Theses and Dissertations from UMD
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New submissions to the thesis/dissertation collections are added automatically as they are received from the Graduate School. Currently, the Graduate School deposits all theses and dissertations from a given semester after the official graduation date. This means that there may be up to a 4 month delay in the appearance of a give thesis/dissertation in DRUM
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Item FUNCTIONAL CHARACTERIZATION OF THE LINK BETWEEN CARBOHYDRATE METABOLISM AND THE PATHOGENESIS OF THE INVASIVE M1T1 GROUP A STREPTOCOCCUS(2016) Valdes, Kayla Maureen; McIver, Kevin S; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The Group A Streptococcus (GAS), or Streptococcus pyogenes, is a strict human pathogen that colonizes a variety of sites within the host. Infections can vary from minor and easily treatable, to life-threatening, invasive forms of disease. In order to adapt to niches, GAS utilizes environmental cues, such as carbohydrates, to coordinate the expression of virulence factors. Research efforts to date have focused on identifying how either components of the phosphoenolpyruvate-phosphotransferase system (PTS) or global transcriptional networks affect the regulation of virulence factors, but not the synergistic relationship between the two. The present study investigates the role of a putative PTS-fructose operon encoded by fruRBA and its role in virulence in the M1T1 strain 5448. Growth in fructose resulted in induction of fruRBA. RT-PCR showed that fruRBA formed an operon, which was repressed by FruR in the absence of fructose. Growth and carbon utilization profiles revealed that although the entire fruRBA operon was required for growth in fructose, FruA was the main fructose transporter. The ability of both ΔfruR and ΔfruB mutants to survive in whole human blood or neutrophils was impaired. However, the phenotypes were not reproduced in murine whole blood or in a mouse intraperitoneal infection, indicating a human-specific mechanism. While it is known that the PTS can affect activity of the Mga virulence regulator, further characterization of the mechanism by which sugars and its protein domains affect activity have not been studied. Transcriptional studies revealed that the core Mga regulon is activated more in a glucose-rich than a glucose-poor environment. This activation correlates with the differential phosphorylation of Mga at its PTS regulatory domains (PRDs). Using a 5448 mga mutant, transcriptome studies in THY or C media established that the Mga regulon reflects the media used. Interestingly, Mga regulates phage-encoded DNases in a low glucose environment. We also show that Mga activity is dependent on C-terminal amino acid interactions that aid in the formation of homodimers. Overall, the studies presented sought to define how external environmental cues, specifically carbohydrates, control complex regulatory networks used by GAS, contribute to pathogenesis, and aid in adaptation to various nutrient conditions encountered.Item Transcriptional Activation by the group A streptococcal Mga Regulator(2012) Hause, Lara L.; McIver, Kevin S; Molecular and Cell Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)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.