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. 2015 Mar 17;83(4):1598–1609. doi: 10.1128/IAI.03122-14

FIG 5.

FIG 5

Release of protein A into S. aureus culture supernatants can be inhibited by altering the sorting signal. (A) Protein A on the surface of LAC* spa sbi(pSpA) (black bars) and LAC* spa sbi(pSpAΩSdrESS) (white bars) was detected using FITC-labeled rabbit IgG, and the fluorescence intensity was measured using flow cytometry. Values are expressed as a percentage of the mean fluorescence intensity measured for LAC* spa sbi(pSpA) grown in broth supplemented with ATc (312.5 ng/ml). Bars represent the mean of three independent experiments. Error bars represent the standard errors of the means. ns, not significant (P > 0.05). (B) LAC* spa sbi(pSpA) and LAC* spa sbi(pSpAΩSdrESS) were grown in broth supplemented with ATc (312.5 ng/ml), and culture supernatants were probed with HRP-conjugated rabbit IgG. Size markers are indicated (kDa). (C) Quantification of SpA in culture supernatants of LAC* spa sbi(pSpA) and LAC* spa sbi(pSpAΩSdrESS) by ELISA. Bacteria were grown in broth supplemented with ATc (312.5 ng/ml), and SpA was captured from culture supernatants using chicken anti-SpA polyclonal IgY. Bound SpA was detected using biotin-labeled mouse monoclonal anti-SpA IgG and HRP-conjugated streptavidin in an ELISA. The absorbance at 450 nm was measured, and readings from wells incubated with culture supernatants from LAC* spa sbi(pRMC2) were subtracted from the mean readings for LAC* spa sbi(pSpA) and LAC* spa sbi(pSpAΩSdrESS) to account for background. Values for LAC* spa sbi(pSpA ΩSdrESS) are expressed as a percentage of the values measured for LAC* spa sbi(pSpA). Bars represent the mean of three independent experiments, and error bars represent the standard errors of the means. ***, P < 0.0001.