Structural and functional analysis of RopB: a major virulence regulator in Streptococcus pyogenes

Summary Group A Streptococcus (GAS) is an exclusive human pathogen that causes significant disease burden. Global regulator RopB of GAS controls the expression of several major virulence factors including secreted protease SpeB during high cell density. However, the molecular mechanism for RopB‐dependent speB expression remains unclear. To understand the mechanism of transcription activation by RopB, we determined the crystal structure of the C‐terminal domain of RopB. RopB‐CTD has the TPR motif, a signature motif involved in protein–peptide interactions and shares significant structural homology with the quorum sensing RRNPP family regulators. Characterization of the high cell density‐specific cell‐free growth medium demonstrated the presence of a low molecular weight proteinaceous secreted factor that upregulates RopB‐dependent speB expression. Together, these results suggest that RopB and its cognate peptide signals constitute an intercellular signalling machinery that controls the virulence gene expression in concert with population density. Structure‐guided mutational analyses of RopB dimer interface demonstrated that single alanine substitutions at this critical interface significantly altered RopB‐dependent speB expression and attenuated GAS virulence. Results presented here suggested that a properly aligned RopB dimer interface is important for GAS pathogenesis and highlighted the dimerization interactions as a plausible therapeutic target for the development of novel antimicrobials. Streptococcus pyogenes, also known as group A Streptococcus (GAS), is a strict human pathogen that causes an estimated ∼ 600 million cases of strep throat and 500,000 cases of invasive disease‐related deaths worldwide. Given the lack of a vaccine for human, there is an urgent need to identify novel therapeutic targets to treat GAS infections. GAS produces a secreted toxin, SpeB, which is important for spread of infection and host tissue destruction. Using a multidisciplinary approach, we demonstrated that speB expression is controlled by intercellular communication machinery that includes the intracellular receptor RopB and its cognate peptide signals. Structural studies and structure‐aided mutational analyses of RopB identified intersubunit interactions that are critical for RopB function. Findings from this study not only advanced the mechanistic understanding of a virulence regulating signaling pathway but also pave the foundation for the development of novel therap