Transcriptional Activation by the group A streptococcal Mga Regulator

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Hause, Lara L.
McIver, Kevin S
Streptococcus pyogenes (Group A Streptococcus, GAS) is a Gram-positive obligate human pathogen that causes a range of diseases at many different tissue sites. The ability of this organism to colonize and persist within these various niches of the body correlates with broad changes in gene expression. Mga, the multiple gene regulator of GAS, is an important global transcriptional regulator of virulence genes that encode factors promoting adhesion, host cell invasion and immune evasion. Mga directly activates these genes by binding to specific promoter sites that range from 45 to 60 nucleotides in size based on DNAseI footprint analysis; however identified Mga binding sites share less than 50% DNA sequence similarity, making the identification of a consensus Mga binding site difficult. We have identified nucleotides necessary for Mga binding in the Mga-regulated Pemm promoter from the clinically relevant M1 MGAS5005 strain of GAS. Random and directed mutations were assessed for effects on transcription in vivo and DNA binding in vitro. This screen identified predominately Gs and Cs, in two clusters at the 3' and 5' end that suggest that Mga binds DNA as a dimer and reduced the Pemm binding site to 35 bp. However directed mutagenesis in other binding sites found that these interactions were not necessarily conserved. These experiments also sought to establish a method to study genome-wide DNA binding and can successfully enrich for Mga-regulated genes. Protein-protein interactions with RNA polymerase are another key component to activate transcription. Functional in vitro transcription assays and in vitro co-purification assays were performed to determine if Mga interacts with either the alpha C terminal domain or domain 4 of sigma. While Mga does appear to make protein-protein contacts with the holoenzyme, they do not occur through either domain alone. The dimerization of Mga through its EIIB domain was established by analytical ultracentrifugation. In vitro transcription assays linked phosphorylation by the phosphoenolpyruvate transferase system to the down regulation of Mga activity. By understanding how Mga interacts with essential elements of its promoters, this study seeks to define Mga's role in regulating virulence in this important human pathogen.