Contributions of the N- and C-terminal domains of surfactant protein d to the binding, aggregation, and phagocytic uptake of bacteria

Abstract

Collectins play important roles in host defense against infectious microorganisms. We now demonstrate that the serum collectins mannose-binding lectin (MBL) and conglutinin have less ability to bind to, aggregate, and enhance neutrophil uptake of several strains of gram-negative and gram-positive bacteria than pulmonary surfactant protein D (SP-D). Collectins are composed of four major structural domains (i.e., N-terminal, collagen, and neck and carbohydrate recognition domains). To determine which domains of SP-D are responsible for its greater bacterial binding or aggregating activity, activities of chimeric collectins containing the N-terminal and collagen domains of SP-D coupled to the neck recognition domains and carbohydrate recognition domains (CRD) of MBL or conglutinin (SP-D/Cong(neck+CRD) and SP-D/MBL(neck+CRD)) were tested. The SP-D/Cong(neck+CRD) and SP-D/MBL(neck+CRD) chimeras bound to and aggregated the bacteria more strongly than did wild-type MBL or conglutinin. SP-D/MBL(neck+CRD) also enhanced neutrophil uptake of bacteria more so than MBL. Hence, the SP-D N-terminal and/or collagen domains contribute to the enhanced bacterial binding and aggregating activities of SP-D. In prior studies, SP-D/Cong(neck+CRD) and SP-D/MBL(neck+CRD) had increased ability to bind to influenza virus compared not only with that of conglutinin or MBL but with that of wild-type SP-D as well. In contrast, the chimeras had either reduced or unchanged ability to bind to or aggregate bacteria compared to that of wild-type SP-D. Hence, although replacement of the neck recognition domains and CRDs of SP-D with those of MBL and conglutinin conferred increased viral binding activity, it did not favorably affect bacterial binding activity, suggesting that requirements for optimal collectin binding to influenza virus and bacteria differ.

FIG. 1.

Diagram of wild-type and chimeric collectins.

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.

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.

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).

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.

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.

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.

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.