Limits...
Group B Streptococcus suppression of phagocyte functions by protein-mediated engagement of human Siglec-5.

Carlin AF, Chang YC, Areschoug T, Lindahl G, Hurtado-Ziola N, King CC, Varki A, Nizet V - J. Exp. Med. (2009)

Bottom Line: A key GBS virulence factor is its capsular polysaccharide (CPS), displaying terminal sialic acid (Sia) residues which block deposition and activation of complement on the bacterial surface.We report the unexpected discovery that certain GBS strains may bind one such receptor, hSiglec-5, in a Sia-independent manner, via the cell wall-anchored beta protein, resulting in recruitment of SHP protein tyrosine phosphatases.We conclude that protein-mediated functional engagement of an inhibitory host lectin receptor promotes bacterial innate immune evasion.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics, University of California, San Diego, La Jolla, CA 92093, USA.

ABSTRACT
Group B Streptococcus (GBS) is a leading cause of invasive bacterial infections in human newborns. A key GBS virulence factor is its capsular polysaccharide (CPS), displaying terminal sialic acid (Sia) residues which block deposition and activation of complement on the bacterial surface. We recently demonstrated that GBS Sia can bind human CD33-related Sia-recognizing immunoglobulin (Ig) superfamily lectins (hCD33rSiglecs), a family of inhibitory receptors expressed on the surface of leukocytes. We report the unexpected discovery that certain GBS strains may bind one such receptor, hSiglec-5, in a Sia-independent manner, via the cell wall-anchored beta protein, resulting in recruitment of SHP protein tyrosine phosphatases. Using a panel of WT and mutant GBS strains together with Siglec-expressing cells and soluble Siglec-Fc chimeras, we show that GBS beta protein binding to Siglec-5 functions to impair human leukocyte phagocytosis, oxidative burst, and extracellular trap production, promoting bacterial survival. We conclude that protein-mediated functional engagement of an inhibitory host lectin receptor promotes bacterial innate immune evasion.

Show MeSH

Related in: MedlinePlus

The GBS β protein mediates attachment to hSiglec-5-Fc in a Sia-independent manner. (A and B) WT GBS serotype III (A) and Ia (B) and their isogenic Sia-deficient ΔNeuA mutants were incubated with hSiglec-9-Fc and hSiglec-5-Fc, respectively, followed by PE-conjugated secondary antibodies, and analyzed by flow cytometry for Sia-dependent binding of hSiglecs. Similar analysis was performed with WT GBS serotype III (A) and Ia (B) pretreated with or without trypsin to determine protein-dependent binding of hSiglecs. (C) Western blot analysis of GBS whole cell lysates from GBS serotype Ia and III ΔNeuA mutants with hSiglec-Fc chimeras as probes. The analysis identified an ∼125-kD specific hSiglec-5 binding protein in the Ia strain. (D) Analysis of GBS extracts prepared under basic conditions (pH 9.7) also identified an ∼125-kD protein that binds hSiglec5. (E) AUS treatment (50 mU/ml for 2 h) does not affect binding of the 125-kD protein to hSiglec5. (F) WT GBS serotype Ia and isogenic mutant ΔBac (lacking the β protein), or the plasmid complemented mutant ΔBac + pBac, were incubated with hSiglec-5-Fc and PE-conjugated secondary antibody and analyzed by flow cytometry. All experiments were performed in duplicate and repeated three (A and B) or four (C–F) times with similar results. Representative experiments are shown.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC2722167&req=5

fig1: The GBS β protein mediates attachment to hSiglec-5-Fc in a Sia-independent manner. (A and B) WT GBS serotype III (A) and Ia (B) and their isogenic Sia-deficient ΔNeuA mutants were incubated with hSiglec-9-Fc and hSiglec-5-Fc, respectively, followed by PE-conjugated secondary antibodies, and analyzed by flow cytometry for Sia-dependent binding of hSiglecs. Similar analysis was performed with WT GBS serotype III (A) and Ia (B) pretreated with or without trypsin to determine protein-dependent binding of hSiglecs. (C) Western blot analysis of GBS whole cell lysates from GBS serotype Ia and III ΔNeuA mutants with hSiglec-Fc chimeras as probes. The analysis identified an ∼125-kD specific hSiglec-5 binding protein in the Ia strain. (D) Analysis of GBS extracts prepared under basic conditions (pH 9.7) also identified an ∼125-kD protein that binds hSiglec5. (E) AUS treatment (50 mU/ml for 2 h) does not affect binding of the 125-kD protein to hSiglec5. (F) WT GBS serotype Ia and isogenic mutant ΔBac (lacking the β protein), or the plasmid complemented mutant ΔBac + pBac, were incubated with hSiglec-5-Fc and PE-conjugated secondary antibody and analyzed by flow cytometry. All experiments were performed in duplicate and repeated three (A and B) or four (C–F) times with similar results. Representative experiments are shown.

Mentions: We tested WT GBS isolates expressing sialylated CPS of serotypes Ia (strain A909) and III (strain COH1), isogenic Sia-negative mutants of each strain generated by allelic replacement of the CMP-Neu5Ac-synthase gene (ΔNeuA), and GBS pretreated with trypsin to remove cell surface proteins, for binding to chimeras of hSiglec5 (hSiglec-5-Fc) and hSiglec-9 (hSiglec-9-Fc) by flow cytometry. Corroborating our previous ELISA-based findings (Carlin et al., 2007), serotype III GBS bound to hSiglec-9-Fc and serotype Ia bound to hSiglec-5 (Fig. 1, A and B). The GBS serotype III strain bound to hSiglec-9-Fc in a Sia-specific manner because this interaction was lost for the isogenic Sia-negative ΔNeuA mutant but unaltered by trypsin treatment (Fig. 1 A). In contrast, attachment of the GBS serotype Ia strain to hSiglec-5-Fc was maintained in the Sia-deficient mutant and eliminated by trypsin treatment (Fig. 1 B), linking binding to a trypsin-sensitive protein rather than the anticipated sugar–lectin interaction.


Group B Streptococcus suppression of phagocyte functions by protein-mediated engagement of human Siglec-5.

Carlin AF, Chang YC, Areschoug T, Lindahl G, Hurtado-Ziola N, King CC, Varki A, Nizet V - J. Exp. Med. (2009)

The GBS β protein mediates attachment to hSiglec-5-Fc in a Sia-independent manner. (A and B) WT GBS serotype III (A) and Ia (B) and their isogenic Sia-deficient ΔNeuA mutants were incubated with hSiglec-9-Fc and hSiglec-5-Fc, respectively, followed by PE-conjugated secondary antibodies, and analyzed by flow cytometry for Sia-dependent binding of hSiglecs. Similar analysis was performed with WT GBS serotype III (A) and Ia (B) pretreated with or without trypsin to determine protein-dependent binding of hSiglecs. (C) Western blot analysis of GBS whole cell lysates from GBS serotype Ia and III ΔNeuA mutants with hSiglec-Fc chimeras as probes. The analysis identified an ∼125-kD specific hSiglec-5 binding protein in the Ia strain. (D) Analysis of GBS extracts prepared under basic conditions (pH 9.7) also identified an ∼125-kD protein that binds hSiglec5. (E) AUS treatment (50 mU/ml for 2 h) does not affect binding of the 125-kD protein to hSiglec5. (F) WT GBS serotype Ia and isogenic mutant ΔBac (lacking the β protein), or the plasmid complemented mutant ΔBac + pBac, were incubated with hSiglec-5-Fc and PE-conjugated secondary antibody and analyzed by flow cytometry. All experiments were performed in duplicate and repeated three (A and B) or four (C–F) times with similar results. Representative experiments are shown.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2722167&req=5

fig1: The GBS β protein mediates attachment to hSiglec-5-Fc in a Sia-independent manner. (A and B) WT GBS serotype III (A) and Ia (B) and their isogenic Sia-deficient ΔNeuA mutants were incubated with hSiglec-9-Fc and hSiglec-5-Fc, respectively, followed by PE-conjugated secondary antibodies, and analyzed by flow cytometry for Sia-dependent binding of hSiglecs. Similar analysis was performed with WT GBS serotype III (A) and Ia (B) pretreated with or without trypsin to determine protein-dependent binding of hSiglecs. (C) Western blot analysis of GBS whole cell lysates from GBS serotype Ia and III ΔNeuA mutants with hSiglec-Fc chimeras as probes. The analysis identified an ∼125-kD specific hSiglec-5 binding protein in the Ia strain. (D) Analysis of GBS extracts prepared under basic conditions (pH 9.7) also identified an ∼125-kD protein that binds hSiglec5. (E) AUS treatment (50 mU/ml for 2 h) does not affect binding of the 125-kD protein to hSiglec5. (F) WT GBS serotype Ia and isogenic mutant ΔBac (lacking the β protein), or the plasmid complemented mutant ΔBac + pBac, were incubated with hSiglec-5-Fc and PE-conjugated secondary antibody and analyzed by flow cytometry. All experiments were performed in duplicate and repeated three (A and B) or four (C–F) times with similar results. Representative experiments are shown.
Mentions: We tested WT GBS isolates expressing sialylated CPS of serotypes Ia (strain A909) and III (strain COH1), isogenic Sia-negative mutants of each strain generated by allelic replacement of the CMP-Neu5Ac-synthase gene (ΔNeuA), and GBS pretreated with trypsin to remove cell surface proteins, for binding to chimeras of hSiglec5 (hSiglec-5-Fc) and hSiglec-9 (hSiglec-9-Fc) by flow cytometry. Corroborating our previous ELISA-based findings (Carlin et al., 2007), serotype III GBS bound to hSiglec-9-Fc and serotype Ia bound to hSiglec-5 (Fig. 1, A and B). The GBS serotype III strain bound to hSiglec-9-Fc in a Sia-specific manner because this interaction was lost for the isogenic Sia-negative ΔNeuA mutant but unaltered by trypsin treatment (Fig. 1 A). In contrast, attachment of the GBS serotype Ia strain to hSiglec-5-Fc was maintained in the Sia-deficient mutant and eliminated by trypsin treatment (Fig. 1 B), linking binding to a trypsin-sensitive protein rather than the anticipated sugar–lectin interaction.

Bottom Line: A key GBS virulence factor is its capsular polysaccharide (CPS), displaying terminal sialic acid (Sia) residues which block deposition and activation of complement on the bacterial surface.We report the unexpected discovery that certain GBS strains may bind one such receptor, hSiglec-5, in a Sia-independent manner, via the cell wall-anchored beta protein, resulting in recruitment of SHP protein tyrosine phosphatases.We conclude that protein-mediated functional engagement of an inhibitory host lectin receptor promotes bacterial innate immune evasion.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics, University of California, San Diego, La Jolla, CA 92093, USA.

ABSTRACT
Group B Streptococcus (GBS) is a leading cause of invasive bacterial infections in human newborns. A key GBS virulence factor is its capsular polysaccharide (CPS), displaying terminal sialic acid (Sia) residues which block deposition and activation of complement on the bacterial surface. We recently demonstrated that GBS Sia can bind human CD33-related Sia-recognizing immunoglobulin (Ig) superfamily lectins (hCD33rSiglecs), a family of inhibitory receptors expressed on the surface of leukocytes. We report the unexpected discovery that certain GBS strains may bind one such receptor, hSiglec-5, in a Sia-independent manner, via the cell wall-anchored beta protein, resulting in recruitment of SHP protein tyrosine phosphatases. Using a panel of WT and mutant GBS strains together with Siglec-expressing cells and soluble Siglec-Fc chimeras, we show that GBS beta protein binding to Siglec-5 functions to impair human leukocyte phagocytosis, oxidative burst, and extracellular trap production, promoting bacterial survival. We conclude that protein-mediated functional engagement of an inhibitory host lectin receptor promotes bacterial innate immune evasion.

Show MeSH
Related in: MedlinePlus