Figure 2.

WTA-specific IgM supports immune-mediated clearance of S. aureus in vitro and in mouse infection experiments

(A) Complement C3b deposition on S. aureus N315 wild-type (WT) or staphylococcal protein A-deficient (Δspa) bacteria, pre-opsonized with 4497-IgG2 (0.3–270 nM) or 4497-IgM (0.01–10 nM). Error bars indicate SD of the mean of biological triplicates. Multiple unpaired t tests with Holm-Šídák correction were used to compare antibody-mediated C3b deposition on N315 WT and Δspa for either IgM or IgG2.

(B) Relative complement C3b deposition on SpA-deficient S. aureus (Newman Δspa/sbi), pre-opsonized with 4497-IgG2 (10 nM) or 4497-IgM (1 nM) in presence of recombinant protein A (SpA, 0.15–100 nM). C3b deposition in the absence of SpA was set at 100%; the dotted line represents background C3b deposition without antibody opsonization. Error bars indicate SEM of the mean of biological triplicates. Differences between 4497-IgM and 4497-IgG2 were assessed using a two-way ANOVA with Šídák correction.

(C) Neutrophil opsonophagocytic killing (OPK) of S. aureus N315 WT, in presence of 10 nM 4497-IgM, 10 nM 4497-IgG2, or no antibody control. Bacterial survival has been normalized over inoculum (dotted line at 100%); data are shown from three independent donors (mean + SD). Statistical analysis was performed using a one-way ANOVA with Tukey correction.

(D) Spleen CFU counts from mice (n = 10–15 per group) at 24 h post infection with S. aureus N315 WT, passively immunized with 30 μg 4497-IgM, anti-TNP-IgM, or PBS control. Data were pooled from three independent experiments. Statistical analysis was performed using a Kruskal-Wallis test with Dunn’s correction. ns, non-significant, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. See also Figure S3.