Theses and Dissertations from UMD
Permanent URI for this communityhttp://hdl.handle.net/1903/2
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
More information is available at Theses and Dissertations at University of Maryland Libraries.
Browse
3 results
Search Results
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 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 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.