Theses and Dissertations at Montana State University (MSU)
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Item Analysis of the role of iron uptake mechanisms and addition of iron-doped apatite nanoparticles in phage infections in Staphylococcus aureus and Mycobacterium smegmatis(Montana State University - Bozeman, College of Letters & Science, 2018) Rost, Linda Christina; Chairperson, Graduate Committee: Greg FrancisAntibiotic resistance has become a significant global public health issue, and phage therapy could serve as an adjuvant to traditional antibiotics. Phages are viruses that kill bacteria and produce more phages. Iron-doped apatite nanoparticles (IDANPs) have been shown to increase phage killing of bacteria. However, the mechanism of JB and Yodasoda infection of bacteria, and mechanism by which IDANPs increase phage infections, is unknown. Based on the iron composition of the IDANP, as well as extensive literature describing Staphylococcus aureus having aggressive iron uptake systems, it was hypothesized that IDANPs may affect such systems, and thereby be involved in the subsequent increase of phage-mediated bacterial death. In this work, the relationship between bacterial exposure to iron and subsequent phage infectivity was established, and IDANP effect on plaque size was determined. S. aureus cells were grown in various iron treatments, plaque assays were performed. There was a strong, positive relationship between iron treatments and plaque counts. The plaque counts were 29% higher in the 0.0004g/L iron treatment, 34% higher in 0.0008g/L, 60% higher in 0.0016 g/L and 82% higher in 0.0032g/L. When S. aureus and M. smegmatis cells were treated with IDANPs, plaque sizes were significantly larger, which may indicate increased infection in adjacent cells. Plaque sizes from IDANP-treated cells continued to increase in size as plates were incubated over 24, 48 and 96 hours. Plaque sizes also increased in size in the control cells in some time frames. S. aureus cells were also grown in 0.0016g/L iron treatment and treated with IDANPs, and there was a 65% increase in plaque counts. In higher iron treatments, it was difficult to achieve a lawn of cells to perform plaque assays. Cell growth was measured by performing serial dilutions and determining CFU/mL. There was no significant difference between cells grown in M9 minimal media or treated with IDANPs. Cells were also grown in the different iron treatments over three hours, with or without IDANPs. Less growth was observed in high iron treatments, but the differences were not significant. Cell growth was relatively slower in high iron levels in overnight treatments, and the results were significantly different. These data can be used to elucidate the relationship of iron uptake and phage killing, and therefore allow conjectures as to whether or not iron uptake mechanisms may be involved in the IDANP effect. Further research in this field can provide opportunities to develop reliable alternatives to antibiotics to treat bacterial infections.Item Investigations into the activity of synthetic & natural products against methicillin-resistant Staphylococcus aureus(Montana State University - Bozeman, College of Letters & Science, 2016) Weaver, Alan James, Jr.; Chairperson, Graduate Committee: Martin Teintze; Joyce B. Shepard, Royce A. Wilkinson, Robert L. Watkins, Sarah K. Walton, Amanda R. Radke, Thomas J. Wright, Milat B. Awel, Catherine Cooper, Elizabeth Erikson, Mohamed E. Labib, Jovanka M. Voyich and Martin Teintze were co-authors of the article, 'Antibacterial activity of Tham trisphenylguanide against methicillin-resistant Staphylococcus aureus' in the journal 'PLoS ONE' which is contained within this thesis.; Abigail Van Vuren, Rakesh, Richard E. Lee, Valerie Copié and Martin Teintze were co-authors of the article, 'Exposure of methicillin-resistant Staphylococcus aureus to low levels of the antibacterial Tham-3 phi G generates a small colony drug-resistant phenotype' submitted to the journal 'PLoS ONE' which is contained within this thesis.; Amanda L Fuchs was an author, and Brian P. Tripet, Martin Teintze, Mary Cloud B. Ammons and Valerie Copié were co-authors of the article, 'Allicin identified as the principal antimicrobial compound in 1,000-year-old Bald's eyesalve' submitted to the journal 'International journal of antimicrobial agents' which is contained within this thesis.; Abigail Van Vuren, Martin Teintze, Valerie Copié and Jovanka Voyich were co-authors of the article, 'Treatment of MRSA with 18-beta-glycyrrhetinic acid reduces cell-to-cell interactions & increases production of staphyloxanthin' submitted to the journal 'Antimicrobial agents & chemotherapy' which is contained within this thesis.The studies herein investigated and characterized synthetic and natural products having efficacy against methicillin-resistant Staphylococcus aureus, which has become a significant threat to both hospital and community environments due to rapid drug resistance development. THAM-3 Phi G is a synthetic compound that showed initial promise as a novel antibacterial against S. aureus (MIC 2 mg/L) through membrane disruption. However, following sub-lethal dosing with THAM-3 Phi G, S. aureus was shown to develop resistance through a small colony variant phenotype, which was defined through 1D 1H NMR metabolomics. Natural products from age-old remedies having efficacy against S. aureus were also investigated in this study. Bald's Eyesalve has shown efficacy against S. aureus; however, the active antibacterial agent(s) remained unknown. Through molecular size and solvent fractionation, activity was isolated to the small (< 3 kDa), non-polar molecule fraction which lost activity following cysteine treatment. Following NMR spectral analysis, the organosulfur garlic-derived compound, allicin, was identified as the active antimicrobial agent. GRA is a natural product found in licorice root, which was used in ancient Chinese medicine. GRA is known to have efficacy against S. aureus and to downregulate key virulence genes. Prolonged exposure of S. aureus to GRA revealed significant increases in the pigment staphyloxanthin. Furthermore, NMR metabolomics of short-term treatments revealed a dysregulation of the TCA cycle, which collectively suggests that treatment of S. aureus with GRA results in oxidative stress. The efficacy of GRA against S. aureus biofilms was also investigated and showed GRA to be ineffective at reducing biofilm CFUs; however, GRA effected biofilm stability. Planktonic studies revealed significant reductions in cell-to-cell interactions beginning at 7.8 mg/L GRA based on optical density measurements and microscopy. Therefore, GRA may serve as part of a novel therapeutic method for treating chronic wound infections. Collectively, these studies utilized NMR to define metabolic phenotypes of bacteria in response to drug treatment and to resolve the active agent in a complex mixture of an age-old remedy. While S. aureus was able to overcome the antibacterial activity of THAM-3 Phi G, the studies of natural products from age-old remedies may provide future treatment options that require further investigation.Item Expression and purification of two CRISPR-CAS proteins, Csm3 and Csm5 from Mycobacterium tuberculosis(Montana State University - Bozeman, College of Letters & Science, 2015) Hashimi, Marziah; Chairperson, Graduate Committee: C. Martin LawrenceOne third of the World's population is infected with tuberculosis (TB). TB disease is caused by bacterium called Mycobacterim tuberculosis, which is a facultative intracellular parasite that is transferred through the air from one person to another in close contact. A six month course of four antimicrobial drugs is the only current treatment for drug-sensitive TB. However, multi-drug resistance TB is difficult to treat. Phage therapy might be one answer as a treatment for multi-drug resistance TB. In order for phage therapy to have a chance against TB, the immune system of bacteria, known as CRISPR/Cas needs to be inhibited. Our lab has taken a structural and biochemical approach to try to understand the CRISPR/Cas system in M. tuberculosis. We have cloned, expressed, and purified individual Csm proteins from the H37Rv M. tuberculosis strain. Two Csm protein, Csm3 and Csm5 were successfully purified to homogeneity in yields suitable for structure and biochemical studies. While to date, each has failed to produce crystals, the ability to the express and purify each of these proteins will allow further biochemical characterization of Csm3 and Csm5.Item SrbA-regulation of ergosterol biosynthesis in Aspergillus fumigatus : gateway to azole resistance & hypoxia adaptation(Montana State University - Bozeman, College of Letters & Science, 2013) Blosser, Sara Jean; Chairperson, Graduate Committee: Robert Cramer; Robert A. Cramer was a co-author of the article, 'Srebp-dependent triazole susceptibility in Aspergillus fumigatus is mediated through direct transcriptional regulation of erg11A (cyp51A)' in the journal 'Antimicrobial agents and chemotherapy' which is contained within this thesis.; Brittney Hendrickson, Nora Grahl, Bridget M. Barker and Robert A. Cramer were co-authors of the article, 'Two C4-sterol methyl oxidases (erg25) catalyze ergosterol intermediate demethylation and impact environmental stress adaptation in Aspergillus fumigatus' submitted to the journal 'Molecular microbiology' which is contained within this thesis.; Robert A. Cramer was a co-author of the article, 'Removal of C4-methyl sterol accumulation in an Srebp-null mutant of Aspergillus fumigatus restores hypoxia growth' submitted to the journal 'PLoS pathogens' which is contained within this thesis.Aspergillus fumigatus is a human fungal pathogen and the primary cause of Invasive Aspergillosis (IA). A rise in susceptible patient populations has dramatically increased the incidence of IA, and led to the emergence of triazole antifungal drug resistance. Triazoles target Erg11, an enzyme involved in ergosterol biosynthesis. Ergosterol biosynthesis has been widely targeted for antifungal drug development, but little is known about this pathway in A. fumigatus. We have identified a transcription factor, SrbA, which mediates triazole susceptibility, growth in hypoxia and low iron, and virulence during IA. Transcriptional studies identify ergosterol biosynthesis as one of the major genetic targets of SrbA, including erg11 and erg25. In this study, we examined the mechanism of Delta srbA triazole susceptibility. Construction of an erg11A conditional expression strain in the Delta srbA background restored erg11A transcript levels and, consequently, wild-type sensitivity to fluconazole and voriconazole. However, pniiAerg11A-Delta srbA did not restore hypoxia growth or the total ergosterol defect of Delta srbA. Increased accumulation of C4-methyl sterols indicates that the Erg25-step of ergosterol biosynthesis is defective in these strains. A. fumigatus encodes for two C4-demethylases, erg25A and erg25B. Erg25A serves in a primary role over Erg25B, as Delta erg25A accumulates more C4-methyl sterol intermediates than Delta erg25B. That both erg25 genes retain function, and are not limited to a singular substrate is unique in the eukaryotic kingdom. Genetic deletion of both erg25 genes is lethal, and single deletion of these genes revealed alterations in ergosterol biosynthesis. Delta erg25A displayed moderate sensitivity to hypoxia, reactive oxygen species (ROS), and dithiothreitol, but was not required for virulence in a murine model of IA. Erg25 assists in the ability of A. fumigatus to grow in hypoxia, as construction of a strain that constitutively expresses erg25A in the Delta srbA background restored the hypoxia growth defect of Delta srbA. This restoration revealed substantial insufficiencies in pflavA-erg25A-Delta srbA when adapting to hypoxia, as this strain was hypersensitive to cell wall perturbation and ROS. Additionally, restoration of erg25A impacted triazole antifungal susceptibility of Delta srbA, demonstrating a complex feedback system involved in ergosterol biosynthesis. These results demonstrate SrbA's involvement in a dynamic stress adaptation program mediated in part through ergosterol biosynthesis.