AN INVESTIGATION ON THE MOLECULAR BASIS FOR DIMER FORMATION OF A BACTERIOPHAGE ENDOLYSIN POSSESSING ANTIMICROBIAL ACTIVITY AGAINST STREPTOCOCCUS PNEUMONIAE

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dc.contributor.advisorNelson, Daniel Cen_US
dc.contributor.authorAlreja, Adit Bipinen_US
dc.contributor.departmentBiologyen_US
dc.contributor.publisherDigital Repository at the University of Marylanden_US
dc.contributor.publisherUniversity of Maryland (College Park, Md.)en_US
dc.date.accessioned2024-02-14T06:47:52Z
dc.date.available2024-02-14T06:47:52Z
dc.date.issued2023en_US
dc.description.abstractThe 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.en_US
dc.identifierhttps://doi.org/10.13016/ezeb-clwz
dc.identifier.urihttp://hdl.handle.net/1903/31760
dc.language.isoenen_US
dc.subject.pqcontrolledMicrobiologyen_US
dc.subject.pqcontrolledBiologyen_US
dc.subject.pqcontrolledMolecular biologyen_US
dc.subject.pquncontrolledantibiotic resistanceen_US
dc.subject.pquncontrolledbacteriophageen_US
dc.subject.pquncontrolledendolysinen_US
dc.subject.pquncontrolledpneumococcusen_US
dc.titleAN INVESTIGATION ON THE MOLECULAR BASIS FOR DIMER FORMATION OF A BACTERIOPHAGE ENDOLYSIN POSSESSING ANTIMICROBIAL ACTIVITY AGAINST STREPTOCOCCUS PNEUMONIAEen_US
dc.typeDissertationen_US

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