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 AN INVESTIGATION ON THE MOLECULAR BASIS FOR DIMER FORMATION OF A BACTERIOPHAGE ENDOLYSIN POSSESSING ANTIMICROBIAL ACTIVITY AGAINST STREPTOCOCCUS PNEUMONIAE(2023) Alreja, Adit Bipin; Nelson, Daniel C; Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The global rise of antibiotic resistance casts a shadow on treating infectious disease. An alternative to the use of antibiotics is bacteriophage-derived peptidoglycan hydrolases called endolysins. Endolysins, produced at the end of a bacteriophage replication cycle, cause bacterial cell lysis and virion release. When applied exogenously as recombinant proteins, they are also capable of cleaving the Gram-positive bacterial peptidoglycan. Various studies conducted in vitro and in vivo showcase the therapeutic potential of endolysins as the next generation of antimicrobials. Streptococcus pneumoniae is the most common cause of a variety of infections ranging from otitis media to invasive bloodstream infection (bacteremia) and meningitis (brain infection). While pneumococcal vaccination programs have proven to be effective, the high rates of antibiotic resistance reported for S. pneumoniae has led to the CDC classifying it as a “serious” threat. One of the most studied endolysins targeting S. pneumoniae is Cpl-1. This thesis represents an investigation into the molecular basis for dimer formation of the Cpl-1 endolysin which displays antibacterial activity against S. pneumoniae. In addition to disproving a long-accepted mechanism of dimerization of Cpl-1 in the presence of choline, we have conclusively identified the residue involved in the formation of the Cpl-1 dimer. Our findings led to the discovery of a novel C-terminal consensus sequence shared by all pneumococcal endolysins that informs their propensity to form dimers in the presence of choline. Next, through a bioinformatics approach we identified a new endolysin containing this C-terminal consensus sequence, produced it, named it SP-CHAP, and showed that it forms a dimer in the presence of choline, indicative of the widespread dimerization phenomenon associated with pneumococcal endolysins. Of interest, SP-CHAP is the first endolysin with antimicrobial activity against S. pneumoniae that possesses a cysteine, histidine-dependent amidohydrolase/peptidase (CHAP) domain. SP-CHAP was subsequently characterized for its biochemical and antimicrobial properties and benchmarked against Cpl-1. SP-CHAP is active in all physiologically relevant conditions (pH, temperature) against various S. pneumoniae strains and displays no activity towards oral/nasal commensal organisms. This enzyme also displays pneumococcal biofilm eradication ability to a greater extent than Cpl-1, as visualized by confocal microscopy. To further translate the antimicrobial potential of this enzyme, the antimicrobial efficacy of SP-CHAP was validated in a S. pneumoniae mouse nasopharyngeal colonization model. Our results demonstrate the therapeutic potential of SP-CHAP as an attractive endolysin to treat S. pneumoniae infections and warrant further translational development of this enzyme.Item Structure-Guided Engineering of a Multimeric Bacteriophage-Encoded Endolysin PlyC(2019) Shang, Xiaoran; Nelson, Daniel; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Emerging antibiotic resistance has become a global health threat. One alternative to antibiotics is bacteriophage-encoded endolysins. Endolysins are peptidoglycan hydrolases produced at the end of the bacteriophage replication cycle resulting in bacterial cell lysis and progeny bacteriophage release. Endolysins are also capable of destroying the Gram-positive bacterial peptidoglycan when applied externally as recombinant proteins. These enzymes typically consist of an enzymatically active domain (EAD) and a separate cell wall binding domain (CBD). Studies have shown therapeutic efficacy of endolysins in vitro and in vivo, with no resistance developed to date. An endolysin from the streptococcal C1 phage, known as PlyC, has the highest activity of any endolysin reported. It also has a unique multimeric structure consisting of one activity subunit (PlyCA) harboring two synergistically acting catalytic domains, GyH and CHAP, and eight identical binding subunits (PlyCB) forming an octameric ring. Groups A, C, and E streptococci as well as Streptococcus uberis are sensitive to the lytic activities of PlyC. In order to harness the potent activity of PlyC for use against other bacteria, we sought to change/extend the host range of PlyC by engineering PlyCB and PlyCA, respectively. We first used a structure-guided mutagenesis method to obtain the single PlyCB monomer subunit, PlyCBK40A E43A (PlyCBm), aiming to study the binding mechanism of PlyCB. Via fluorescence microscopy and binding assays, we determined that PlyCBm retained the host range of the octamer with a much lower binding affinity, which suggests the PlyCB octamer binds concurrently to a specific epitope on the bacterial surface resulting in a tight, stable interaction. Thus, it is not feasible to change/extend the PlyC host range via engineering PlyCB. Next, we proposed a novel design to engineer PlyCA. We successfully created two chimeric endolysins, ClyX-1 and ClyX-2, possessing the synergistic activity of the GyH and CHAP catalytic domains, but extended the host range to include, Streptococcus pneumoniae, Group B streptococci, Streptococcus mutans, and Enterococcus faecalis, all strains previously insensitive to PlyC. Finally, we tested a novel hypothesis that a positively charged catalytic domain could display lytic activity in a CBD-independent manner resulting in a broad host range. Using the PlyC CHAP domain as a model, we converted the net surface charge of the CHAP domain from negative three to positive one through positive seven. Notwithstanding the range of charges, our mutant CHAP domains did not show lytic activity in a CBD-independent manner, suggesting that other factors, like surface charge distribution, need to be considered in such a way of engineering.Item IDENTIFICATION AND ENGINEERING BACTERIOPHAGE ENDOLYSINS FOR INACTIVATION OF GRAM-POSITIVE SPORE-FORMING BACILLI(2018) Etobayeva, Irina V.; Nelson, Daniel C.; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)This dissertation concentrates on the study of the antibacterial potential of bacteriophage-encoded endolysins derived from phages that infect the Gram-positive Bacillus cereus sensu lato group. Bacteriophage-encoded endolysins are peptidoglycan hydrolases that have been identified as important factors in the phage life cycle. Endolysins are encoded by phage late genes during an intracellular infection cycle to lyse the bacterial cell wall from within and allow phage progeny release. Endolysins derived from phages of Gram-positive bacterial hosts are equipped with an enzymatic domain that hydrolyzes conserved covalent bonds in bacterial peptidoglycan, and a cell wall binding domain that ensures proper attachment of endolysins to bacilli. In this study three novel endolysins, PlyP56, PlyN74, and PlyTB40 have been discovered and identified. The biochemical analysis shows that all three endolysins have relatively broad antimicrobial activity against organisms of the B. cereus group with the optimal lytic activity at physiological pH (pH 7.0–8.0), over a broad temperature range (4–55°C), and at low concentrations of NaCl (<50 mM). The domain shuffling engineering studies were undertaken to observe enhancements of bacteriolytic properties of chimeric lysins that retained their specificity to B. cereus species. Finally, these studies have identified a new development in lysis of peptidoglycan of Gram-positive B. cereus group of organisms by phage-encoded endolysins. When grown to stationary phase, bacilli, especially, in overnight cultures become more resistant to lysis despite the evidence that the cell wall domains bind the bacterial surface. In light of these findings, I hypothesize that B. cereus group of species have evolved complex behaviors to interact with phage by modulating surface associated secondary polymers throughout the maturation of the bacilli in order to render them more resistant to the lytic action of phage encoded endolysins, which, contributes to bacterial survival from phage infection.