Theses and Dissertations from UMD

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Subject: search.filters.subject.Streptococcus pyogenes

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    THE STANDALONE REGULATOR ROFA OF STREPTOCOCCUS PYOGENES EXHIBITS CHARACTERISTICS OF A PRD-CONTAINING VIRULENCE REGULATOR
    (2024) Hart, Meaghan Taylor; McIver, Kevin S; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    Streptococcus pyogenes (Group A Streptococcus; GAS) is a human pathogen estimated to cause nearly 790 million cases of disease annually at diverse tissue sites. To successfully infect these sites, GAS must detect nutrient availability and adapt accordingly. One mechanism employed to detect and import carbohydrates is the phosphoenolpyruvate transferase system ‎ (PTS), which mediates both carbohydrate uptake and metabolic gene regulation. Gene regulation by the PTS can occur through phosphorylation of transcriptional regulators at conserved PTS-regulatory domains (PRDs). GAS has several stand-alone regulators that contain PRDs, with corresponding regulons encoding both metabolic genes and important virulence factors. These regulators form a family called PRD-Containing Virulence Regulators (PCVRs). RofA is a putative member of this family and is known to regulate the expression of genes important for virulence. It was hypothesized that RofA is phosphorylated by the PTS in response to carbohydrate levels to coordinate appropriate virulence gene expression. In this dissertation, the RofA regulon was determined in strain 5448, a representative strain of the globally disseminated M1T1 serotype. The pilus and capsule operons were consistently dysregulated across growth in the absence of RofA. This correlated with increased capsule production and decreased adherence to primary keratinocytes. Purified RofA-His was phosphorylated in vitro by the general PTS components EI and HPr, and phosphorylated species of RofA-FLAG were detected in vivo late in stationary phase in a glucose-dependent manner. Together, these findings support the hypothesis that RofA is a PCVR that may couple sugar detection and utilization with GAS virulence gene regulation. Additionally, a bioluminescent construct was generated for allelic exchange into any S. pyogenes strain. Allelic exchange of this construct into WT 5448 yielded strains that were highly bioluminescent, grew to a similar density as WT, and survived as well as WT when challenged with human neutrophils. This tool could be used to study the contribution of specific proteins on in vivo virulence in a non-invasive manner, including RofA and RofA phosphorylation.
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    CHARACTERIZING THE ROLE OF THE PHOSPHOENOLPYRUVATE-DEPENDENT PHOSPHOTRANSFERASE SYSTEM ENZYME II LOCI IN THE PATHOGENESIS OF THE GROUP A STREPTOCOCCUS
    (2017) Sundar, Ganesh; McIver, Kevin S; Molecular and Cell Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    The Group A Streptococcus (GAS, Streptococcus pyogenes) is a Gram-positive human pathogen that must adapt to unique host environments in order to survive. Links between sugar metabolism and virulence have been demonstrated in GAS, where mutants in the phosphoenolpyruvate-dependent phosphotransferase system (PTS) exhibited Streptolysin S (SLS)-mediated hemolysis during exponential growth. This early onset hemolysis correlated with an increased lesion size and severity in a murine soft tissue infection model when compared with parental M1T1 MGAS5005. To identify the PTS components responsible for this phenotype, we insertionally inactivated the 14 annotated PTS EIIC-encoding genes in the GAS MGAS5005 genome to functionally characterize each EIIC. It was found that a few EIIs had a limited in uence on PTS sugar metabolism, whereas others were promiscuous. The mannose-speci c EII locus exhibited the most in uence on PTS sugar metabolism. Importantly, the mannose-speci c EII also acted to prevent the early onset of SLS-mediated hemolysis. These roles were not identical in two different M1T1 GAS strains, highlighting the versatility of the PTS to adapt to strain-speci c needs. This is further illustrated by the fructose-speci c EII, which is important for survival in whole human blood for MGAS5005, but not 5448. The mannose-speci c EII can transport glucose in other pathogens, but the route of glucose utilization is unknown in GAS. MGAS5005 mutants were generated in a non-PTS glucose transporter (GlcU) and a glucokinase (NagC) of an annotated non-PTS glucose metabolic pathway. Since ∆ptsI, ∆nagC, and ∆glcU all grow to some extent in glucose, it is evident that glucose can be metabolized both by PTS and non-PTS routes. . However, the route of glucose utilization affects overall pathogenesis, as ∆nagC survives like WT in whole human blood, whereas ptsI is unable to survive. Subcutaneous infection of mice with ∆nagC did not exhibit increased lesion size, although these lesions are more severe than MGAS5005 due to the early onset of hemolysis. Overall this suggests that the routes of glucose metabolism greatly in uence SLS-mediated hemolysis. These results highlight that PTS carbohydrate metabolism plays an important role for GAS pathogenesis in both the skin and whole human blood, through the actions of EIIs.
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    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.
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    Characterization of the TrxSR Two-Component Signal Transduction System of Streptococcus pyogenes and its Role in Virulence Regulation
    (2011) Gold, Kathryn; McIver, Kevin; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    The Gram-positive group A streptococcus (GAS) is a strict human pathogen, which causes a wide variety of infections, ranging in severity from minor to life threatening. In order to cause such a diverse array of diseases, GAS utilizes two-component signal transduction systems (TCS) to coordinately regulate sets of virulence genes in response to changing host conditions. The present study investigates the role of the TrxSR TCS in the regulation of virulence of the GAS. Using an insertional inactivation mutation in TrxR in serotype M1 MGAS5005, transcriptome studies established that TrxR activates transcription of Mga-regulated virulence genes, a separate non-TCS regulatory pathway controlling factors important for immune evasion and colonization. Transcriptional reporter fusions of Pmga to firefly luciferase revealed that the TrxR regulation occurs through the Pmga promoter. Additionally, electrophoretic mobility shift assays using purified His-MBP tagged TrxR established specific binding of TrxR to Pmga, although the interaction appeared to be transient. To determine the importance of signal transduction for TrxR-mediated regulation of the Mga regulon and virulence, an in vitro reconstitution assay was performed with purified TrxR and TrxS. Using both wild type and mutated forms of the TrxSR proteins, we demonstrated that TrxSR is a functional two-component phosphorelay system. Interestingly, phosphorylation of TrxR did not appear to be critical for DNA binding and regulation, since a TrxR D55A mutation did not change the expression of TrxR regulated genes in GAS based on EMSA and qPCR. In order to investigate whether there is a functional conservation of TrxR's involvement in GAS virulence regulation, mutations were made in serotype M4 and M49 strains representing either throat only or generalist strains. We have determined that TrxR regulates mga and Mga-regulated genes (emm, arp) in the M4 and M49 backgrounds, suggesting conservation of TrxR's role in virulence regulation. Overall, TrxSR represents a functional TCS that appears to directly regulate the Mga virulence regulon independent of phosphorelay. Furthermore, the functional conservation of TrxR regulation of Mga in other serotypes suggests a conserved role for its involvement in virulence regulation in GAS.