Fig 1.
Streptococcal species cleave human C3.
A) Cleavage of human C3 by representative strains of Groups A (GAS-M1), C (S. zoo and S. equi) 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, C3αscpA (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 C3αscpA (white arrow). Concentration of each protease inhibitor used is indicated. The serine protease Pefabloc was sufficient to inhibit streptococcal cleavage of C3.
Fig 2.
ScpA is necessary and sufficient for cleavage of human C3.
A-C) Cleavage of human complement factors A) C3, B) C3a and C) C5a by whole cell pellets (4x106 cfu) of GAS-M1 vs GAS-M1ΔscpA, GAS-M89 vs GAS-M89ΔscpA and rScpA (100 ng). Only bacterial strains expressing ScpA were able to cleave these complement factors. Cleaved moieties (C3αscpA, C3ascpA, C5ascpA) are indicated by a white arrow in each panel. D) C3αscpA was subjected to N-terminal sequencing. ScpA was demonstrated to cleave C3 between Ala741 and Ser742, 7aa N-terminal to the physiological C3 convertase cleavage site. E) Schematic representation of C3 α and β chains and the internal ScpA cleavage site. C3 is cleaved by C3 convertase to release C3b (grey bar) and C3a (white bar). The vertical black line between them shows the physiological C3 convertase cleavage site. ScpA cleaves C3 at an alternative site (red dotted line, black arrow) to release unique cleavage products, C3ascpA and C3αscpA (red text).
Fig 3.
Rapid cleavage of complement factors by streptococcal ScpA.
Rate of rScpA cleavage of complement components was assessed at 37°C and visualized by SDS-PAGE and Coomassie staining. A) Cleavage of C3 by rScpA at 1:1 molar ratio. C3αscpA cleavage product (white arrow) was detectable after incubation with rScpA for 30 minutes, and became more pronounced by 60 minutes. B) Cleavage of C3a by rScpA at 10:1 molar ratio. C3a appeared to be completely cleaved to C3ascpA (white arrow) following 0.5 minutes incubation with rScpA. C) Cleavage of C5a by rScpA at 10:1 molar ratio. C5ascpA cleavage product (white arrow) was visible following 0.5 minutes incubation with rScpA. Complete cleavage was observed within 60 minutes.
Fig 4.
ScpA activity impairs C3 deposition on the GAS surface.
A) C3 deposition on GAS-M1 and GAS-M1ΔscpA 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 [52]. C) Visualization of serum-mediated degradation of C3α chain. Degradation of physiological C3bα (right hand panel) and equivalent streptococcal ScpA cleavage product C3αscpA (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 C3αscpA moieties by 1% C3-depleted serum was visualized by SDS-PAGE. Degradation of both C3αscpA and C3bα was observed. D) The rate of degradation of physiological C3bα and the equivalent streptococcal cleavage product C3αscpA was compared following quantification by densitometry and visualized as a line graph.
Fig 5.
ScpA activity impairs the host neutrophil response to GAS.
A) Survival of GAS-M1 and GAS-M1ΔscpA 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) [28] prior to incubation with either GAS-M1 or GAS-M1ΔscpA. 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-M1ΔscpA 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-M1ΔscpA. 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.
Fig 6.
ScpA enhances streptococcal pathogenesis in wild type and complement deficient mice.
A) C3 deposition on GAS-M1 and GAS-M1ΔscpA 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-M1ΔscpA 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-M1ΔscpA in C3-/-/C5-/-mice. Quantitative culture of bacteria recovered from spleen, liver and blood of infected mice.
Fig 7.
ScpA requires serum factors to mediate GAS adhesion to epithelial and endothelial cells.
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-M1ΔscpA 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).
Fig 8.
Catalytic activity of ScpA is not required for pathogenesis in vivo.
A+B) Cleavage of human complement factors A) C3a and B) C5a by whole cell pellets (4x106 cfu) of GAS-M89ΔpC, GAS-M89ΔpCscpA and GAS-M89ΔpCcat2. Only strain GAS-M89ΔpCscpA, expressing catalytically active ScpA was able to cleave either complement factor. Cleaved moieties (C3ascpA and C5ascpA) are indicated by a white arrow in each panel. C+D) Characterization of GAS-M89ΔpC, GAS-M89ΔpCscpA and GAS-M89ΔpCcat2 dissemination in a murine model of soft tissue infection in wildtype CD57/BL6 mice (n = 5/group). Quantitative culture of tissue homogenates (C) from the site of infection (thigh) to (D) the draining inguinal lymph node (LN). Line depicts median value. (Mann Whitney U, No difference (ns) = p>0.05, * = p<0.05).
Table 1.
Strains used in this study.