to the eight strains
belonging to the O3 and O5 serotypes. There were also serotypic
differences in SP-D-mediated bacterial agglutination attributable to
differences in the LPS. For example, purified LPS of the O1 serotype
was a much less effective inhibitor of SP-D-dependent agglutination of
K50-3OF (O3) than LPS of the O3 serotype (Table I).
Our data indicate that the core oligosaccharide domain is necessary for
binding to at least some components of Klebsiella LPS. The
R-LPS forms of all four serotypes examined show similar interactions
with SP-D (
Figs. 1–3), consistent with the conserved structure of the
core domain among various serotypes. In addition, hydrolytic cleavage
of the O-polysaccharide and core oligosaccharide from the S-LPS
molecules abrogated SP-D binding to all components of K50-3OF (O3) and
K21a/3 (O1) LPS, but unmasked epitopes for the binding of Ab to lipid A
(Fig. 1). However, there are several observations that indicate that
the O-polysaccharide domain determines these serotype-dependent
differences in SP-D activity. For example, the dose-dependence of SP-D
binding to R-LPS was similar for O3 and O1 S-LPS (Fig. 2), suggesting
similar affinities for the conserved core oligosaccharides of both
serotypes. In contrast, there were obvious differences in the
dose-dependency of SP-D binding to the S-LPS forms, with negligible
binding to the S-LPS forms of the O1 LPS. Because the size
distributions of the S-LPS forms visualized by silver staining or
blotting were similar and overlapping, we infer that the differential
binding of SP-D to S-LPS from the O1 and O3 serotypes reflects
differences in the composition of the repeating units rather than
simply differences in the length of the O-polysaccharide chains.
Formally, there are at least three possible explanations for the serotype-dependent differences in the interaction of SP-D with S-LPS. First, the O-Ag of LPS from O1 and O4 (but not the O3 and O5) serotypes might somehow "repel" SP-D, or more effectively mask sites in the core. Second, the O3 and O5 Ags might directly mediate binding of SP-D to LPS in lieu of interactions with the core, which is masked by the attached O-polysaccharide. Third, the O-Ags of the O3 and O5 serotypes might bind weakly to SP-D, stabilizing residual interactions of the collectin with the subjacent core oligosaccharide. These possibilities could theoretically be distinguished by comparing the binding of SP-D to purified core and purified O-Ags devoid of contaminating core sugars. However, no biochemical or genetic methods have been described for the isolation of pure O-polysaccharide, devoid of core.
Nevertheless, there is strong circumstantial evidence for direct
interactions of SP-D with the reactive O-Ags. Most importantly, the
common structures of the O3 and O5 Ags—but not the O1 and O4
Ags—contain potential saccharide ligands for SP-D, particularly
mannose. The O3 and O5 Ags consist exclusively of repeating units of
-1,2-linked mannose (39), and D-mannose is a known,
albeit relatively weak, competitor of SP-D binding to a variety of
glycoconjugates including LPS (18, 41). Mannose is also
highly represented in a number of other complex glycoconjugates known
to bind to SP-D. These include the high mannose oligosaccharides of the
influenza A virus hemagglutinin (42), commercial
preparations of yeast mannan (19), the gpA glycoprotein of
Pneumocystis carinii (43), and the
lipoarabinomannan of Mycobacterium tuberculosis
(44). In contrast, all the repeating units of the O1 and
O4 Ags lack mannose, but contain galactose (39), a sugar
that reacts very weakly with SP-D (41). CRD-dependent
binding to yeast mannan is of particular interest (19)
because these complex polymers consist of repeating units of mannose in
-1,2, -1,3, and -1,6 linkage similar to the reactive
O-polysaccharides. Notably, the interactions are efficiently inhibited
by known competing sugars such as maltose or mannose. Contributions of
-glucans, which contaminate many commercial preparations of mannan,
cannot be entirely excluded. However, mutant yeast devoid of -glucan
but expressing mannan still show significant binding (43).
Thus, SP-D can interact with mannose-rich polysaccharides closely
related to those expressed by the reactive O-serotypes.
There are currently no available isogenic mutants with switched O-Ag genes and the selective exchange of mannose-rich and galactose-rich O-polysaccharides. Nevertheless, it is impressive that for 16 independent isolates there is complete concordance between SP-D binding and known O-Ag structure, i.e., all isolates with mannose-rich O-Ags bind efficiently to SP-D, while none of the isolates from galactose-rich O-serotypes show this interaction. This likely occurs in the face of numerous other significant clonal variations in cell wall structure among the individual isolates of a given O-serotype.
As shown in Table I, the presence of a nonreactive O1-polysaccharide
can markedly inhibit bacterial agglutination. In earlier experiments,
we found that the extent of O-substitution of nonencapsulated
Klebsiella (in retrospect, a reactive O3 serotype) was
dramatically decreased when the cells were grown under vigorously
aerated vs static conditions (21). This was accompanied by
a marked decrease in the minimum amount of SP-D required to cause
macroscopic aggregation. Thus, there is an apparent inverse
relationship between the number of repeating units of a reactive O-Ag
and the efficiency of SP-D-dependent agglutination. If the reactive
O-Ag were to function as an independent, high-affinity ligand,
increasing the numbers of mannose-containing repeating units might be
expected to enhance or have no effect on agglutination. Although
agglutination is a complex binding phenomenon, these observations
suggest that both the reactive and nonreactive O-polysaccharides
decrease SP-D binding to the core.
Given the combined observations, we speculate that any binding of SP-D to reactive O-Ags is relatively weak, consistent with the comparatively weak interactions of SP-D with D-mannose. If the interactions of SP-D with the O3 or O5 Ags are weak, it is possible that they are stabilized by residual interactions with the core oligosaccharide, even though the latter interactions are too weak to mediate SP-D binding in the presence of a nonreactive O-Ag. In this regard, recent computer docking studies suggest that SP-D can favorably interact with internal—as well as terminal—glucose residues (45). Thus, the presence of O-Ag should not preclude interactions of SP-D with glucose (or heptose) residues in the subjacent core oligosaccharide.
Serotype-dependent inhibition of epithelial adhesion
Our data also suggest a new potential mode of SP-D-mediated
antibacterial host defense. In particular, SP-D can inhibit the binding
of certain nonencapsulated Gram-negative bacteria to lung epithelial
cells. Thus, the anti-adhesion activity of SP-D may represent
another mechanism through which this important molecule functions in
innate immunity. The minimum concentration of SP-D required to
reproducibly inhibit the epithelial adhesion of the O1 serotype is
close to some estimates of alveolar SP-D concentration (3 µg/ml)
(46), whereas the observed minimum inhibitory
concentration for the O3 serotype is severalfold lower. However, such
calculations are based on estimates of hypophase volume and alveolar
surface area, and reflect the contributions of many different alveolar
and distal airway compartments. If normal SP-D concentrations are
higher, as suggested by calculations based on some human lavage studies
(46), serotypic differences might not be evident except in
clinical settings where the local concentration of active SP-D is
decreased (e.g., with smoking or lung injury). As indicated above, an
additional complexity relates to the effects of "environmental
conditions" on the length of reactive or nonreactive O-Ags expressed
by bacteria at specific sites within the respiratory tract.
The effect of SP-D on epithelial adhesion is dependent on the lectin activity of the CRD and requires the multimerization of the SP-D subunits. In this regard, trimeric subunits are inactive as inhibitors of epithelial adhesion, but can block the inhibitory effect of dodecamers. Interactions of bacteria with collagenous sequences or N-linked sugars were excluded with the single-arm mutant and nonglycosylated dodecamers. Previous studies have shown that the single arm mutant is inactive as a bacterial or viral agglutinin (1, 40). In addition, the minimum concentration of SP-D required to agglutinate the O1 and O3 organisms was comparable to the minimum concentration required to significantly inhibit the adhesion K21a/3 (O1) and K50-3OF (O3) to A549 cells, respectively. Thus, we hypothesize that the inhibitory effect involves bacterial aggregation with interference of adhesin-mediated attachment of bacteria to the epithelial cell surface, a stage that is essential for the infectious process (12).
Interestingly, trimeric subunits significantly inhibited the antiadhesive activity of dodecamers at relatively low concentrations. This probably reflects competitive binding to SP-D ligands required for bridging interactions and bacterial agglutination. In any case, the phenomenon is of considerable potential clinical significance given that the relative amounts of collectin trimers can vary among individuals and in the setting of certain lung disorders (2, 47, 48). Thus, increased concentrations of trimers might decrease the effectiveness of the observed antiadhesive activity of SP-D. Recent studies have shown that recombinant, trimeric SP-D neck + CRD domains can neutralize certain fungi and respiratory viruses in animal models, suggesting its potential usefulness as a therapeutic agent (49, 50). However, our observation suggests a potential limitation of such strategies given that exogenous trimers could potentially favor colonization by other microbial pathogens.
SP-D is not the only constituent of the innate immune system that is able to recognize specific O-Ag structures. For example, the mannose-rich O-Ag of Salmonella was shown to be more potent than structures lacking the O-polysaccharide in activating the alternative pathway of complement (51, 52). Other potential mannose-binding molecules within the lung include the macrophage MR and SP-A. Although SP-A has been reported to interact with the lipid A domain, it has also been shown to interact with mannose-containing capsular glycoconjugates (6). Thus, a more complete profiling of the reactivity of various host defense lectins with specific serotypes of Gram-negative bacteria is indicated.
If SP-D or other lectins with similar specificity contribute to the
lung’s defense against Klebsiella, then strains expressing
SP-D reactive O-polysaccharides are expected to be isolated at lower
frequency from patients with K. pneumoniae. In fact, the
statistical analysis of the isolates bearing SP-D reactive and
nonreactive O-Ags is entirely consistent with this possibility (Table II). Given the above, we speculate that in otherwise healthy
individuals the levels of SP-D, together with other host defense
lectins with similar saccharide selectivity, are sufficient for the
clearance of Klebsiella from the respiratory tract. When the
levels of these host defense molecules (or their functional oligomers)
fall below a threshold, strains bearing nonreactive O-Ags can escape
host recognition, contributing to the development of an infection.
Recent evidence that SP-D is also expressed in the human upper
respiratory tract (53), the usual initial site of
bacterial colonization, makes this an attractive hypothesis. The
finding that SP-D is expressed in low amounts at extrapulmonary sites
(53), including the urinary tract, also raises the
possibility of more generalized roles in antibacterial host
defense.
|
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Erika Crouch, Department of Pathology and Immunology, Washington University School of Medicine, Mail Stop 90-31-649, 216 South Kingshighway Boulevard, St. Louis, MO 63110. E-mail address: crouch{at}path.wustl.edu
3 Abbreviations used in this paper: SP-A, surfactant protein A; SP-D, surfactant protein D; R-LPS, rough LPS; S-LPS, smooth LPS; CRD, carbohydrate recognition domain; MR, mannose receptor; RrSP-D, recombinant wild-type rat SP-D dodecamer; RrSP-Dser15,20, recombinant rat SP-D trimer; RrSP-Dala72, recombinant rat SP-D dodecamer lacking N-linked sugars; HBS, HEPES-buffered saline; UTI, urinary tract infection.
Received for publication March 29, 2002. Accepted for publication July 12, 2002.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
-lactamase production, O-serotypes and serum resistance properties in Klebsiella isolates from different European countries. Clin. Microbiol. Infect. 7:286.
1–2-linked D-glycero-D-manno-heptopyranose: chemical and antigenic structure of lipopolysaccharides from Klebsiella pneumoniae ssp. pneumoniae rough strain R20 (O1-:K20-). J. Biol. Chem. 273:7006.This article has been cited by other articles:
|
|
 |
|
  S. Matalon, K. Shrestha, M. Kirk, S. Waldheuser, B. McDonald, K. Smith, Z. Gao, A. Belaaouaj, and E. C. Crouch Modification of surfactant protein D by reactive oxygen-nitrogen intermediates is accompanied by loss of aggregating activity, in vitro and in vivo FASEB J, May 1, 2009; 23(5): 1415 - 1430. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Sahly, S. Navon-Venezia, L. Roesler, A. Hay, Y. Carmeli, R. Podschun, C. Hennequin, C. Forestier, and I. Ofek Extended-Spectrum {beta}-Lactamase Production Is Associated with an Increase in Cell Invasion and Expression of Fimbrial Adhesins in Klebsiella pneumoniae Antimicrob. Agents Chemother., September 1, 2008; 52(9): 3029 - 3034. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Sahly, Y. Keisari, E. Crouch, N. Sharon, and I. Ofek Recognition of Bacterial Surface Polysaccharides by Lectins of the Innate Immune System and Its Contribution to Defense against Infection: the Case of Pulmonary Pathogens Infect. Immun., April 1, 2008; 76(4): 1322 - 1332. [Full Text] [PDF]
|
|
|
|
|
|
E. Kostina, I. Ofek, E. Crouch, R. Friedman, L. Sirota, G. Klinger, H. Sahly, and Y. Keisari Noncapsulated Klebsiella pneumoniae Bearing Mannose-Containing O Antigens Is Rapidly Eradicated from Mouse Lung and Triggers Cytokine Production by Macrophages following Opsonization with Surfactant Protein D Infect. Immun., December 1, 2005; 73(12): 8282 - 8290. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. P. Moran, W. Khamri, M. M. Walker, and M. R. Thursz Role of surfactant protein D (SP-D) in innate immunity in the gastric mucosa: evidence of interaction with Helicobacter pylori lipopolysaccharide Innate Immunity, December 1, 2005; 11(6): 357 - 362. [Abstract] [PDF]
|
|
|
|
|
|
W. Khamri, A. P. Moran, M. L. Worku, Q. N. Karim, M. M. Walker, H. Annuk, J. A. Ferris, B. J. Appelmelk, P. Eggleton, K. B. M. Reid, et al. Variations in Helicobacter pylori Lipopolysaccharide To Evade the Innate Immune Component Surfactant Protein D Infect. Immun., November 1, 2005; 73(11): 7677 - 7686. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. Crouch, Y. Tu, D. Briner, B. McDonald, K. Smith, U. Holmskov, and K. Hartshorn Ligand Specificity of Human Surfactant Protein D: EXPRESSION OF A MUTANT TRIMERIC COLLECTIN THAT SHOWS ENHANCED INTERACTIONS WITH INFLUENZA A VIRUS J. Biol. Chem., April 29, 2005; 280(17): 17046 - 17056. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Ni, D. J. Evans, S. Hawgood, E. M. Anders, R. A. Sack, and S. M. J. Fleiszig Surfactant Protein D Is Present in Human Tear Fluid and the Cornea and Inhibits Epithelial Cell Invasion by Pseudomonas aeruginosa Infect. Immun., April 1, 2005; 73(4): 2147 - 2156. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. K. van de Wetering, A. van Remoortere, A. B. Vaandrager, J. J. Batenburg, L. M. G. van Golde, C. H. Hokke, and J. J. van Hellemond Surfactant Protein D Binding to Terminal {alpha}1-3-Linked Fucose Residues and to Schistosoma mansoni Am. J. Respir. Cell Mol. Biol., November 1, 2004; 31(5): 565 - 572. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. F. Head, T. R. Mealy, F. X. McCormack, and B. A. Seaton Crystal Structure of Trimeric Carbohydrate Recognition and Neck Domains of Surfactant Protein A J. Biol. Chem., October 31, 2003; 278(44): 43254 - 43260. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
