Group B streptococcal capsular sialic acids interact with siglecs (immunoglobulin-like lectins) on human leukocytes

Abstract

Group B Streptococcus (GBS) is classified into nine serotypes that vary in capsular polysaccharide (CPS) architecture but share in common the presence of a terminal sialic acid (Sia) residue. This position and linkage of GBS Sia closely resembles that of cell surface glycans found abundantly on human cells. CD33-related Siglecs (CD33rSiglecs) are a family of Sia-binding lectins expressed on host leukocytes that engage host Sia-capped glycans and send signals that dampen inflammatory gene activation. We hypothesized that GBS evolved to display CPS Sia as a form of molecular mimicry limiting the activation of an effective innate immune response. In this study, we applied a panel of immunologic and cell-based assays to demonstrate that GBS of several serotypes interacts in a Sia- and serotype-specific manner with certain human CD33rSiglecs, including hSiglec-9 and hSiglec-5 expressed on neutrophils and monocytes. Modification of GBS CPS Sia by O acetylation has recently been recognized, and we further show that the degree of O acetylation can markedly affect the interaction between GBS and hSiglec-5, -7, and -9. Thus, production of Sia-capped bacterial polysaccharide capsules that mimic human cell surface glycans in order to engage CD33rSiglecs may be an example of a previously unrecognized bacterial mechanism of leukocyte manipulation.

FIG. 1.

All GBS serotypes described to date contain a terminal α2,3-linked Sia on their CPSs. The repetitive subunits of the CPS of GBS serotypes Ia, Ib, II, III, and V were previously published as Ia/Ib (18), II (19), III (39), and V (38). The repetitive subunits of each serotype are covalently joined end to end to form the CPS. The structure of the serotype VI repetitive subunit is not known but consists of a 2:2:1 molar ratio of glucose, galactose, and N-acetylneuraminic acid, respectively (36).

FIG. 2.

Binding of various group B Streptococcus serotypes to hCD334rSiglecs. All GBS serotypes bind to at least one hCD33rSiglec, while several serotypes interact with multiple hCD33rSiglecs. FITC-GBS were added to hSiglec-Fc chimera-coated wells, and the efficiency of binding was tested. The values represent the means from at least three separate experiments ± standard deviations.

FIG. 3.

Serotype III GBS engages hSiglec-9 through Sias present on the CPS. FITC-GBS (serotype III) were treated with trypsin in order to degrade bacterial surface proteins or with exogenous sialidase to remove all cell surface sialic acid and tested for their ability to bind hSiglec-9-Fc chimeras in comparison to serotype III ΔNeuA. The graphs show mean percent binding ± standard deviation for one representative experiment that was repeated three times with similar results. P values are from two-tailed t tests.

FIG. 4.

Binding of WT serotype III GBS, its isogenic ΔNeuD mutant, and the ΔNeuD mutant complemented with pNeuD-K123A to hSiglec-5, -7, and -9. Values represent the means from three separate experiments ± standard deviations. P values are from two-tailed t tests comparing each sialylated GBS group with the nonsialylated negative control. O-Ac, O acetylation.

FIG. 5.

Sialylated GBS adheres to CHO cells expressing hSiglecs in a Sia- and Siglec-specific manner. (A) FITC-labeled serotype III Sia-negative ΔNeuA (top row), sialylated WT serotype Ia (middle row), and sialylated WT serotype III (bottom row) GBS were allowed to adhere to untransfected CHO cells (first column) or to CHO cells expressing either hSiglec-9 (second column) or hSiglec-5 (third column). (B) Adherence of sialylated serotype III FITC-GBS to CHO cells expressing hSiglec-9 in the presence or absence of 10 μg/ml hSiglec-9-specific Sia-blocking or Sia-nonblocking antibodies. All images were captured using an inverted microscope with an appropriate fluorescent filter set and CCD digital camera. Images are representative of three separate experiments with similar results.

FIG. 6.

GBS attached to the surface of human neutrophils colocalizes with hSiglec-9. FITC-GBS were added to isolated purified human neutrophils and allowed to interact for 5 min, after which cells were fixed and hSiglec-9 was labeled using fluorescent antibodies. Cells were mounted onto slides, and images were acquired using DeltaVision deconvolution microscopy and a CCD camera. Three images representing the nucleus (A) (blue Hoechst stain), bacteria (B) (green FITC), and hSiglec-9 (C) (red phycoerythrin) are shown separately and then combined in one merged image (D). The areas of colocalization between FITC-GBS (green) and hSiglec-9 (red) appear yellow in the merged image. The experiment was repeated three times, and this image is representative of many GBS-neutrophil interactions.