Cell Biology & Molecular Genetics Theses and Dissertations
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Item INVESTIGATING THE COMPETITION BETWEEN COMPONENTS OF DUAL-FUNCTION RNA(2021) Aoyama, Jordan James Masuo; Storz, Gisela T; Molecular and Cell Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Non-coding RNAs (ncRNAs) and small proteins have both emerged as important regulators of gene expression. Dual-function RNAs encode a small protein and have a separate function as a regulatory RNA. Although first discovered in bacteria, dual-function RNAs have now been identified and characterized in eukaryotes as well. These RNAs allow two activities of a single gene to regulate targets at multiple levels. The work described here explores how two novel and one synthetic dual-function RNA act and how competition between the components of a dual-function RNA impacts their functions. AzuCR is a 164-nucleotide E. coli RNA that was previously shown to encode a 28 amino acid protein (AzuC). This work demonstrates that the AzuC small protein impacts glycerol metabolism, with the small protein increasing activity of GlpD, an essential enzyme in glycerol catabolism, while the RNA base pairs with and represses galE mRNA, a gene essential for galactose metabolism. The second dual-function RNA studied in this work is Spot 42, a 109-nucleotide RNA known to base pair with and repress mRNAs encoding proteins involved in the metabolism of non-preferred carbon sources. Although Spot 42 is a well-characterized base pairing small RNA (sRNA) in E. coli, this work shows it also encodes a 15-amino acid protein, SpfP. SpfP was found to bind to cAMP receptor protein (CRP) and block activation of some target genes. For both AzuCR and Spot 42, the coding sequence for the small protein overlaps the base pairing region, and we have observed that translation interferes with base pairing activity suggesting competition between the sRNA and mRNA activities. Finally, a synthetic dual-function RNA was constructed from the Escherichia coli sRNA MgrR and the mRNA for the small protein MgtS. Various versions of this hybrid molecule are used to probe how the organization of components is important for the proper functioning of a dual-function RNA. These three studies highlight the complexities of regulation by dual-function RNAs and provide insights into how these molecules coordinate two different activities to carry out regulatory roles in the cell.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 HIV Reverse Transcriptase Fidelity And Inhibition Are Modulated By Divalent Cations In A Concentration-Dependent Manner In Vitro(2016) Achuthan, Vasudevan; DeStefano, Jeffrey; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Human immunodeficiency virus (HIV) rapidly evolves through generation and selection of mutants that can escape drug therapy. This process is fueled, in part, by the presumably highly error prone polymerase reverse transcriptase (RT). Fidelity of polymerases can be influenced by cation co-factors. Physiologically, magnesium (Mg2+) is used as a co-factor by RT to perform catalysis, however, alternative cations including manganese (Mn2+), cobalt (Co2+), and zinc (Zn2+) can also be used. I demonstrate here that fidelity and inhibition of HIV RT can be influenced differently, in vitro, by divalent cations depending on their concentration. The reported mutation frequency for purified HIV RT in vitro is typically in the 10-4 range (per nucleotide addition), making the enzyme several-fold less accurate than most polymerases. Paradoxically, results examining HIV replication in cells indicate an error frequency that is ~10 times lower than the error rate obtained in the test tube. Here, I reconcile, at least in part, these discrepancies by showing that HIV RT fidelity in vitro is in the same range as cellular results, in physiological concentrations of free Mg2+ (~0.25 mM). At low Mg2+, mutation rates were 5-10 times lower compared to high Mg2+ conditions (5-10 mM). Alternative divalent cations also have a concentration-dependent effect on RT fidelity. Presumed promutagenic cations Mn2+ and Co2+ decreases the fidelity of RT only at elevated concentrations, and Zn2+, when present in low concentration, increases the fidelity of HIV-1 RT by ~2.5 fold compared to Mg2+. HIV-1 and HIV-2 RT inhibition by nucleoside (NRTIs) and non-nucleoside RT inhibitors (NNRTIs) in vitro is also affected by the Mg2+ concentration. NRTIs lacking 3'-OH group inhibited both enzymes less efficiently in low Mg2+ than in high Mg2+; whereas inhibition by the “translocation defective RT inhibitor”, which retains the 3ʹ-OH, was unaffected by Mg2+ concentration, suggesting that NRTIs with a 3ʹ-OH group may be more potent than other NRTIs. In contrast, NNRTIs were more effective in low vs. high Mg2+ conditions. Overall, the studies presented reveal strategies for designing novel RT inhibitors and strongly emphasize the need for studying HIV RT and RT inhibitors in physiologically relevant low Mg2+ conditions.Item Building a map of the dynamic ribosome(2015) Gulay, Suna; Dinman, Jonathan D.; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Our understanding of the static structure of the 80S eukaryotic ribosome has been enhanced by the emergence of high resolution cryo-electron microscopy and crystallography data over the past 15 years. However our understanding of the dynamic nature of the ribosome has lagged. High-throughput Selective 2'-Hydroxyl Acylation analyzed by Primer Extension (hSHAPE) is easily amenable for interrogation of rRNA dynamics. Here we report an improved method of hSHAPE data analysis and apply it to translation initiation and elongation complexes of the yeast ribosome to identify the changes in rRNA flexibility that occur during these processes. Most importantly, we have obtained complete analyses of tRNA binding and intersubunit bridge dynamics, as well as overall expansion segment dynamics, as the ribosome progresses through the translation elongation cycle. The results from these analyses suggest that (1) the yeast P site tRNA binding site is a "hybrid" between the prokaryotic and mammalian P sites, (2) there may be substates of intersubunit rotation, (3) expansion segments may have roles in accommodation. We are also able to identify a network of information pathways that connect elongation factor binding sites to all tRNA binding sites, five intersubunit bridges and two expansion segments. Future directions of this project will focus on improving the visualization of our data to better reflect the highly dynamic nature of the yeast ribosome and to reveal the underlying causes of the observed rRNA flexibility changes.Item Engineering Enhanced Structural Stability to the Streptococcal Bacteriophage Endolysin PlyC(2014) Heselpoth, Ryan Daniel; Nelson, Daniel C; Molecular and Cell Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Antibiotic misuse and overuse has prompted bacteria to rapidly develop resistance, thereby hindering the efficacy of these chemotherapeutics. Due to antibiotic resistant strains expeditiously disseminating, antimicrobial resistance has been labeled as one of the greatest threats to human health globally. An emerging alternative antimicrobial strategy involves using bacteriophage-derived enzymes, termed endolysins. Endolysins are peptidoglycan hydrolases that liberate lytic bacteriophage virions late in the infection cycle by cleaving critical covalent bonds in the bacterial cell wall. As a result, the high intracellular osmotic pressure induces cell lysis. Antimicrobial strategies have been devised involving the extrinsic application of recombinant endolysins to susceptible Gram-positive pathogens. The efficacy of these enzymes has been validated in vitro and in vivo, with no resistance observed to date. One such example is the streptococcal-specific endolysin PlyC. This endolysin is currently the most bacteriolytically-active and possesses the ability to lyse human and animal pathogens known to cause serious health complications. Unfortunately, like numerous other endolysins, PlyC is relatively unstable and accordingly has short shelf life expectancy. With a long-term goal of using endolysins for industrial applications, furthering the development of a thermolabile translational antimicrobial with a short shelf life is ambitious. The main objective of this dissertation is to develop and validate bioengineering strategies for thermostabilizing bacteriolytic enzymes. Using PlyC as the model enzyme, we first used a rationale-based computational screening methodology to identify stabilizing mutations to a thermosusceptible region of the catalytic subunit, PlyCA. One mutation, T406R, caused a 2.27°C increase in thermodynamic stability and a 16 fold improvement in kinetic stability. Next, we developed a substantiated novel directed evolution protocol that involves randomly incorporating mutations into a bacteriolytic enzyme followed by a screening process that effectively identifies mutations that are stabilizing. Finally, applying multiple rounds of directed evolution to PlyC allowed for the identification of a thermostabilizing mutation, N211H, which increased the thermodynamic stability by 4.10°C and kinetic stability 18.8 fold. Combining the N211H and T406R mutations was additive in terms of thermal stability, with thermodynamic and kinetic stability enhancements of 7.46°C and 28.72 kcal/mol activation energy (EA) of PlyCA unfolding, respectively.Item Exopolysaccharide Analysis and EPS Depolymerases as Possible Biofilm Control Strategies(2014) Bales, Patrick; Nelson, Daniel; Molecular and Cell Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Bacteria form biofilms by adhering to surfaces and secreting high molecular weight macromolecules. When in the biofilm mode of growth, bacteria possess increased resistance to the action of antimicrobials and the immune system. By gaining an increased understanding of the structure of the biofilm extrapolymeric substance (EPS) and investigating ways to break up the EPS matrix, more effective treatment of biofilm-related infections can be achieved. In this thesis, the isolation and characterization of the polysaccharide portion of the EPS of several bacterial species is reported. The identification of 14 possible biofilm-degrading enzymes is described. One of these enzymes, HexNW, is shown to be highly thermostable and effective as a biofilm treatment.Item MODULATION OF HIV-1 REVERSE TRANSCRIPTASE AND FAMILY A DNA POLYMERASE PRIMER-TEMPLATE BINDING(2014) Fenstermacher, Katherine Joan; DeStefano, Jeffrey J; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Polymerases are enzymes used by all cellular and viral organisms to replicate their genomes. The human immunodeficiency virus (HIV) polymerase, reverse transcriptase (RT), uses a single-stranded RNA template to create double-stranded DNA during the course of the viral life cycle. Successful reverse transcription relies on the speed of catalysis and the ability of the enzyme to stay bound to the template during synthesis. I demonstrate that both of these properties can be modulated by the presence of different divalent cations, fundamentally altering the behavior of HIV RT. In the presence of 2 mM Mg2+, the HIV RT primer-template complex has a half-life of 1.7±1.0 min, incorporating nucleotides at a maximum rate of 3.5 nucleotides (nt) per second (average speed 1.4±0.4 nt/sec). Substituting 2 mM Mg2+ with 400 μM Zn2+ dramatically slows the speed of catalysis (maximum 0.1 nt/sec, average 0.022±0.003 nt/sec) and promotes primer-template complexes that last hours (half-life of 220±60 min). These profound changes to the enzyme's function critically inhibit reverse transcription, even in the presence of optimal concentrations of Mg2+. In addition to the cation composition during reverse transcription, previous studies have demonstrated that the sequence of the primer-template substrate can also affect the duration of a RT-primer-template complex. In light of this discovery, I investigated the tendency of two Family A DNA polymerases, the Thermus aquaticus DNA polymerase (Taq pol) and the Klenow fragment from Escherichia coli DNA polymerase I (Klenow), to selectively and tightly bind primer-template complexes. Using Primer-Template Systematic Evolution of Ligands by Exponential Enrichment (PT SELEX), I determined that both Taq pol and Klenow tightly bind to sequences containing regions that match the initiation and melting domains of promoters for the structurally similar bacteriophage T7-like RNA polymerases. This suggests a shared sequence preference that might be present in all Family A DNA polymerases, derived from a common ancestor. I plan to exploit this primer-template binding preference to advance biotechnologies utilizing these enzymes.Item Recessive Osteogenesis Imperfecta: Prevalence and Pathophysiology of Collagen Prolyl-3-Hydroxylation Complex Defects(2014) Cabral, Wayne Anthony; Mount, Stephen M; Marini, Joan C; Molecular and Cell Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Osteogenesis Imperfecta (OI) is a clinically and genetically heterogeneous heritable bone dysplasia occurring in 1/15,000-20,000 births. OI is a collagen-related disorder, with the more prevalent dominant forms caused by defects in the genes encoding the α1 and α2 chains of type I collagen (COL1A1 and COL1A2). Rare recessive forms of OI are caused by deficiency of proteins required for collagen post-translational modifications or folding. We identified deficiency of components of the ER-resident collagen prolyl 3-hydroxylase (P3H) complex as a cause of recessive OI. The P3H complex, consisting of prolyl 3-hydroxylase 1 (P3H1), cartilage-associated protein (CRTAP) and cyclophilin B/peptidyl-prolyl isomerase B (CyPB/PPIB), modifies the α1(I) P986 and α2(I) P707 residues of type I collagen. The most common P3H complex defects occur in LEPRE1, the gene encoding P3H1, and over one-third of these cases are due to a founder mutation we identified among individuals of West African and African American descent. Our screening of contemporary cohorts revealed that 0.4% of African Americans and nearly 1.5% of West Africans are carriers for this mutation, predicting a West African frequency of recessive OI due to homozygosity for this mutation at 1/18,260 births, equal to de novo dominant OI. Furthermore, haplotype analysis of affected families was consistent with a single founder for this mutation, occurring 650-900 years ago (1100-1350 C.E.). Patients deficient in P3H1 and CRTAP have consistent bone phenotypes and collagen biochemistry. However, the rare cases of CyPB deficiency have variable findings distinct from P3H1/CRTAP. To clarify the OI mechanism of CyPB deficiency, we generated a Ppib knock-out mouse. In the absence of CyPB, only residual collagen prolyl 3-hydroxylation is detectable in KO cells and tissues. The delay in collagen folding in KO cells is further increased upon cyclophilin inhibition, supporting CyPB's role as an isomerase and the presence of redundancy for collagen ER PPIases. Site-specific alterations of collagen post-translational modification, particularly at residues involved in helical crosslinking, suggest that CyPB is critical to the function of collagen hydroxylases, especially LH1. Thus our studies indicate novel roles for CyPB, separate from the P3H complex, which directly and indirectly regulate collagen biosynthesis and bone development.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.Item MUTATIONAL ANALYSIS OF POLIOVIRUS PROTEIN 3AB TO IDENTIFY REGIONS CRITICAL TO NUCLEIC ACID CHAPERONE ACTIVITY(2013) Gangaramani, Divya; DeStefano, Jeffrey J; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Poliovirus 3AB protein was the first picornavirus protein demonstrated to have nucleic acid chaperone activity. Current results demonstrate that chaperone activity requires the C-terminal 22 amino acid (3B region (also referred to as VPg), amino acid 88-109) of the protein as mutations in this region abrogated nucleic acid binding and chaperone activity. Protein 3B alone had no chaperone activity as determined by established assays testing chaperone activity including: the ability to stimulate nucleic acid hybridization in a primer-template annealing assay, or helix-destabilization in a nucleic acid unwinding assay, or aggregation of nucleic acids. In contrast, the putative 3AB C-terminal cytoplasmic domain (N terminal amino acid 81-109, 3B + the last 7 C-terminal amino acids of 3A, termed 3B+7 in this report) possessed strong activity in these assay, albeit at much higher concentrations than 3AB. Results from several mutations in 3B+7 are described and a model proposing that 3B+7 possesses the "intrinsic" chaperone activity of 3AB while the 3A N-terminal region (amino acid 1-58) and/or membrane anchor domain (amino acid 59-80) serve to increase the effective concentration of the 3B+7 region leading to the potent chaperone activity of 3AB. Two mutations with reduced chaperone activity in vitro, K81A and F83A in 3AB were tested in tissue culture. Viruses with these mutations produced near wildtype and minute plaques, respectively. F83A gave rise to revertants with either wildtype 3AB sequences or additional nearby compensatory mutations. Translation and polyprotein processing were not affected by these mutations but RNA synthesis compared to wildtype, was slightly lower for K81A and significantly lower for F83A. This data suggests that mutations that decrease chaperone activity of 3AB may lead to decreased RNA synthesis, although the exact steps that are affected need to be determined.