Involvement of lipid rafts and actin remodeling in erythrocyte invasion by <i>S. pneumoniae</i>.

<p>Erythrocytes were pretreated with or without 5βCD or 20 µM cytochalasin D for 30 minutes at 4°C, then <i>S. pneumoniae</i> cells were added and incubated for 1 hour at 37°C in a 5% CO₂ atmosphere. The numbers of invaded bacteria were determined as described in the Experimental Procedures section. The experiments were performed 3 times and data shown represent the mean of 6 wells from a representative experiment. S.D. values are represented by vertical lines. *<i>P</i><0.005; **<i>P</i><0.05.</p

Effects of erythrocytes and iron ions on <i>S. pneumoniae</i> growth.

<p><i>A</i>. Growth of <i>S. pneumoniae</i> strains R6 and D39, and <i>S. aureus</i> strain Cowan-I in the presence of human erythrocytes with or without an iron chelator. Bacterial cells were incubated for 2, 4, and 6 hours at 37°C in a 5% CO₂ atmosphere. <i>B</i>. Growth of <i>S. pneumoniae</i> strains R6 and D39, and <i>S. aureus</i> strain Cowan-I in erythrocyte intracellular solution (erythrocyte lysates without membrane) with or without an iron chelator for 2, 4, and 6 hours at 37°C in a 5% CO₂ atmosphere. <i>C</i>. Growth of <i>S. pneumoniae</i> strains R6 and D39, and <i>S. aureus</i> strain Cowan-I in RPMI 1640 medium with or without an iron chelator for 2, 4, and 6 hours at 37°C in a 5% CO₂ atmosphere. The experiments were performed 3 times and data shown represent the mean of 3 wells from a representative experiment. S.D. values are represented by vertical lines.</p

<i>S. pneumoniae</i> invasion of human erythrocytes.

<p><i>A</i>. Gram staining of cultures of blood obtained from a patient with invasive pneumococcal pneumonia. A blood sample was obtained from a splenectomized patient with pneumococcal bacteremia and meningitis. We observed that some of the <i>S. pneumoniae</i> cells in the sample adhered to or invaded erythrocytes. <i>B</i>. SEM analysis of <i>S. pneumoniae</i> in blood. <i>S. pneumoniae</i> cells (arrows) were incubated in human whole blood for 30 minutes at 37°C. Strains R6 and D39 adhered to (a, c) and invaded (b, d) erythrocytes in human blood. <i>C</i>. Confocal fluorescence microscopic analysis of <i>S. pneumoniae</i> strains R6 (a, b) and D39 (c, d) incubated with human erythrocytes for 30 minutes at 37°C. (a, c) Erythrocytes were visualized using Alexa Fluor 594 Phalloidin. <i>S. pneumoniae</i> organisms were stained using SYTOX green. (b) Boxed areas from panel (a), along with x–z and y-z projections. (d) 3D analysis of image from panel (c) showing erythrocytes invaded by <i>S. pneumoniae</i>. <i>D</i>. Rate of <i>S. pneumoniae</i> invasion of erythrocytes. The numbers of invaded bacteria were determined as described in the Experimental Procedures section. *Significant difference (<i>P</i><0.005) between mean values, as determined with a Mann-Whitney <i>U</i>-test. The experiments were performed 3 times and data are shown as the mean of 6 wells from a representative experiment. S.D. values are represented by vertical lines. <i>E</i>. Histopathological examinations of infected mice lung tissues. Tissues were excised from sites of infection after 72 hours, then fixed, embedded in paraffin, and stained with hematoxylin-eosin solution. (a) and (b) were obtained from individual mice. Arrows indicate association of <i>S. </i><i>pneumoniae</i> with erythrocytes. (c) Numbers of bacteria associated with erythrocytes per field. Data shown represent the mean of 10 fields from a representative mouse. S.D. values are represented by vertical lines.</p

Erythrocytes inhibit pneumococcal growth by reactive oxygen species-related mechanism.

<p><i>S. pneumoniae</i> cells (∼1×10² CFU, 10 µl) were added to erythrocytes (5×10⁹ cells/ml, 190 µl) with or without 1 mM 2,2′-bipyridyl (iron chelator), 1 mM S-ethyl-ITU (nitric oxide synthase inhibitor), 100 µM EUK8 (synthetic catalytic free radical scavenger), or 150 µM MnTBAP (superoxide dismutase mimetic) for 2 hours at 37°C in a 5% CO₂ atmosphere. Next, each mixture was serially diluted and plated on TS blood agar. Following incubation, CFU values were determined. *Significant difference (<i>P</i><0.005) between mean values, as determined with a Mann-Whitney <i>U</i>-test. The experiments were performed 3 times and data are shown as the mean of 6 wells from a representative experiment. S.D. values are represented by vertical lines.</p

PCR primers used in this study.

<p>PCR primers used in this study.</p

Erythrocyte intracellular solution inhibits pneumococcal growth in presence of erythrocytes with the membrane.

<p><i>A.</i> Growth of <i>S. pneumoniae</i> strain D39 in RPMI 1640 medium with or without human hemoglobin and/or an iron chelator for 2, 4, and 6 hours at 37°C in a 5% CO₂ atmosphere. <i>B.</i> Growth of <i>S. pneumoniae</i> strain D39 in erythrocyte lysates with or without the erythrocyte membrane and/or an iron chelator for 2, 4, and 6 hours at 37°C in a 5% CO₂ atmosphere. The experiments were performed 3 times and data shown represent the mean of 3 wells from a representative experiment. S.D. values are represented by vertical lines.</p

Erythrocytes inhibit killing of <i>S. pneumoniae</i>.

<p><i>A</i>. Inhibition of killing by neutrophils. <i>S. pneumoniae</i> cells (R6∶2.2×10² CFU/well, D39∶1.4×10² CFU/well) were incubated with human neutrophils (1×10⁵ cells/well), then erythrocytes (5.0×10⁷ cells/well) and/or 10% human blood serum (HBS) or heat-inactivated HBS were added to the mixture. Viable CFU were counted following 1, 2, and 3 hours of incubation. <i>B.</i> Inhibition of killing by H₂O₂. <i>S. pneumoniae</i> cells were incubated in 0%, 0.03%, or 0.30% H₂O₂-RPMI 1640, then viable CFU were counted following 1, 2, and 3 hours of incubation. The experiments were performed 3 times and data shown represent the mean of 6 wells from a representative experiment. S.D. values are represented by vertical lines.</p

H₂O₂ produced by <i>S</i>. <i>sanguinis</i> induced release of NETs from infected neutrophils.

<p>Neutrophils were infected with the <i>S</i>. <i>sanguinis</i> strains at an MOI of 10. Exogenously added H₂O₂ (1 mM) or PMA (200 nM) served as a control. Following incubation for 3 h, cells were reacted with SYTOX Green (50 nM), then observed with a fluorescent microscope. Bar, 20 μm.</p

Bacterial association with human neutrophils.

<p>Human neutrophils were pre-incubated for 30 min, then infected with the WT, KO, or Wr strain at an MOI of 10. Following incubation for 0.5 or 1 h, cells were fixed with methanol and visualized by Giemsa staining. Using phase-contrast microscopy, 10 random fields (x 1000) were observed and the amount of bacteria demonstrating phagocytosis by neutrophils was determined by counting bacterial chains. (A) Representative images. (B) The numbers of bacterial chains associated with neutrophils at 0.5 and 1 h after infection are shown as the mean ± SD of 5 independent experiments. Statistically significant differences were evaluated using one-way ANOVA and Tukey’s multiple comparison test. *<i>p</i><0.01, **<i>p</i><0.05.</p

Extracellular DNA from neutrophils increased by streptococcal H₂O₂.

<p>Neutrophils were infected with the <i>S</i>. <i>sanguinis</i> strains at an MOI of 10 with or without catalase (100 units/ml), and cultured for 1–3 h. Neutrophils treated with 200 nM of PMA served as a control. Cells were reacted with SYTOX Green and fluorescent intensity was determined. Data are presented as the mean ± SD of triplicate samples from 3 independent experiments. Statistically significant differences were evaluated using two-way ANOVA and Tukey’s multiple comparison test. *<i>p</i><0.01.</p