Binding of hSAP to ST2, ST4, and ST23F Strains of S. pneumoniae

<div><p>(A) Results of whole-cell ELISAs presented as maximum ODs after incubation with different concentrations of hSAP (given below each column as μg/ml), using the E. coli O111:B4 strain as a negative control. Error bars represent SDs, and asterisks mark significant <i>p</i>-values for comparisons of results for S. pneumoniae in 20 μg/ml hSAP versus medium alone (2-tailed <i>t</i> tests, *<i>p</i> < 0.01, ***<i>p</i> < 0.0001).</p><p>(B) Examples of flow cytometry histograms demonstrating hSAP binding to the surface of the three different S. pneumoniae strains, the E. coli O111:B4 strain, and the S. pyogenes H372 strain (positive control) after incubation in human serum.</p><p>(C) Effect of different concentrations of EDTA on SAP binding to the ST2 S. pneumoniae strain in human serum. Error bars represent SDs, and asterisks mark significant <i>p</i>-values for comparisons of results for EDTA versus serum alone (2-tailed <i>t</i> tests, ***<i>p</i> < 0.0001).</p><p>(D) Effect of addition of 100 mM PC on SAP and CRP binding to the ST2 S. pneumoniae strain in human serum. Grey columns, results for human serum; white columns, results for human serum in the presence of PC. Addition of 100 mM bovine serum albumin had no effect on SAP binding (unpublished data). Error bars represent SDs, and <i>p</i>-values are indicated above the columns.</p><p>(E) Examples of flow cytometry histograms demonstrating inhibition of hSAP binding to the surface of the ST2 S. pneumoniae strains by addition of 100 mM PC to human serum.</p></div

Effect of SAP on Phagocytosis of S. pneumoniae

<div><p>(A–C) Phagocytosis (presented as proportion of HL60 cells associated with fluorescent bacteria) of (A) ST2, (B) ST4, and (C) ST23F after incubation in different dilutions of serum from wild-type (circles) or <i>Apcs^−/−</i> (squares) mice. Results for incubation in HBSS are shown by the triangle symbol, and for the ST2 strain the results for a 50:50 mix of serum from wild-type and <i>Apcs^−/−</i> mice (diamonds) are also included.</p><p>(D) Example of a flow cytometry histogram of phagocytosis of ST2 S. pneumoniae by HL60 cells after incubation in HBSS or serum from wild-type or <i>Apcs^−/−</i> mice.</p><p>(E) Effect of addition of 5 or 50 μg/ml exogenous hSAP on phagocytosis of the ST2 S. pneumoniae strain in serum from <i>Apcs^−/−</i> mice.</p><p>(F) Effect of addition of hSAP (50 μg/ml) on phagocytosis of the ST2 S. pneumoniae strain in serum from <i>Apcs</i>^−/−<i>.C1qa</i>^−/− mice. Grey column, results for <i>Apcs</i>^−/−<i>.C1qa</i>^−/− serum; white column, results for <i>Apcs</i>^−/−<i>.C1qa</i>^−/− serum in the presence of hSAP. For panels (A–C), (E), and (F), asterisks mark significant <i>p</i>-values for comparisons of results for wild-type or mixed serum to <i>Apcs^−/−</i> serum (2-tailed <i>t</i> tests, *<i>p</i> < 0.01, **<i>p</i> < 0.001, ***<i>p</i> < 0.0001). All error bars represent SDs and when not visible are too small to be seen outside the symbol.</p></div

Clearance of the ST2 S. pneumoniae Strain from Wild-Type and <i>Apcs</i>^−/− Mice Inoculated Intravenously with 1.0 × 10⁶ cfu

<div><p>Each data point represents log₁₀ cfu/ml results for a single mouse, with the bar showing the median for each group.</p><p>(A) Results for blood 2 h after inoculation.</p><p>(B) Results for blood 4 h after inoculation.</p><p>(C) Results for spleen homogenates 4 h after inoculation. Data is obtained from one experiment that is representative of two separate experiments.</p><p><i>p</i>-Values for Mann–Whitney <i>U</i> comparisons between wild-type and <i>Apcs</i>^−/− mice are given below the title for each panel.</p></div

Effects of SAP on C3b Deposition on S. pneumoniae Measured Using Flow Cytometry

<div><p>(A) Time course of the proportion of ST2 S. pneumoniae bacteria positive for C3b after incubation in serum from wild-type and <i>Apcs^−/−</i> mice. For the comparison of results for wild-type versus <i>Apcs^−/−</i> mice, <i>p</i> < 0.001 at all time points from 1 to 20 min.</p><p>(B and C) Proportion of ST4 (B) and ST23F (C) S. pneumoniae bacteria positive for C3b after incubation for 20 min in serum from wild-type and <i>Apcs^−/−</i> mice.</p><p>(D and E) Examples of flow cytometry histograms of C3b deposition on ST2 (D) and ST23F (E) S. pneumoniae strains after incubation in PBS or serum from wild-type or <i>Apcs^−/−</i> mice.</p><p>(F) Effect on the proportion of ST2 S. pneumoniae positive for C3b of addition of 10 μg/ml hSAP to serum from <i>Apcs^−/−</i> mice.</p><p>(G) Effect on the proportion of ST2 S. pneumoniae positive for C3b of addition of an equal volume of serum from wild-type mice to serum from <i>Apcs^−/−</i> mice.</p><p>(H) Effect of addition of hSAP (50 μg/ml, white column) on C3b deposition on the ST2 S. pneumoniae strain in serum from <i>Apcs</i>^−/−<i>.C1qa</i>^−/− mice. Grey columns, results for <i>Apcs</i>^−/−<i>.C1qa</i>^−/− serum; white columns, results for <i>Apcs</i>^−/−<i>.C1qa</i>^−/− serum in the presence of hSAP.</p><p>For panels (A–C) and (F–H), error bars represent SDs, and in (A) when not visible are too small to be seen outside of the symbol. <i>p</i>-Values are calculated using 2-tailed <i>t</i> tests.</p></div

Role in virulence of the different strains using a sepsis model of infection.

<p>(A) Virulence represented as a competitive index (CI) of <i>lytB</i>, <i>lytC</i> and <i>lytB lytC</i> strains compared to their parental D39 wild-type strain. Error bars represent SDs of the means. For the comparisons of the results of the CI of the <i>lytC</i> strain vs. <i>lytB</i> **, <i>P</i><0.01 (two-tailed Student's <i>t</i> test). For the comparisons of the double <i>lytB lytC</i> strain vs. the single <i>lytB</i> or <i>lytC</i> strains ***, <i>P</i><0.001 and *, <i>P</i><0.05 respectively (two-tailed Student's <i>t</i> test). (B) CIs of <i>lytB</i> strain compared to strain D39 and CIs of <i>lytB lytC</i> mutants compared to <i>lytC</i> strain to assess the combined effect of LytB and LytC in sepsis. **, <i>P</i><0.01 for the comparison of both CIs (two-tailed Student's <i>t</i> test).</p

C3b deposition on <i>lytB</i>, <i>lytC</i> and <i>lytB lytC</i> D39 strains using a flow cytometry assay.

<p>(A) Proportion of C3b deposition at 30°C. (B) Example of a flow cytometry histogram for C3b deposition at 30°C. (C) Proportion of C3b deposition at 37°C. (D) Example of a flow cytometry histogram for C3b deposition at 37°C. Error bars represent the SDs and asterisks indicate statistical significance compared to the wild-type strain (two-tailed Student's <i>t</i> test; *, <i>P</i><0.05; **, <i>P</i><0.01; ***, <i>P</i><0.001). <i>P</i><0.001 (at 30°C and 37°C) for the comparison of the results for <i>lytB lytC</i> versus the single mutants. For the results for all defective strains compared to wild-type strain at both temperatures, <i>P</i><0.001 (one-way ANOVA with Dunnett's post hoc test). (E) Restoration of the C3b levels on <i>lytB</i>, <i>lytC,</i> and <i>lytB lytC</i> mutants in the presence of 2 µg of the indicated purified CWHs. Open bars indicate C3b deposition without the addition of exogenous proteins (–), whereas blackened bars represent C3b levels in the presence of LytB, LytC or a mixture of both proteins (+).</p

Opsonophagocytosis mediated by human neutrophils using a flow cytometry assay.

<p>(A) Phagocytosis of D39 strain and <i>lytB</i>, <i>lytC</i> and <i>lytB lytC</i> strains incubated in 20% human serum and expressed as percent fluorescent indices relative to the results for the wild-type D39 strain. (B) Example of a flow cytometry histogram for phagocytosis. (C) Restoration of the levels of phagocytosis of <i>lytB</i>, <i>lytC</i> and <i>lytB lytC</i> mutants in the presence of 2 µ]g of the indicated purified CWHs. Open bars represent phagocytosis without addition of proteins. Grey bar shows phagocytosis of wild-type strain in the presence of LytB and LytC proteins. Striped, blackened, and hatched bars indicate, respectively, phagocytosis of <i>lytB</i>, <i>lytC</i>, or <i>lytB lytC</i> mutants in the presence of LytB, LytC, or a mixture of both proteins, respectively. Error bars represent SDs and asterisks represent statistical significance compared to the wild-type strain (two-tailed Student's <i>t</i> test; *, <i>P</i><0.05; **, <i>P</i><0.01; ***, <i>P</i><0.001). <i>P</i><0.0001 for the overall comparison in phagocytosis (one-way ANOVA with a post hoc Dunnett test). For the comparison of the phagocytosis of the different mutant strains compared to the phagocytosis in the presence of exogenous proteins (<i>P</i><0.05 for <i>lytB</i> and <i>P</i><0.01 for <i>lytC</i> or <i>lytB lytC</i> mutants).</p

Nasopharyngeal colonization by clinical <i>S. pneumoniae</i> isolates and CWHs mutants in a mouse model.

<p>(A) Colonization curve of mice inoculated intranasally with <i>S. pneumoniae</i> TIGR4 (triangles) and D39 (circles). Number of pneumococci recovered from the nasopharynx of the infected mice is expressed as Log₁₀ CFU ml⁻¹. (B) Results of the nasopharyngeal colonization levels at 24 h from mice infected with TIGR4 (open bars) or D39 (solid bars) and the correspondent <i>lytB</i>, <i>lytC</i> and <i>lytB lytC</i> mutant strains. Error bars represent the SDs, and asterisks mark results that are statistically significant compared with wild-type strains (two-tailed Student's <i>t</i> test; *, <i>P</i><0.05; **, <i>P</i><0.01; ***, <i>P</i><0.001). For the <i>lytB lytC</i> double mutant strain vs. <i>lytB</i> or <i>lytC</i> mutants, <i>P</i> values are: <i>P</i><0.01 for TIGR4 and <i>P</i><0.001 for D39 (Student's unpaired <i>t</i> test, 2-tailed). <i>P</i><0.001 for the overall comparison (one-way ANOVA with a post hoc Dunnett test). (C) Nasopharyngeal colonization levels at 120 h from mice infected with D39 and the correspondent <i>lytB</i>, <i>lytC,</i> and <i>lytB lytC</i> mutants. Error bars represent the SDs, and asterisks mark results that are statistically significant compared with the wild-type strain (two-tailed Student's <i>t</i> test; ***, <i>P</i><0.001). <i>P</i><0.001 for the overall comparison (one-way ANOVA with a post hoc Dunnett test).</p

Impact of mutations in the genes encoding LytB and LytC on pneumococcal pneumonia.

<p>(A to C) CIs of <i>lytB</i>, <i>lytC</i> and the double <i>lytB lytC</i> strains compared to their parental D39 wild-type strain in BALF (A), lungs (B), and blood (C) after intranasal inoculation of a mixed culture of the corresponding mutant with the wild-type strain. Error bars represent SDs of the mean and asterisks indicate results that are statistically significant compared to those for the <i>lytB</i> strain which was outcompeted (two-tailed Student's <i>t</i> test; *, <i>P</i><0.05; **, <i>P</i><0.01; ***, <i>P</i><0.001). (D) CIs of <i>lytB</i> vs. D39 (open bars) and <i>lytB lytC</i> vs. <i>lytC</i> (blackened bars) in BALF, lungs, and blood after intranasal infection. **, <i>P</i><0.01 for the comparison of both CIs at the corresponding sites of infection (two-tailed Student's <i>t</i> test).</p

Phagocytosis mediated by murine alveolar macrophages.

<p>(A) Adhesion of D39 wild-type strain and <i>lytB</i>, <i>lytC</i> and <i>lytB lytC</i> strains to alveolar macrophages. (B and C) Phagocytosis of the wild-type D39 and CWHs defective strains by alveolar macrophages. Error bars represent the SDs and asterisks indicate statistical significance compared to the wild-type strain (two-tailed Student's <i>t</i> test; *, <i>P</i><0.05; **, <i>P</i><0.01; ***, <i>P</i><0.001). <i>P</i><0.05 and <i>P</i><0.01 for the comparison of the results in adhesion and phagocytosis for <i>lytB lytC</i> vs. <i>lytB</i> or <i>lytC</i> respectively. <i>P</i><0.001 for the overall comparison in phagocytosis (one-way ANOVA with a post hoc Dunnett test).</p