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 Investigating the Distribution of CRISPR Adaptive Immune Systems Among Prokaryotes(2019) Weissman, Jake; Johnson, Philip L.F.; Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Just as larger organisms face the constant threat of infection by pathogens, so too do bacteria and archaea. In response, prokaryotes employ a diverse set of strategies to simultaneously cope with their viral and physical environments. Here I explore the ecology and evolution of the CRISPR adaptive immune system, a powerful form of protection against viruses that is the only known example of adaptive immunity in prokaryotes. CRISPR systems are widespread across diverse bacterial and archaeal lineages, suggesting that CRISPR effectively defends against viruses in a broad array of environments. Nevertheless, this defense system is nearly absent in many bacterial groups, and in many environments. I focus on understanding these patterns in CRISPR incidence and the ecological drivers behind them. First, I identify the ecological conditions that favor the adoption of a CRISPR-based defense strategy. I develop a phylogenetically-conscious machine learning approach to build a predictive model of CRISPR incidence using data on over 100 phenotypic traits across over 2600 species and discovered a strong but hitherto-unknown negative interaction between CRISPR and aerobicity. I then consider the multiplicity of CRISPR arrays on a genome, testing whether or not selection favors redundancy in immunity. I use a comparative genomics approach, looking across all prokaryotes to demonstrate that on average, organisms are under selection to maintain more than one CRISPR array. I then explain this surprising result with a theoretical model demonstrating that a trade-off between memory span and learning speed could select for paired “long-term memory” and “short-term memory” CRISPR arrays. Finally, I provide a theoretical examination of the phenomenon of immune loss, specifically in the context of CRISPR immunity. In doing so, I propose an additional mechanism to answer the perennial question: “How do bacteria and bacteriophage coexist stably over long time-spans?” I show that the regular loss of immunity by the bacterial host can produce host-phage coexistence more reliably than other mechanisms, pairing a general model of immunity with an experimental and theoretical case study of CRISPR-based immunity.Item AN INVESTIGATION ON A BACTERIOPHAGE ENDOLYSIN POSSESSING ANTIMICROBIAL ACTIVITY AGAINST ANTIBIOTIC-RESISTANT STAPHYLOCOCCUS AUREUS(2016) Linden, Sara Beth; Nelson, Daniel C; Molecular and Cell Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Staphylococcus aureus is one of the most common causes of nosocomial (i.e. hospital-acquired) infection. Significantly, over 90% of S. aureus strains are resistant to penicillin, and since the mid-1980’s, methicillin-resistant S. aureus (MRSA) strains have become prevalent in hospitals worldwide, with resistance rates approaching 70%. In the U.S. alone, MRSA is responsible for over 100,000 invasive life threatening infections, such as necrotizing fasciitis, and causes 20,000 deaths annually. More worrisome, a variant known as community-acquired MRSA (CA-MRSA) is spreading in schools, gymnasiums, and even professional sports teams, where it infects otherwise healthy adolescents and young adults. Vancomycin is often considered the last antibiotic of choice against MRSA and other Gram-positive pathogens. However, rates of vancomycin-resistant enterococci (VRE) have already reached 30% and it is widely believed that emergence of vancomycin-resistant S. aureus (VRSA) is due to gene transfer during co-colonization of MRSA and VRE. Thus, alternative antimicrobial approaches are desperately needed. Endolysins, or peptidoglycan hydrolases, are phage-derived enzymes that actively lyse bacterial cells upon direct contact and may be considered such an alternative option. Moreover, the inability of bacteria to evolve resistance to endolysins is due to the specificity of the N-terminal catalytic domain, which cleaves a conserved peptidoglycan bond, and the C-terminal cell wall binding domain, which binds a cell surface moiety. This thesis represents an investigation into the endolysin PlyGRCS, which displays potent bacteriolytic activity against all antibiotic-resistant strains of S. aureus tested. This enzyme is active in physiologically relevant conditions (pH, NaCl, temperature), and its activity is greatly enhanced in the presence of calcium. PlyGRCS is the first endolysin with a single catalytic domain that cleaves two distinct sites in the peptidoglycan. Unlike antibiotics, PlyGRCS displays anti-biofilm activity, preventing, removing, and killing biofilms grown on abiotic and biotic surfaces. Engineering efforts were made to create an enzyme with a variable binding domain, which unfortunately displayed less activity than the wild type endolysin in the conditions tested. The antimicrobial efficacy of PlyGRCS was validated in a mouse model of S. aureus septicemia. The results from this study indicate that the endolysin PlyGRCS is a revolutionary therapeutic that should be further pursued for subsequent translational development.Item Genomic analysis of bacteriophages from non-O157 shiga toxin-producing Escherichia coli(2015) Tang, Shuai; Meng, Jianghong; Food Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Shiga toxin-producing Escherichia coli (STEC) is the fiercest pathotype among all diarrheic E. coli. STEC O157:H7 has been a predominant serotype for STEC in the United States. However, an increasing number of cases of infections by STEC other than O157 have been reported in recent years. Shiga toxin (Stx) is the most important virulence factor of STEC and is encoded by stx, which is introduced into STEC genome by bacteriophages through gene transduction. A detailed understanding about Stx bacteriophages is necessary to reveal the emergence and pathogenicity of STEC. The very unstable genomes of Stx bacteriophages result in a dynamic phenotypic versatility including virulence, host cell repertoire and tolerance to adversities. Sequencing technology enables us to generate genomic sequence data of bacteriophages at an affordable cost. The project aimed at obtaining genomic DNA sequences of Stx bacteriophages of non-O157 STEC isolates of diverse serotypes. Thirteen bacteriophages were successfully induced from 83 STEC isolates of serotypes O74, O111, O121, O130, O163, O179 and O183. The bacteriophage DNA samples were collected and sequenced using MiSeq Desktop Sequencer (Illumina®, Inc). Automatically assembled sequences were manually compared to E. coli genome sequence available from NCBI (NC_000913.3) to verify the reliability of the sequencing results. Nine verified bacteriophage sequences were aligned to two Stx bacteriophage genomes of NCBI (NC_000924.1 and NC_018846.1) and visible alignment results were obtained. A phylogenetic relationship of the nine phages and the two reference sequences was constructed and gene profiles of each sample sequences were identified. The comparative analysis indicated that recombination events occurred in probacteriophages showed traces. Similarity of bacteriophage genomes correlated to the serotypes of host bacteria based on the comparison of phylogenetic tree and STEC serotypes. Gene identification results showed that nucleotide variance does not show region specificity, silent mutations are frequent in housekeeping genes and virulence genes are conservative among phage samples.Item Probing the Internalization Mechanism of a Bacteriophage-encoded Endolysin that can Lyse Extracellular and Intracellular Streptococci(2013) Shen, Yang; Nelson, Daniel C; Molecular and Cell Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Bacteriophage-encoded peptidoglycan hydrolases, or endolysins, have been investigated as an alternative to antimicrobials due to their ability to lyse the bacterial cell wall upon contact. However, pathogens are often able to invade epithelial cells where they can repopulate the mucosal surface after antibiotic or endolysin prophylaxis. Thus, there is growing interest in endolysins that can be engineered, or inherently possess, a capacity to internalize in eukaryotic cells such that they can target extracellular and intracellular pathogens. Previously, one streptococcal specific endolysin, PlyC, was shown to control group A Streptococcus localized on mucosal surfaces as well as infected tissues. To further evaluate the therapeutic potential of PlyC, a streptococci/human epithelial cell co-culture model was established to differentiate extracellular vs. intracellular bacteriolytic activity. We found that a single dose (50 μg/ml) of PlyC was able to decrease intracellular streptococci by 96% compared to controls, as well as prevented the host epithelial cells death. In addition, the internalization and co-localization of PlyC with intracellular streptococci was captured by confocal laser scanning microscopy. Further studies revealed the PlyC binding domain alone, termed PlyCB, with a highly positive-charged surface, was responsible for entry into epithelial cells. By applying site-directed mutagenesis, several positive residues (Lys-23, Lys-59, Arg-66 and Lys-70&71) of PlyCB were shown to mediate internalization. We then biochemically demonstrated that PlyCB directly and specifically bound to phosphatidic acid, phosphatidylserine and phosphatidylinositol through a phospholipid screening assay. Computational modeling suggests that two cationic residues, Lys-59 and Arg-66, form a pocket to help secure the interaction between PlyC and specific phospholipids. Internalization of PlyC was found to be via caveolae-mediated endocytosis in an energy-dependent process with the subsequent intracellular trafficking of PlyC regulated by the PI3K pathway. To the best of our knowledge, PlyC is the first endolysin reported that can penetrate through the eukaryotic lipid membrane and retain biological binding and lytic activity against streptococci in the intracellular niche.