Functional Domains in the Multigene Regulator of the Group A Streptococcus
Vahling, Cheryl M.
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The group A streptococcus (GAS) is a human pathogen capable of causing a broad range of symptoms from mild erythema to severe tissue destruction. The multigene regulator of the GAS, or Mga, has been shown to bind DNA and activate transcription of virulence genes implicated in colonization and immune evasion in response to environmental conditions. Several different avenues of investigation were undertaken in the present study with an overall goal of identifying and characterizing functional domains within the protein. The first section of this study uses a naturally occurring mutant, previously identified to be deficient in transcriptional activation, as a guide to map functional residues within Mga. Electrophoretic mobility shift assays using purified proteins determined that the defect was not a result of the inability to bind DNA, so gain-of-function mutants were used to restore transcriptional activity to the mutant and pinpoint the residues necessary for full activation. In the second section, a genetic screen was undertaken to identify novel domains within the protein. From this screen, a domain was found within the extreme N-terminus. Sequence homology revealed several proteins in other pathogenic treptococci that shared this motif. As a result, it was named CMD-1 for conserved Mga domain 1, while the group of proteins was subsequently designated as the Mga family of putative virulence gene regulators. Alanine scanning mutagenesis demonstrated the importance of CMD-1 for transcriptional activation in several members of this family. This established that the results yielded from investigations of Mga could be broadened beyond the GAS to provide global insight into possible mechanisms of virulence regulation in other pathogenic streptococci. Finally, an in silico analysis of Mga was performed, revealing several areas with differing degrees of structural similarity to known domains of other bacteria including a receiver domain and a PTS-regulatory domain. Each region was explored to determine if it was functionally active in Mga. Since both domains have been implicated in forming higher order structures, the oligomeric state of Mga was also determined. Overall, characterization of Mga has helped unravel the mechanisms of virulence regulation in pathogenic streptococci.