Multimerization of the virulence-enhancing group A Streptococcus transcription factor RivR is required for regulatory activity

Abstract

ABSTRACT Group A Streptococcus (GAS) (Streptococcus pyogenes) causes more than 700 million human infections each year. The significant morbidity and mortality rates associated with GAS infections are in part a consequence of the ability of this pathogen to coordinately regulate virulence factor expression during infection. RofA-like protein IV (RivR) is a member of the Mga-like family of transcriptional regulators, and previously we reported that RivR negatively regulates transcription of the hasA and grab virulence factor-encoding genes. Here, we determined that RivR inhibits the ability of GAS to survive and to replicate in human blood. To begin to assess the biochemical basis of RivR activity, we investigated its ability to form multimers, which is a characteristic of Mga-like proteins. We found that RivR forms both dimers and a higher-molecular-mass multimer, which we hypothesize is a tetramer. As cysteine residues are known to contribute to the ability of proteins to dimerize, we created a library of expression plasmids in which each of the four cysteines in RivR was converted to serine. While the C68S RivR protein was essentially unaffected in its ability to dimerize, the C32S and C377S proteins were attenuated, while the C470S protein completely lacked the ability to dimerize. Consistent with dimerization being required for regulatory activity, the C470S RivR protein was unable to repress hasA and grab gene expression in a rivR mutant. Thus, multimer formation is a prerequisite for RivR activity, which supports recent data obtained for other Mga-like family members, suggesting a common regulatory mechanism.