(a) Schematic illustrating the primary structure of SpA and of SpAED with the immunoglobulin binding domains (IgBDs, designated E, A, B, C and D), region X (Xr), LysM domain and LPXTG sorting signal. Cleavage sites for signal peptidase and sortase A are indicated. The amino acid sequence of the SpA signal peptide is displayed. YSIRK/GXXS motif residues are printed in red. (b) The structural genes for SpAED and its variants were cloned into pOS1, expressed from the spa promoter in S. aureus WY110 (∆spa ∆sbi) and secretion of SpAED was analyzed by immunoblotting with SpA-specific antibody in culture supernatant (S) and lysostaphin-digested bacterial pellet (P) samples. Signal peptide bearing SpAED precursors are labeled ‘Pre’ on the side of each blot; ‘Mat’ denotes mature protein without signal peptide. The calculated molecular weight (MW) of the variant precursors are: SpAED, 16.78 kD; SpAED/ΔYSIRK,16.13 kD; SpAED/ΔGIAS, 16.45 kD; SpAED/ΔIA, 16.59 kD; SpAED/ΔG15, 16.72 kD; SpAED/ΔS18, 16.69 kD; SpAED/ΔG15ΔS18, 16.63 kD; SpAED/Y7A, 16.68 kD; SpAED/I9S, 16.75 kD; SpAED/R10A, 16.69 kD; SpAED/K11A, 16.72 kD; SpAED/G15L, 16.83 kD; SpAED/S18L, 16.8 kD; SpAED/G15L/S18L, 16.86 kD. The MW of SpAED mature protein is 13.15 kD. Sortase A (SrtA, MW 23.54 kD) immunoblot serves as loading control. (c) Percent secretion of wild-type SpAED and its variants was quantified from triplicate experiments as the intensity of immunoblotting signals in the supernatant (S) divided by the sum signals in (S + P) fractions × 100. Statistical significance was analyzed with one-way ANOVA comparing each variant with wild-type and p values were recorded: WT vs. ∆GIAS, p=0.031; WT vs. ∆IA, p=0.0032; WT vs. R10A, p=0.0116; WT vs. S18L, p=0.0172. * denotes p<0.05, ** denotes p<0.01.