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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.

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Related in: MedlinePlus

GBS expressing β protein colocalize with hSiglec-5 on U937 leukocytes and neutrophils, increasing bacterial attachment but impairing their phagocytosis (A) Colocalization of FITC-WT GBS (green) with hSiglec 5 (red) on the cell surface of U937 cells (blue nuclei) not observed in parallel studies with the isogenic FITC-labeled β protein–deficient mutant, as visualized by deconvolution microscopy. Bar, 5 µm. (B) FITC-labeled GBS were allowed to interact with U937 for 60 min at 4°C (to prevent phagocytosis). Adherence was measured by flow cytometry. (C) Primary human neutrophils were incubated with live GBS-FITC on ice to allow cell–cell interactions but block phagocytosis. The percentage of neutrophils with adherent bacteria was measured using flow cytometry. (D) To calculate phagocytic uptake of GBS, bacteria were added to U937 at MOI 10:1 for 30 min, washed, antibiotics added for 2 h to kill extracellular bacteria, and then cells lysed and dilutions plated to enumerate intracellular CFU. Where indicated, U937 cells were preincubated with anti–Siglec-5 Ab (1-A5) for 50 min, which results in endocytosis of the receptor, leaving ∼10% of original levels of Siglec-5 on the surface before addition of GBS. Horizontal bars represent the mean. Each experiment was performed in triplicate and repeated three times with similar results. Representative images (A) or pooled data (Manders coefficient; B–D) are shown. Error bars represent standard deviation. Statistical analysis was performed using one-way ANOVA with Tukey's post-test.
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fig3: GBS expressing β protein colocalize with hSiglec-5 on U937 leukocytes and neutrophils, increasing bacterial attachment but impairing their phagocytosis (A) Colocalization of FITC-WT GBS (green) with hSiglec 5 (red) on the cell surface of U937 cells (blue nuclei) not observed in parallel studies with the isogenic FITC-labeled β protein–deficient mutant, as visualized by deconvolution microscopy. Bar, 5 µm. (B) FITC-labeled GBS were allowed to interact with U937 for 60 min at 4°C (to prevent phagocytosis). Adherence was measured by flow cytometry. (C) Primary human neutrophils were incubated with live GBS-FITC on ice to allow cell–cell interactions but block phagocytosis. The percentage of neutrophils with adherent bacteria was measured using flow cytometry. (D) To calculate phagocytic uptake of GBS, bacteria were added to U937 at MOI 10:1 for 30 min, washed, antibiotics added for 2 h to kill extracellular bacteria, and then cells lysed and dilutions plated to enumerate intracellular CFU. Where indicated, U937 cells were preincubated with anti–Siglec-5 Ab (1-A5) for 50 min, which results in endocytosis of the receptor, leaving ∼10% of original levels of Siglec-5 on the surface before addition of GBS. Horizontal bars represent the mean. Each experiment was performed in triplicate and repeated three times with similar results. Representative images (A) or pooled data (Manders coefficient; B–D) are shown. Error bars represent standard deviation. Statistical analysis was performed using one-way ANOVA with Tukey's post-test.

Mentions: GBS–U937 monocyte interactions were visualized using fluorescent deconvolution microscopy with a hSiglec-5–specific antibody, a fluorescently tagged (red) secondary antibody to label hSiglec-5, and Hoechst DNA stain (blue) to visualize cell nuclei. WT GBS expressing β protein colocalized with clear aggregations of hSiglec-5 present on the U937 cell surface (Fig. 3 A). Increased quantities of hSiglec-5 at the interface of the ΔBac mutant and the U937 cell surface were not observed; consequently, the Manders coefficient for colocalization of bacteria and hSiglec-5 was significantly less in the ΔBac mutant compared with the WT GBS strain (Fig. 3 A).


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)

GBS expressing β protein colocalize with hSiglec-5 on U937 leukocytes and neutrophils, increasing bacterial attachment but impairing their phagocytosis (A) Colocalization of FITC-WT GBS (green) with hSiglec 5 (red) on the cell surface of U937 cells (blue nuclei) not observed in parallel studies with the isogenic FITC-labeled β protein–deficient mutant, as visualized by deconvolution microscopy. Bar, 5 µm. (B) FITC-labeled GBS were allowed to interact with U937 for 60 min at 4°C (to prevent phagocytosis). Adherence was measured by flow cytometry. (C) Primary human neutrophils were incubated with live GBS-FITC on ice to allow cell–cell interactions but block phagocytosis. The percentage of neutrophils with adherent bacteria was measured using flow cytometry. (D) To calculate phagocytic uptake of GBS, bacteria were added to U937 at MOI 10:1 for 30 min, washed, antibiotics added for 2 h to kill extracellular bacteria, and then cells lysed and dilutions plated to enumerate intracellular CFU. Where indicated, U937 cells were preincubated with anti–Siglec-5 Ab (1-A5) for 50 min, which results in endocytosis of the receptor, leaving ∼10% of original levels of Siglec-5 on the surface before addition of GBS. Horizontal bars represent the mean. Each experiment was performed in triplicate and repeated three times with similar results. Representative images (A) or pooled data (Manders coefficient; B–D) are shown. Error bars represent standard deviation. Statistical analysis was performed using one-way ANOVA with Tukey's post-test.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig3: GBS expressing β protein colocalize with hSiglec-5 on U937 leukocytes and neutrophils, increasing bacterial attachment but impairing their phagocytosis (A) Colocalization of FITC-WT GBS (green) with hSiglec 5 (red) on the cell surface of U937 cells (blue nuclei) not observed in parallel studies with the isogenic FITC-labeled β protein–deficient mutant, as visualized by deconvolution microscopy. Bar, 5 µm. (B) FITC-labeled GBS were allowed to interact with U937 for 60 min at 4°C (to prevent phagocytosis). Adherence was measured by flow cytometry. (C) Primary human neutrophils were incubated with live GBS-FITC on ice to allow cell–cell interactions but block phagocytosis. The percentage of neutrophils with adherent bacteria was measured using flow cytometry. (D) To calculate phagocytic uptake of GBS, bacteria were added to U937 at MOI 10:1 for 30 min, washed, antibiotics added for 2 h to kill extracellular bacteria, and then cells lysed and dilutions plated to enumerate intracellular CFU. Where indicated, U937 cells were preincubated with anti–Siglec-5 Ab (1-A5) for 50 min, which results in endocytosis of the receptor, leaving ∼10% of original levels of Siglec-5 on the surface before addition of GBS. Horizontal bars represent the mean. Each experiment was performed in triplicate and repeated three times with similar results. Representative images (A) or pooled data (Manders coefficient; B–D) are shown. Error bars represent standard deviation. Statistical analysis was performed using one-way ANOVA with Tukey's post-test.
Mentions: GBS–U937 monocyte interactions were visualized using fluorescent deconvolution microscopy with a hSiglec-5–specific antibody, a fluorescently tagged (red) secondary antibody to label hSiglec-5, and Hoechst DNA stain (blue) to visualize cell nuclei. WT GBS expressing β protein colocalized with clear aggregations of hSiglec-5 present on the U937 cell surface (Fig. 3 A). Increased quantities of hSiglec-5 at the interface of the ΔBac mutant and the U937 cell surface were not observed; consequently, the Manders coefficient for colocalization of bacteria and hSiglec-5 was significantly less in the ΔBac mutant compared with the WT GBS strain (Fig. 3 A).

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