Figure 1.
SMSB4 reduces the blood killing of S. aureus strain Xen29 in whole blood (A) and pyoderma isolates of S. aureus (B).
SMSB4 promotes bacteria growth in a concentration dependent manner (C) similarly to CVF (D). S. aureus Xen29 or pyoderma isolates MRSA strains (HS16, M34), MSSA strains (HS56, M5) were harvested from mid-log growth phase culture. Bacteria (1×105 cfu/ml) were challenged with whole blood pre-treated with either 100 µg/ml SMSB4, positive control 10 µg/ml CVF, negative controls 100 µg/ml BSA or GVB2+ buffer only. S. aureus cells in PBS only without blood was also included to illustrate that the reduction in bacteria number was due to blood killing (A). Numbers of bacteria were counted as cfu/ml at various time points (A) or at 3 h (B, C, D). Bacterial recovery was calculated as a percentage of the challenge dose. Results are shown as means ± SEM from three independent experiments. The statistical significance of differences between samples was estimated using two way ANOVA with Tukey’s multiple comparison test. **, p<0.01; ***, p<0.001; ****, p<0.0001, ns, not significant (B).
Figure 2.
SMSB4 reduces phagocytosis of S. aureus by neutrophils.
FITC-labeled bacteria (105 cfu) was opsonized either with 20% NHS or heated serum and challenged with neutrophils (106 cells). Serum samples were pre-treated for 30 min at 37°C with 50 µg/ml SMSB4, 50 µg/ml BSA (A), or various concentrations of SMSB4 or BSA (B). Uptake of FITC by neutrophils was measured at various time points over 1 h (A) or at 40 min (B). Results are shown as means ± SEM from three independent experiments.
Figure 3.
SMSB4 reduces deposition of C3b (A) and MAC complex (SC5b-9) formation (B).
The wells of 96-well microtiter plates were coated with 100 µl aliquots of bacterial cell suspensions containing 5×106 cfu/ml of S. aureus. Wells were then incubated with 10% NHS which has been pre-treated with increasing concentrations of either SMSB4 or BSA. Antibodies were detected by ELISA using primary human specific antibodies, followed by HRP-conjugated secondary antibodies, and fluorescence was detected at 490 nm. Results are shown as means ± SEM from three independent experiments.
Figure 4.
Effect of SMSB4 on the depositions of C4b (A), C1q, MBL and properdin (B) on S. aureus cells.
The wells of 96-well microtiter plates were coated with 100 µl aliquots of bacterial cell suspensions containing 5×106 cfu/ml of S. aureus. Wells were then incubated with 10% NHS which has been pre-treated with increasing concentrations of either SMSB4 or BSA. Antibodies were detected by ELISA using primary human specific antibodies, followed by HRP-conjugated secondary antibodies, and fluorescence was detected at 490 nm. Results are shown as means ± SEM from three independent experiments. The statistical significance of differences between BSA and SMSB4 treated samples were estimated using two way ANOVA with Sidak’s multiple comparison test. **, p<0.01; ***, p<0.001; ns, not significant (B).