Fig. 1: Opsonophagocytic killing activity (OPKA) of anti-AdcA serum against different S. aureus strains.

A schematic representation of rabbit immunization (A) and mechanism of OPKA (B). Antigen-specific antibodies and complement proteins opsonise bacteria and facilitate the uptake of the antibody-bacteria complex by phagocytes (PMN). C OPKA against S. aureus strains MW2, (D) LAC, and (E) Reynolds were tested with sera raised against the recombinant AdcA from E. faecium, used at total IgG concentrations ranging from 1 to 0.5 mg/mL (grey bars). The effectiveness of opsonophagocytic killing by the anti-AdcA rabbit sera (MW2 - C, LAC – D, and Reynolds - E) was compared to that by the pre-immune rabbit sera (white bars). Statistical significance was tested by the unpaired two-tailed T test with a 95% confidence interval with a Bonferroni post hoc test using pre-immune and terminal immune sera at the same concentration. Bars and whiskers denote mean values ± standard errors of the mean of replicates within one assay. *P ≤ 0.025, **P ≤ 001, ***P ≤ 0.001. F Schematic representation of an OPIA assay. Antibodies, once engaged by their antigens (inhibitors) are unable to opsonise, leading to a decrease in bacterial killing. G OPIA of anti-AdcA rabbit serum performed by incubation of serum with recombinant AdcA protein and tested against S. aureus MW2. Antibodies raised against the AdcA at 0,5 mg/mL of total IgG concentration were pre-incubated with different amounts of AdcA (black bars) in a range from 0.14 to 3.56 µM. Pre-immune serum (P) at 0,5 mg/mL of total IgG concentration was used as a negative control, and anti-AdcA sera (C) without incubation with AdcA, at 0.5 mg/mL of total IgG concentration was used as a positive control. Statistical significance of inhibition was performed by the One-way analysis of variances test, followed by the Dunnett’s multiple comparison post hoc test. Bars and whiskers denote mean values ± standard errors of the mean of replicates within one assay. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.