Theses and Dissertations from UMD
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Item 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.Item Characterization of luminous bacteria as a biosensing element for detection of acrylamide in food.(2014) Ganjavi, Maryam; Lo, Y. Martin; Food Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)World Health Organization (WHO) has called for further research into acrylamide, a known carcinogen and neurotoxin in animals, following emergency consultations to review data from studies that revealed the presence of acrylamide in starch-based foods fried and baked at high temperatures. The presence of acrylamide in food is recognized as a major concern in humans based on the ability to induce cancer and heritable mutations in laboratory animals. The objective of this study was to characterize the cellular-level damage of acrylamide by bioluminescence stress fingerprinting. Five genetically engineered strains containing selected stress-responsive E. coli promoters fused to the luxCDABE reporter were employed. One of the strains containing DNA damage responsive promoter, DPD2222, was also employed in conjunction with alkaline and neutral comet assay to assess respective single- and double-stranded DNA damages. Results showed that Luminous E. coli DPD2222 containing DNA damage responsive promoter, recA, yielded the highest response followed by luminous E. coli DPD2234 which contained protein damage responsive promoter, grpE. Moreover, acrylamide stress response of the cells up to 14 days old was the same as that of the overnight culture. Furthermore, it was revealed that the E. coli DPD2222 and DPD2234 were capable of detecting acrylamide between 1 and 10,000 μg/L upon contact, with the response signals proportional to acrylamide concentration. The most severe cellular damage of these two strains was achieved after 100 min of contact, as indicated by the highest signals. In addition, temperature-wise, 37 °C resulted in the most significant light emission of E. coli DPD2222. No single strand break was observed at acrylamide concentrations <1000 μg/L by using alkaline comet assay. However, neutral comet assay performed at acrylamide concentrations >10,000 μg/L (ambient temperature) showed a more severe DNA double strand breaks in the cells, so did 1000 μg/L acrylamide at 37 °C. Therefore, it is evident that exposure of bioluminescence sensing cells to acrylamide causes severe DNA damage at either high concentration at room temperature or reduced concentration at body temperature. Quantitative and fingerprint assessment of acrylamide damage could be achieved by optimizing bioluminescence cells constructed with different stress-responsive reporter plasmids.