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

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 Addition of 100 mM bovine serum albumin had no effect on SAP binding (unpublished data).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

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

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

ScpA activity impairs the host neutrophil response to GAS.

<p>A) Survival of GAS-M1 and GAS-M1_ΔscpA in whole human blood was quantified by the classical Lancefield assay to determine resistance to neutrophil-mediated killing. Data represent six individual blood donors (each data point is mean of 4 technical replicates), line denotes median value (Mann Whitney U, ** = p<0.001). B) The role of C5a signaling on GAS survival in whole human blood was quantified by Lancefield assay. Whole blood was pre-treated with C5aR1 inhibitor PMX205 (1 μM) [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006493#ppat.1006493.ref028" target="_blank">28</a>] prior to incubation with either GAS-M1 or GAS-M1Δ_scpA. Each graph represents an independent donor (mean+/- SD 4 technical replicates, Mann Whitney U, * = p<0.05). C) Neutrophil-mediated uptake of fluorescent GAS-M1 and GAS-M1_ΔscpA was quantified following incubation with purified human neutrophils. Each graph represents percentage of neutrophils bearing fluorescent GAS from an independent donor (mean+/- SD 4 technical replicates, Mann Whitney U, * = p<0.05). D) Neutrophil activation was assessed by quantification of surface expression of CD11b on purified neutrophils following incubation with GAS-M1 or GAS-M1_ΔscpA. Each graph represents an independent neutrophil donor (mean+/- SD 4 technical replicates, Mann Whitney U, * = p<0.05). E) Quantification of neutrophil migration along a chemokine gradient. Absolute number of purified human neutrophils migrating towards C3a or C5a +/- pre-treatment with rScpA was quantified following 30 minute incubation at 37°C. Each graph represents an independent neutrophil donor (4 technical replicates (mean +/- SD), Mann Whitney U, * = p<0.05). F-G) Neutrophil activation by C3a and C5a +/- pre-treatment with rScpA was quantified in a calcium mobilization assay. Calcium transients in fluo-4-AM labelled neutrophils were elicited by C3a or C5a, and calcium derived MFI was recorded on a FACSCalibur from 0–120 seconds. F) Overall MFI of all neutrophils at 120 seconds were compared. Each graph represents an independent neutrophil donor (4 technical replicates (mean +/- SD), Mann Whitney U, * = p<0.05). G) Calcium flux over 120 seconds was visualized following kinetic analysis. Data presented as a histogram of one donor, representative of 3 donors.</p

Streptococcal species cleave human C3.

<p>A) Cleavage of human C3 by representative strains of Groups A (GAS-M1), C (<i>S</i>. <i>zoo</i> and <i>S</i>. <i>equi</i>) and G (GGS) streptococci. Bacterial pellets (4x10⁶ cfu) were incubated with human C3 for 16 hours, 37°C. Specific cleavage of the C3α chain was visualized following SDS-PAGE and resulted in release of a 100 kDa product, C3α^scpA (white arrow). No cleavage of the β chain was observed. B) The impact of a panel of protease inhibitors on C3 cleavage by GAS-M1 cell pellets was assessed. GAS-M1 pellets (4x10⁶ cfu) were incubated with human C3 for 16 hours at 37°C in the presence of individual protease inhibitors and compared for ability to generate cleavage fragment C3α^scpA (white arrow). Concentration of each protease inhibitor used is indicated. The serine protease Pefabloc was sufficient to inhibit streptococcal cleavage of C3.</p

ScpA requires serum factors to mediate GAS adhesion to epithelial and endothelial cells.

<p>A+B) Adhesion of A) GAS-M1 and B) GAS-M89 and isogenic ΔscpA strains to A549 epithelial cells was compared by quantitative culture (30 min incubation). Data represent mean+/-SD of 3 experimental replicates (T-test * < 0.05). C+D) Adhesion of C) GAS-M1 and D) GAS-M89 and isogenic ΔscpA strains to primary human HUVEC was compared by quantitative culture (30 min incubation). Data represent mean+/-SD of 3 experimental replicates (T-test * < 0.05, ** < 0.001). E) Adherence of rScpA protein to paraformaldehyde-fixed human HUVEC. Bound rScpA was quantified following incubation with anti-ScpA mouse serum and goat-anti mouse HRP antibody and detection with TMB. Adhesion was compared in the presence and absence of 10% FCS. Data represent mean+/-SD of 3 experimental replicates (T-test * < 0.05). F) Adhesion of GAS-M1 and GAS-M1_ΔscpA to the murine endothelial cell line MCEC-1 was compared by quantitative culture (30 min incubation). Data represent mean+/-SD of 3 experimental replicates (T-test * < 0.05).</p

ScpA activity impairs C3 deposition on the GAS surface.

<p>A) C3 deposition on GAS-M1 and GAS-M1_ΔscpA surface was compared following incubation with 0% to 50% human serum. Deposition of C3 on the bacterial surface was quantified using FITC-conjugated anti-C3. Data represent mean +/- SD of 3 serum donors. C3 deposition was not seen when 50% heat-inactivated (HI) serum was used. B) GAS-M89 and isogenic ΔscpA strains were incubated with 50% human serum for 30 minutes, at 37°C. Deposition of C3 on the bacterial surface was quantified using FITC-conjugated anti-C3. Data represent mean +/- SD of 3–4 technical replicates produced from 3 serum donors. (One-tailed T-test * = p<0.05). In order to combine the percentage of C3-positive bacteria and the binding intensity, C3 deposition is presented as fluorescence index (FI), calculated as the proportion of positive bacteria expressed as a percentage multiplied by the gMFI [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006493#ppat.1006493.ref052" target="_blank">52</a>]. C) Visualization of serum-mediated degradation of C3α chain. Degradation of physiological C3bα (right hand panel) and equivalent streptococcal ScpA cleavage product C3α^scpA (left hand panel) in serum was assessed over 60 minutes. Purified human C3 or C3b were incubated with rScpA for 16 hours, at 37°C. The relative degradation of resulting C3bα and C3α^scpA moieties by 1% C3-depleted serum was visualized by SDS-PAGE. Degradation of both C3α^scpA and C3bα was observed. D) The rate of degradation of physiological C3bα and the equivalent streptococcal cleavage product C3α^scpA was compared following quantification by densitometry and visualized as a line graph.</p

ScpA enhances streptococcal pathogenesis in wild type and complement deficient mice.

<p>A) C3 deposition on GAS-M1 and GAS-M1_ΔscpA surface was compared following incubation with 50% murine serum. Deposition of C3 on the bacterial surface was quantified using FITC-conjugated anti-mouse C3. Data are presented as fluorescence index (FI), calculated as the proportion of positive bacteria expressed as a percentage multiplied by the gMFI (line depicts median of four technical replicates Mann Whitney U, * = p<0.05). B-D) Characterization of GAS-M1 and GAS-M1_ΔscpA dissemination from the site of infection (thigh) to the draining inguinal lymph node (LN) in a murine model of soft tissue infection. Comparison of dissemination in B) wildtype C57BL/6 mice (n = 8/group), C) C5^-/- mice (C57BL/6 background) (n = 9/group) and D) C3^-/-/C5^-/- mice (C57BL/6 background) (n = 8/group). Line depicts median value. (Mann Whitney U, No difference (ND) = p>0.05, * = p<0.05). E) Systemic spread of GAS-M1 and GAS-M1_ΔscpA in C3^-/-/C5^-/-mice. Quantitative culture of bacteria recovered from spleen, liver and blood of infected mice.</p

Strains used in this study.

<p>Strains used in this study.</p