Figure 3.

De-repression of cepA in a low producer of Streptococcus pyogenes cell envelope proteinase (SpyCEP), by covR/S mutation. A, Western blot analysis for SpyCEP in culture supernatant and cell wall extract from wild type (WT) emm75 pharyngeal isolate and ΔcovR/S mutant grown to early-log (EL), mid-log (ML) and late-log (LL) phases of growth. B, Coomassie blue-stained sodium dodecyl sulphate-polyacrylamide gel electrophoresis showing IL-8 cleavage after incubation with supernatant from WT or ΔcovR/S mutant. IL-8 incubated in Todd-Hewitt broth alone was used as a control. As a positive control for cleavage, IL-8 was incubated with supernatant from the high SpyCEP producer, H292. C, No. of copies of cepA transcript per 10,000 copies of gyrA transcript measured by real-time polymerase chain reaction comparing WT (black bars) and ΔcovR/S mutant (white bars) at EL, ML and LL phase. Each bar denotes the mean of 3 independent samples measured in triplicate (+standard deviation). The effect of the CovR/S mutation was reproduced in another pharyngeal emm2 isolate.