Fig. 4.
Autolysin function is important for maintaining optimal surface levels of S. aureus protein A (Spa). (A) ELISA-based (Abs450nm) detection of IgG-antibody adhesion to Spa exposed on the surface of immobilized whole cells of JE2 (sbi::Tn), complestatin-treated (0.5 and 1.0 µg/mL) JE2-sbi::Tn and JE2-atl H265A_E1129A cells (∆spa). A mutant lacking Spa (JE2-∆spa ∆sbi) was included as a negative control. (B) Immunoblot analysis of Spa abundance in lysed whole cells, (C) culture supernatants, (D) cytoplasm fractions, (E) cell wall fractions, and (F) membrane fractions. For each immunoblot, data points represent the average of three technical replicates for a single biological replicate ± the SD. Representative immunoblots from one biological replicate are shown. P-values were calculated using the two-tailed unpaired Student’s t test comparing each mutant with the wild-type strain (JE2). Statistically significant decreases in adhesion and normalized intensity compared with JE2 were denoted as P ≤ 0.05*, P ≤ 0.01**, P ≤ 0.001***, and P ≤ 0.0001****. Protein abundance was normalized to total protein using a stain-free approach, related to SI Appendix, Fig. S10.