Contributions of the N- and C-Terminal Domains of Surfactant Protein D to the Binding, Aggregation, and Phagocytic Uptake of Bacteria

Reagents.Formyl-methionyl-leucyl-phenylalanine, cytochalasin B, horseradish peroxidase-Type II, scopoletin, Ficoll, dextran, trypan blue, and the mannose-, glucose-, or galactose-bovine serum albumin (BSA) conjugates were purchased from Sigma Co. (St. Louis, Mo.), and Hypaque was obtained from Winthrop Pharmaceuticals (Des Plaines, Ill.). Dulbecco's phosphate-buffered saline (PBS) was purchased from Flow Laboratories (Costa Mesa, Calif.).

Collectin preparations.Table 1 summarizes the various recombinant collectin preparations used in this paper. All preparations were expressed in CHO-K1 cells as previously described (3, 8). Wild-type recombinant collectins included recombinant rat and human SP-Ds (RrSP-D and RhSP-D), recombinant bovine conglutinin (RbConglutinin), and recombinant human MBL (RhMBL). The detailed characterization of these preparations was reported in the references listed in Table 1. RbConglutinin, RrSP-D, and RhSP-D consisted of the purified dodecamer fraction, which was isolated by gel filtration chromatography under nondenaturing conditions. Natural bovine serum conglutinin and human serum MBL were also used in some experiments (as noted in Results), purified as described previously (6), and graciously provided by Jens Jensenius (University of Aarhus, Aarhus, Denmark). Unlike the other collectins used in these studies, natural and recombinant human MBL are composed principally of octadecamers (16, 33).

Two mutated recombinant forms of RrSP-D were also used in these experiments: RrSP-D_CDM and RrSP-D_ala72. RrSP-D_CDM is a trimeric form of SP-D that lacks the entirety of the collagen domain (28). RrSP-D_CDM was the gracious gift of Dennis Voelker (National Jewish Medical and Research Center, Denver, Colo.). RrSP-D_ala72 was generated by substituting alanine for serine at position 72, thereby disrupting the consensus for N-linked glycosylation at Asn 70. RrSP-D_ala72 dodecamers were prepared as previously described (2).

Two chimeric collectins were also employed (see Fig. 1). The first consisted of the N-terminal and collagen domains of RrSP-D joined to the neck domain and CRD of conglutinin (termed SP-D/Cong_neck+CRD). The SP-D/Cong_neck+CRD chimera was constructed by thermal cycling with overlap extension and was expressed in CHO-K1 cells (10). The secreted protein was isolated by GlcNAc affinity chromatography, and the SP-D/Cong_neck+CRD dodecamers were purified by gel filtration chromatography under nondenaturing conditions. The second chimera consists of the N-terminal and collagen domains of recombinant human SP-D (RhSP-D) joined to the neck domain and CRD of human MBL (SP-D/MBL_neck+CRD) (36). The secreted protein was isolated from the medium by saccharide affinity chromatography on mannose-agarose. The dodecameric and trimeric forms of the SP-D/MBL_neck+CRD chimera were then purified by gel filtration chromatography.

Biotinylation of collectins (or antibodies) was performed by incubation of collectins with Immunopure NHS-LC-Biotin (Pierce, Rockford, Ill.), at a ratio of 2:1 of biotin to collectin by weight, for 2 h at room temperature in the dark. Excess biotin was then removed by dialysis overnight. Biotinylation did not alter the ability of the collectins to induce bacterial aggregation or to inhibit hemagglutination activity of influenza virus (data not shown).

Bacterial preparations.Bacterial strains were selected to facilitate comparisons with the published literature (see Table 2). Rough strains of Escherichia coli have been extensively used in previous studies of SP-D interactions with gram-negative bacteria and LPS (9, 22). Likewise, Salmonella enterica serovar Typhimurium has been used in studies of MBL-bacterial interactions. Both gram-negative organisms offer the advantage of a number of well-characterized mutants that vary in the structure of their LPS molecules. Unlabeled and fluorescein isothiocyanate- or Rhodamine-labeled E. coli (K-12 strain) cells were obtained from Molecular Probes (Eugene, Oreg.), as were Zymosan particles. Streptococcus pneumoniae strains were the gracious gift of Alan J. Parkinson (Centers for Disease Control Arctic Investigation Laboratory, Anchorage, Alaska). The TV119, TV160, and SL1102 strains of S. enterica serovar Typhimurium were kind gifts of H. Nikaido (University of California, Berkeley) and were grown in our laboratory. These are rough strains with known LPS oligosaccharide structure (17). The TV119 strain is also known as the Ra strain and has an outer-core oligosaccharide terminating in GlcNAc. The TV160 and SL1102 mutants contain capsular polysaccharide terminating in galactose or inner-core polysaccharides (i.e., 2-keto-3-deoxyoctonic acids), respectively (17). Table 2 summarizes the gram-negative bacterial strains used in this paper.

Live E. coli or S. enterica serovar Typhimurium cells for experiments were prepared by growing bacteria overnight on Luria-Bertani agar, selecting several colonies the morning of experiments, and resuspending these in Luria-Bertani medium. Bacteria were incubated until an optical density at 600 nm (OD₆₀₀) of ∼0.6 was reached (usually ∼2 h at 37°C). Bacteria were then counted with a Hausser counting chamber and were resuspended in PBS. S. pneumoniae strains were grown in sheep blood agar (Remel, Lenexa, Kans.) in low oxygen and then in tryptic soy broth with 10% horse serum. Live S. pneumoniae cells were grown and counted on the day of experiments as described above.

FITC labeling of formalin-fixed bacteria was carried out as previously described (14). The FITC stock was prepared at 1 mg/ml in 1 M sodium carbonate, pH 9.6. Concentrated bacterial or yeast stocks were incubated with FITC (10:1 mixture by volume of bacteria in PBS with FITC stock) for 1 h, followed by dialysis of the mixture for 18 h in PBS. FITC labeling of live bacteria was performed in the same manner except that the bacteria were not dialyzed but were washed in PBS by repeated centrifugation to allow their immediate use in the neutrophil uptake assays. Prior to all assays using bacteria or yeast particles, the suspensions of these organisms were gently sonicated to disrupt aggregates, as monitored by light microscopy.

Assessment of binding of collectins to bacteria.Binding of collectins to bacteria or influenza virus was tested by enzyme-linked immunosorbent assay (ELISA) as described previously (9). Suspensions of formalin-fixed bacteria (∼1 μg [dry weight] of bacteria/ml) were allowed to dry onto 96-well plates, followed by washing and incubation with biotinylated collectins. The presence of bound collectins was detected by using streptavidin conjugated to horseradish peroxidase and 3,3′,5,5′-tetramethylbenzidine substrate (Kierkegaard and Perry, Gaithersburg, Md.). The reaction was stopped by using 1 N H₂SO₄. Optical density was measured with an ELISA reader. A modification of the ELISA was also employed to exclude artifacts resulting from formalin treatment or drying of bacteria. Freshly isolated, live bacteria (∼3 × 10⁶ bacteria/ml) were allowed to settle on ELISA plates overnight at 4°C in liquid buffer, followed by centrifugation of plates at 2,500 rpm in a Sorvall RT7 centrifuge prior to assay to maximize bacterial adhesion. Each experimental condition was tested in duplicate or triplicate wells and was averaged for analysis. The background binding of collectins to wells coated with BSA alone was subtracted from the experimental values. The background binding levels were in the range of 0.03 to 0.2 at OD₄₅₀. More than three separate ELISAs were performed for each variable tested.

Assessment of bacterial aggregation.Bacterial aggregation was examined by several methods. First we assessed aggregation by measuring changes in light transmission through suspensions of bacteria after addition of various concentrations of collectins as previously described (9) by using an SLM/Aminco 8000C (SLM Instruments, Urbana, Ill.) spectrofluorometer. Viable bacteria were grown to an OD₆₀₀ of ∼0.6 on the day of assay and then were counted and resuspended in PBS at a final concentration of 3 × 10⁶ bacteria/ml. Another method for assessing bacterial aggregation involved fluorescence microscopy by using FITC- or Rhodamine-labeled bacteria (9). For this assay, higher concentrations of bacteria (3.3 × 10⁸ bacteria/ml) were used in order to maximize the detection of aggregates and to optimize the binding of the bacteria to neutrophils (see below).

Bacterial colony formation was measured after incubation of live bacteria for aggregation experiments. This was done by taking samples of bacteria that had been incubated with collectins for 30 min at 37°C during the aggregation assay, diluting these bacterial samples in fresh, sterile PBS at 1:1,000 dilution, and plating the samples on LB agar. Plating was performed by taking 50 μl of samples and dispersing on the agar by using sterile glass beads. Colonies were counted after incubation for 24 h at 37°C. Results were expressed as percentages of colonies formed in collectin-treated samples compared to those of bacteria treated in an identical manner with control buffer (e.g., incubated in aggregation assay and plated in identical manner).

Neutrophil preparation.Neutrophils from healthy volunteer donors were isolated to >95% purity as previously described by using dextran precipitation followed by a Ficoll-Hypaque gradient separation for removal of mononuclear cells and hypotonic lysis to eliminate contaminating erythrocytes (13). Cell viability was >98% as determined by trypan blue staining, and cells were used within 2 h of isolation. Cells were subsequently washed three times and resuspended in PBS.

Measurement of bacterial uptake by neutrophils.Bacterial uptake by neutrophils was measured by incubating FITC- or Rhodamine-labeled bacteria with neutrophils, followed by evaluation of cell-associated fluorescence by using a flow cytometer as described previously (9). Bacteria were preincubated with collectins in the manner described above for evaluation of bacterial aggregates under fluorescent microscopy (i.e., 72 μg of bacteria/ml and various concentrations of collectins for 30 min at 37°C). For experiments using live bacteria, bacteria were grown up on the day of assay as described above, incubated with FITC as described for 1 h followed by washing, and then directly incubated with neutrophils. Neutrophils were then incubated with these bacterial samples (5 × 10⁵ neutrophils and 1.2 × 10⁷ bacteria at a ratio of 1 neutrophil to 24 bacteria in 135 μl) for 1 h at 37°C and washed twice in fresh PBS buffer and fixed with 2% paraformaldehyde. The mean cell-associated fluorescence of 2,000 cells was measured by using flow cytometry. These assays were performed after incubation with 0.2 mg of trypan blue/ml for 3 min to eliminate extracellular fluorescence (9).

Statistics.Statistical comparisons were made using Student's paired t test.

Wild-type SP-D causes greater aggregation of S. enterica serovar Typhimurium TV119 or E. coli K-12 than wild-type MBL or conglutinin.Initially FITC- or rhodamine-labeled bacteria were incubated with SP-D, human serum MBL, or bovine serum conglutinin, and aggregate formation was assessed by fluorescence microscopy (Fig. 2). Recombinant human and rat SP-D dodecamers caused marked aggregation of formalin-fixed E. coli K-12 and S. enterica serovar Typhimurium TV119. By contrast, a mutant trimeric form of SP-D (RrSP-D_CDM) lacked aggregating activity in this assay. Human serum MBL or RhMBL caused formation of small aggregates of E. coli K-12 as described by Kawasaki and coworkers (18) (data not shown). Similar small aggregates of S. enterica serovar Typhimurium TV119 were formed by MBL (Fig. 2). Unexpectedly, bovine serum conglutinin caused very little aggregation of E. coli K-12 or S. enterica serovar Typhimurium TV119 (Fig. 2).

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FIG. 2.

Comparison of the ability of bovine conglutinin, RrSP-D, RhSP-D, human serum MBL, or RrSP-D_CDM(SP-D^CDM) to cause aggregation of E. coli K-12 or S. enterica serovar Typhimurium TV119 as assessed by fluorescent microscopy. Samples of rhodamine-labeled E. coli (left panels) or FITC-labeled S. enterica serovar Typhimurium TV119 (right panels) were incubated for 30 min at 37°C with either control buffer or various collectins as indicated, followed by examination of samples under fluorescent microscopy. Pictures are representative of three or more experiments. No (or minimal) aggregation was evident in samples treated with conglutinin or RrSP-D_CDM (a collagen domain deletion form of RrSP-D). MBL caused formation of small aggregates of S. enterica serovar Typhimurium. RrSP-D or RhSP-D (not shown) induced massive aggregation of E. coli, and RhSP-D induced massive aggregation of S. enterica serovar Typhimurium. Control samples were treated with buffer alone without added collectins.

Quantitative comparison of bacterial aggregating activity of MBL and SP-D was obtained by use of light transmission assay (Fig. 3). RhMBL, which is predominantly composed of octadecamers, caused significantly less aggregation of formalin-fixed E. coli K-12 or live S. enterica serovar Typhimurium TV119 than RhSP-D dodecamers (Fig. 3).

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FIG. 3.

RhSP-D or SP-D/MBL_neck+CRD causes greater aggregation of E. coli K-12 (A) or S. enterica serovar Typhimurium TV119 (B) than RhMBL. Bacterial aggregation was assessed by using light transmission assay (see Materials and Methods). (A) The indicated concentration of RhMBL, RhSP-D (dodecameric fraction), or SP-D/MBL_neck+CRD (dodecameric or trimeric fraction) was added at time zero. Dodecamers of RhSP-D and SP-D/MBL_neck+CRD had similar potency in causing aggregation of E. coli. In contrast, even a substantially higher concentration of RhMBL or of the trimeric fraction of SP-D/MBL_neck+CRD caused significantly (P ≤ 0.05) less aggregation than either RhSP-D or SP-D/MBL_neck+CRD dodecamers. (B) RhMBL also caused significantly less aggregation of live S. enterica serovar Typhimurium TV119 than either RhSP-D or SP-D/MBL_neck+CRD dodecamers. Results are means ± SEM of four or more experiments.

The chimeric collectin, SP-D/MBL_neck+CRD, causes significantly greater aggregation of E. coli K-12 and S. enterica serovar Typhimurium TV119 than MBL.SP-D/MBL_neck+CRD contains the N terminus and collagen domain of human SP-D and the neck domain and CRD of human MBL. As shown in Fig. 3, dodecamers of SP-D/MBL_neck+CRD caused significantly greater aggregation of formalin-treated E. coli than RhMBL. Similar results were obtained with live E. coli (data not shown) and with live S. enterica serovar Typhimurium TV119 (Fig. 3B). The degree of aggregation of E. coli by SP-D/MBL_neck+CRD dodecamers was comparable to that induced by RhSP-D dodecamers. After completion of aggregation assays with live bacteria, the ability of collectin- or buffer-treated bacteria to form colonies was also tested. This was done by making dilutions of the bacterial samples and culturing them overnight on LB agar. As shown in Table 3, SP-D/MBL_neck+CRD dodecamers caused significantly greater inhibition of colony growth than RhMBL. This provides further confirmation of the altered ability of SP-D/MBL_neck+CRD dodecamers to interact with bacteria compared to that of RhMBL. The trimeric fraction of SP-D/MBL_neck+CRD caused significantly less aggregation of bacteria than SP-D/MBL_neck+CRD dodecamers even when a significantly higher concentration of SP-D/MBL_neck+CRD was added (Fig. 3 and data not shown).

SP-D/MBL_neck+CRD binds E. coli K-12 and S. enterica serovar Typhimurium TV119 more strongly than MBL.As shown in Fig. 4A, the dodecameric fraction SP-D/MBL_neck+CRD bound to formalin-fixed E. coli significantly more strongly than RhMBL. In contrast, the trimeric fraction of SP-D/MBL_neck+CRD did not bind to bacteria to a significantly greater extent than RhMBL. Of note, at the lower concentrations tested, binding of SP-D/MBL_neck+CRD dodecamers to E. coli was significantly less than binding of dodecamers of RhSP-D in these experiments (Fig. 4B).

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FIG. 4.

RhSP-D or SP-D/MBL_neck+CRD binds to E. coli K-12 and S. enterica serovar Typhimurium TV119 more strongly than RhMBL. Binding to formalin-fixed E. coli was measured by ELISA. (A) SP-D/MBL_neck+CRD dodecamers and RhSP-D bound to E. coli significantly more strongly than either RhMBL or SP-D/MBL_neck+CRD trimers. RhSP-D dodecamers also bound to E. coli to a significantly greater extent than SP-D/MBL_neck+CRD dodecamers. (B) RhMBL bound to S. enterica serovar Typhimurium significantly less strongly than either RhSP-D or SP-D/MBL_neck+CRD dodecamers. RhSP-D dodecamers bound to S. enterica serovar Typhimurium TV119 significantly more strongly than SP-D/MBL_neck+CRD dodecamers. (C) The effect of adding the indicated monosaccharides (160 mM) or EDTA (10 mM) to assay buffer was tested. All monosaccharides and EDTA significantly inhibited binding of either RhSP-D or SP-D/MBL_neck+CRD dodecamers to S. enterica serovar Typhimurium TV119. However, binding of SP-D/MBL_neck+CRD dodecamers was inhibited to a significantly greater extent than binding of RhSP-D dodecamers by the monosaccharides. GlucNAc inhibited binding of RhSP-D dodecamers significantly less than mannose or glucose. Results are means ± SEM of three or more experiments where significant differences are noted (P < 0.05).

RhMBL also bound to live S. enterica serovar Typhimurium TV119 significantly less than RhSP-D or SP-D/MBL_neck+CRD dodecamers (Fig. 4B). For this assay, freshly grown bacteria were allowed to adhere to the ELISA plates without fixation (i.e., by incubation overnight at 4°C followed by centrifugation of plates at 2,500 rpm in a Sorval RT7 centrifuge to maximize bacterial adhesion). Binding of either RhSP-D or SP-D/MBL_neck+CRD dodecamers was essentially abolished by EDTA and was significantly reduced by addition of mannose, glucose, or GlucNAc (Fig. 4C). Of interest, binding of SP-D/MBL_neck+CRD dodecamers was more strongly inhibited than that of RhSP-D by these monosaccharides. Mannose or glucose inhibited binding of RhSP-D more strongly than GlucNAc.

To further assess the specificity of these interactions we examined the ability of these collectins to bind to or aggregate strains of S. enterica serovar Typhimurium that differ in the structure of their LPS, TV160 and TV119. The TV160 strain differs from TV119 in lacking terminal glucose and GlcNAc residues on its outer-core oligosaccharide. The SL1102 strain lacks the outer-core oligosaccharide. Neither RhSP-D nor SP-D/MBL_neck+CRD showed significant binding to the TV160 or SL1102 strains of S. enterica serovar Typhimurium (Table 4). These results are consistent with the possibility that these collectins preferentially bind to glucose or GlcNAc residues in the outer-core oligosaccharide of S. enterica serovar Typhimurium TV119.

SP-D/MBL_neck+CRD dodecamers increase in neutrophil uptake of E. coli K-12 to a greater extent than wild-type MBL.As shown in Table 5, SP-D/MBL_neck+CRD dodecamers also caused significantly greater increase in uptake of FITC-labeled, formalin-treated E. coli K-12 than RhMBL. The trimeric fraction of SP-D/MBL_neck+CRD was less potent at increasing the uptake of E. coli than SP-D/MBL_neck+CRD dodecamers. This difference was statistically significant at the concentration of 1.4 μg/ml of the collectins. SP-D/MBL_neck+CRD dodecamers also caused significantly greater neutrophil uptake of freshly isolated, live E. coli than RhMBL or SP-D/MBL_neck+CRD trimers. SP-D/MBL_neck+CRD dodecamers (4 μg/ml) increased uptake of live E. coli to 319% ± 67% of control, compared to 106% ± 5.6% and 154% ± 36% of control for RhMBL and SP-D/MBL_neck+CRD trimers, respectively (n = 4; P < 0.04 comparing SP-D/MBL_neck+CRD dodecamers to either RhMBL or SP-D/MBL_neck+CRD trimers).

Conglutinin causes less aggregation of E. coli K-12 and S. enterica serovar Typhimurium TV119 than SP-D.As noted above, bovine serum conglutinin did not cause aggregation of formalin-fixed E. coli K-12 or S. enterica serovar Typhimurium TV119 in fluorescent microscopic assays (Fig. 2). This result was unexpected, since conglutinin strongly aggregates influenza virus (15). Similar results were obtained by using light transmission assays for aggregation of the bacteria and RbConglutinin (Fig. 5). Note that freshly isolated, live S. enterica serovar Typhimurium TV119 also was not aggregated by RbConglutinin (Fig. 5).

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FIG. 5.

Comparison of the ability of RrSP-D, RbConglutinin, and SP-D/Cong_neck+CRD to aggregate E. coli K-12 or S. enterica serovar Typhimurium TV119. Collectins (final concentration, 3 μg/ml) were added at time zero followed by monitoring of light transmission through stirred suspensions of formalin-fixed E. coli K-12 (A) or live S. enterica serovar Typhimurium TV119 (B). Results shown are means ± SEM of three or more experiments. In the case of E. coli all of the collectins caused significant increases in light transmission (indicating aggregation of bacteria) compared to that of the control buffer (not shown). However, SP-D/Cong_neck+CRD caused significantly greater aggregation than RbConglutinin, and RrSP-D caused significantly greater aggregation than either RbConglutinin or SP-D/Cong_neck+CRD (P ≤ 0.05 in each case). In the case of S. enterica serovar Typhimurium, SP-D/Cong_neck+CRD and RrSP-D caused a similar degree of aggregation that was significantly greater than that caused by RbConglutinin or control buffer (P < 0.05). RbConglutinin did not cause greater aggregation of S. enterica serovar Typhimurium than control buffer.

SP-D/Cong_neck+CRD causes greater aggregation of E. coli K-12 and S. enterica serovar Typhimurium TV119 than RbConglutinin.SP-D/Cong_neck+CRD aggregated E. coli K-12 and S. enterica serovar Typhimurium TV119 to a significantly greater extent than RbConglutinin (Fig. 5). SP-D/Cong_neck+CRD aggregated S. enterica serovar Typhimurium to an extent similar to that of RrSP-D; however, it aggregated E. coli significantly less than RrSP-D. The reactions showed specificity given that neither RrSP-D nor SP-D/Cong_neck+CRD caused aggregation of the TV160 or SL1102 strains of S. enterica serovar Typhimurium. After 25 min, light transmission through suspensions of the TV160 or SL1102 strains was not significantly greater for samples treated with the collectins than for samples maintained in control buffer (n = 2 for each; data not shown).

Conglutinin binds less efficiently to several strains of bacteria than does SP-D.Since conglutinin has high affinity for GlcNAc and is structurally similar to SP-D, it was unexpected that conglutinin would not aggregate either E. coli K-12 or S. enterica serovar Typhimurium TV119. We therefore compared the ability of conglutinin and SP-D to bind to these bacteria. We found minimal binding of bovine serum conglutinin to several strains of formalin-fixed bacteria, despite strong binding to influenza virus (Fig. 6). Although RbConglutinin showed detectable binding to freshly isolated, live S. enterica serovar Typhimurium TV119 (Fig. 6B) at physiological concentrations with this method, binding was still significantly lower than observed for RrSP-D.

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FIG. 6.

Binding of conglutinin, RrSP-D, or SP-D/Cong_neck+CRD to bacteria or Influenza A virus (IAV) as assessed by ELISA. (A) IAV or formalin-fixed bacteria (S. pneumoniae Type 23, E. coli K-12, or S. enterica serovar Typhimurium TV119, as indicated) were coated onto ELISA plates and were incubated with biotinylated bovine serum conglutinin. Bound conglutinin was detected by using avidin-horseradish peroxidase and peroxidase substrates. Conglutinin bound significantly to IAV (P < 0.05) in calcium-containing buffer but not in buffer containing 10 mM EDTA (IAV/EDTA). (B) Live S. enterica serovar Typhimurium TV119 was coated onto ELISA plates by sedimentation and binding of biotinylated RrSP-D and RbConglutinin, and SP-D/Cong_neck+CRD was assessed by ELISA. All of the collectins showed significant binding to live S. enterica serovar Typhimurium; however, RrSP-D bound significantly more than RbConglutinin or SP-D/Cong_neck+CRD, and SP-D/Cong_neck+CRD bound significantly more than RbConglutinin. Results are means ± SEM of three or more separate ELISAs.

SP-D/Cong_neck+CRD binds S. enterica serovar Typhimurium TV119 more extensively than RbConglutinin.SP-D/Cong_neck+CRD bound to S. enterica serovar Typhimurium TV119 to a greater extent than RbConglutinin but to a lesser extent than RrSP-D (Fig. 6B). Thus, exchange of the N-terminal and collagen domains of RrSP-D for those of conglutinin resulted in significantly increased binding to and aggregation of E. coli K-12 or S. enterica serovar Typhimurium TV119. These results cannot be attributed to differences in the extent of multimerization of RbConglutinin or SP-D/Cong_neck+CRD because both preparations consist of dodecamers (10).

One potentially important difference between the collagen domains of conglutinin and SP-D is the presence of an N-linked oligosaccharide at asparagine 70 of SP-D. Conglutinin lacks N-linked oligosaccharide attachments. To exclude the possibility that the difference in ability to bind to or aggregate bacteria resulted from interactions of bacteria with the N-linked sugar on SP-D, we compared the abilities of RrSP-D_ala72 and wild-type RrSP-D to aggregate E. coli. The RrSP-D_ala72 mutant lacks a consensus sequence (NGS) for N-linked glycosylation (2). RrSP-D_ala72 had a significantly greater bacterial aggregating activity than wild-type SP-D (i.e., mean percentages ± standard errors of the means [SEM] for light transmission 20 min after addition of collectins was 170% ± 7% for RrSP-D_ala72 versus 134% ± 4% for wild-type RrSP-D; n = 6; P < 0.01).

SP-D, or chimeras containing the N-terminal and collagen domains of SP-D, bind to S. pneumoniae significantly more than RhMBL or RbConglutinin. S. pneumoniae strains are common causes of gram-positive infection and are the most common cause of community-acquired bacterial pneumonia. As shown in Fig. 7, RhSP-D or SP-D/MBL_neck+CRD dodecamers bound to a formalin-fixed preparation of S. pneumoniae Type 23 (a prevalent strain) more extensively than RhMBL. The enhanced binding activity of SP-D/MBL_neck+CRD was dependent on formation of higher-order multimers, since SP-D/MBL_neck+CRD trimers did not have greater binding activity than RhMBL. The results were confirmed by using live S. pneumoniae Type 23 (Fig. 8). Although binding of SP-D/MBL_neck+CRD dodecamers to live S. pneumoniae Type 23 was also significantly greater than that of RhMBL, it was slightly, but significantly, less than that for binding of RhSP-D dodecamers. SP-D/Cong_neck+CRD also bound to S. pneumoniae to a significantly greater extent than RbConglutinin (Fig. 8). RbConglutinin also bound significantly less than RrSP-D to several other capsular types of S. pneumoniae (i.e., types 4, 6A, 14, and 19) by using this ELISA method (data not shown).

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FIG. 7.

RhSP-D, RhMBL, or SP-D/MBL_neck+CRD bind to S. pneumoniae significantly more strongly than RhMBL. S. pneumoniae Type 23 (formalin-fixed) cells were coated onto ELISA plates. RhSP-D dodecamers and SP-D/MBL_neck+CRD dodecamers bound to the bacteria significantly more avidly than either RhMBL or SP-D/MBL_neck+CRD trimers.

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FIG. 8.

Comparison of ability of SP-D, conglutinin, MBL, and collectin chimeras to bind to live S. pneumoniae type 23. Results are means ± SEM of three or four ELISA experiments. Background binding of the collectins to wells with blocking buffer alone was subtracted from the results shown. Wild-type RbConglutinin and RhMBL bound to the bacteria to a significantly lower extent than the other collectins tested (∗∗, P < 0.05). RrSP-D and RhSP-D bound to the bacteria significantly more strongly than the SP-D/Cong_neck+CRD or SP-D/MBL_neck+CRD chimera, respectively.