A Staphylococcus aureus Small RNA Is Required for Bacterial Virulence and Regulates the Expression of an Immune-Evasion Molecule
Figure 2
The SprD regulates expression of the Sbi protein at translational level in vivo.
(A) Northern blot analysis of the SprD expression at the E phase (OD600nm: 2) in wild-type N315 (WT), N315 isogenic ΔsprD mutant (Δ),ΔsprD transformed by pCN38ΩsprD (complemented strain ‘ΔSprD’) and also in strains RN4220 and SH1000 transformed by pCN38ΩsprD. (B) Coomassie staining of SDS–PAGE of the exoproteins in N315, RN4220 and SH1000 strains expressing, or not, SprD at the E phase (OD600nm: 2). The arrows point to the reduced levels of a protein when SprD is expressed. (C) Immunoblot analysis with anti-Sbi antibodies of extra- and intracellular proteins in the three S. aureus strains at the E phase (OD600nm: 2). (D) Monitoring the expression of the Sbi protein during S. aureus growth in strains N315 (WT) and ΔsprD (Δ) by immunoblots with anti-Sbi antibodies separated on the same gel. The graph shows the quantification of the Sbi protein levels in both strains relative to the total protein amount (Figure S5). The blue squares represent the ΔsprD and the red triangles represent WT. The superimposable growth curves of the two strains are represented as the dashed lines. (E) Northern blot analysis of the sbi mRNA in wild-type N315 (WT) and ΔsprD mutant (Δ) during bacterial growth. 16S rRNAs are loading controls. The graph shows the quantification of the sbi mRNA levels in both strains relative to 16S rRNA and the colours correspond to panel D. We have measured the sbi mRNA half-life in WT SH1000 strain using rifampicin treatment, which is about 1 min (data not shown).