Sialic acid-binding immunoglobulin-like lectin 7 mediates selective recognition of sialylated glycans expressed on Campylobacter jejuni lipooligosaccharides

Abstract

siglecs are a family of sialic-acid binding immunoglobulin-like lectins mostly expressed by cells of the immune system that have the potential to interact with sialylated glycans expressed not only on host cells but also on certain pathogens. Campylobacter jejuni is a common pathogen of humans that expresses surface lipooligosaccharides (LOS) that can be modified with ganglioside-like terminal structures in the core oligosaccharides. In this study, we examined the interaction of 10 siglecs with LOS purified from four different C. jejuni isolates expressing GM1-like, GD1a-like, GD3-like, and GT1a-like oligosaccharides. Of all siglecs examined, only Siglec-7 exhibited specific, sialic acid-dependent interactions with C. jejuni LOS in solid-phase binding assays. Binding was especially prominent with LOS from the HS:19(GM1(+) GT1a(+)) isolate, with weaker binding with LOS from the HS:19(GD3(+)) isolate. Binding of Siglec-7 was also observed with intact bacteria expressing these LOS structures. Specific binding of HS:19(GM1(+) GT1a(+)) bacteria was demonstrated with Siglec-7 expressed on transfected Chinese hamster ovary cells and with peripheral blood leukocytes, among which HS:19(GM1(+) GT1a(+)) bacteria bound selectively to both natural killer cells and monocytes which naturally express Siglec-7. These results raise the possibility that, in addition to their role in generating autoimmune antibody responses, C. jejuni LOS could interact with Siglec-7 expressed by leukocytes, modulate the host-pathogen interaction, and contribute to the clinical outcome and the development of secondary complications such as Guillain-Barré syndrome.

FIG. 1.

Sialic acid-dependent binding of Siglec-7 to purified LOS from C. jejuni. (A) Schematic representation of C. jejuni LOS structures used in this study. The sialic acid linkages are indicated. (B) Human and murine (m) siglec-Fc proteins were precomplexed with anti-human IgG-alkaline phosphatase and added to plastic microwells coated with LOS purified from the indicated C. jejuni strains. SnR97A is a mutated form of Sn-Fc that is unable to bind sialic acids and was used as a negative control. Relative binding is given in arbitrary fluorescence units (AFU) determined following the addition of a fluorescein diphosphate substrate. *, P < 0.05; **, P < 0.01 (both apply to binding of HS19 isolates versus binding of HS3). (C) Plastic microwells were coated with LOS from the strains indicated in panel B and treated with V. cholerae sialidase before the addition of precomplexed Siglec-7-Fc. **, P < 0.01 for binding of sialidase-treated bacteria versus untreated bacteria. LPS, lipopolysaccharide. (D) Either an irrelevant MAb (7D2 anti-Sn) or anti-Siglec-7 MAb S7.7A was added to the precomplexed Siglec-7 prior to its addition to the indicated LOS. **, P < 0.01 for binding of bacteria in the presence of S7.7A versus binding of bacteria alone. (B through D) Data from one experiment representative of three independent experiments performed.

FIG. 2.

Sialic acid-dependent binding of Siglec-7 to heat-inactivated and live C. jejuni bacteria. (A) Heat-inactivated HS:3 and HS:19(GM1⁺ GT1a⁺) strains were added to precomplexed siglec-Fc proteins in suspension, and binding was measured using a fluorescent substrate. Relative binding is given in arbitrary fluorescence units (AFU). (B) Binding assays were performed with either untreated or sialidase-treated HS:3 or HS:19(GM1⁺ GT1a⁺) strains using CD22-Fc, Siglec-7-Fc or Siglec-10-Fc. (C) The binding properties of live or heat-inactivated (HI) C. jejuni bacteria to Siglec-7-Fc were compared either without or with sialidase treatment of bacteria (left panel) or following the preincubation of precomplexed Siglec-7-Fc with anti-Siglec-7 MAb S7.7A or an irrelevant MAb (right panel). Left panel, **, P < 0.01 for binding of sialidase-treated bacteria versus untreated bacteria. Right panel, **, P < 0.01 for binding of bacteria in the presence of S7.7A versus binding of bacteria alone. Panels A and B show data from one experiment representative of three independent experiments performed. Panel C shows data from a single experiment.

FIG. 3.

Binding of Siglec-7 expressed on CHO cells to C. jejuni bacteria. HS:3 and HS:19(GM1⁺ GT1a⁺) strains were FITC labeled and used in a binding assay with untreated or sialidase-treated CHO-Sig7 cells. Binding of bacteria was assessed by fluorescence microscopy. Bar, 100 μm.

FIG. 4.

Flow cytometric analyses of binding interactions between CHO cells expressing Siglec-7 and C. jejuni bacteria. Untreated or sialidase-treated HS:3 and HS:19(GM1⁺ GT1a⁺) strains were FITC labeled and used in a binding assay with untreated or sialidase-treated CHO-WT cells and CHO-Sig7 cells. (A) Fluorescence-activated cell sorter histograms showing binding of bacteria (gray histograms) are compared with histograms of cells incubated in the absence of bacteria (open histograms). (B) Quantification of bacterial binding to CHO-WT and CHO-Sig7 cells by flow cytometry. Data show means ± 1 standard deviation of binding indices derived from three independent experiments.

FIG. 5.

Siglec-7-expressing NK cells and monocytes recognize C. jejuni HS:19(GM1⁺ GT1a⁺) bacteria in a sialic acid-dependent manner. Untreated or sialidase-treated HS:3 and HS:19(GM1⁺ GT1a⁺) strains were FITC labeled and incubated with human PBMCs (A) or purified NK cells (B). (A) Binding of bacteria was assessed by flow cytometry following labeling with the indicated MAbs: CD3, T cells; CD19, B cells; CD56, NK cells; CD14, monocytes. Values show the bacterial binding indices for each gated population of cells. (B) Binding of bacteria to purified NK cells from two different human donors. Open histograms show background fluorescence in the absence of added bacteria, and shaded histograms show binding signals obtained in the presence of bacteria. Values show the bacterial binding indices for each population of cells.