STRUCTURAL AND FUNCTIONAL STUDIES OF TAILSPIKE PROTEINS FROM ESCHERICHIA COLI O157:H7 PHAGE CBA120

dc.contributor.advisorHerzberg, Osnaten_US
dc.contributor.authorGreenfield, Julia Yudehen_US
dc.contributor.departmentBiochemistryen_US
dc.contributor.publisherDigital Repository at the University of Marylanden_US
dc.contributor.publisherUniversity of Maryland (College Park, Md.)en_US
dc.date.accessioned2019-06-21T05:35:00Z
dc.date.available2019-06-21T05:35:00Z
dc.date.issued2019en_US
dc.description.abstractBacteriophage CBA120, a member of the Ackermannviridae family, was isolated, sequenced, shown to selectively infect the food pathogen E. coli O157:H7. The CBA120 genome encodes four separate tailspike proteins (TSPs), TSP1-4 corresponding to ORFs 210 through 213. TSPs bind and degrade or modify the lipopolysaccharide (LPS) on the bacterial cell surface as part of the adsorption apparatus of tailed bacteriophages. Electron microscopy revealed that phage CBA120 possesses a long contractible tail with distinct star-like structures attached to the baseplate of the tail. Sequence analysis of the four CBA120 TSPs suggests two distinct classes; one class comprising TSP1 and TSP3 corresponds to two-domain structures. The second class comprising TSP2 and TSP4 have longer amino acid sequences and contain additional N-terminal regions responsible for the assembly of the TSP star-like complex. Crystal structure of TSP1 at 1.8 Å resolution and TSP3 at 1.85 Å resolution revealed two-domain homotrimers displaying conserved N-terminal head domain structures and C-terminal receptor binding domains with an overall β-helical fold. Sequence analysis of the assembly regions of TSP2 and TSP4 reveal remote homology to gp10 of phage T4, which is involved in the assembly of the phage baseplate. The crystal structure of TSP2 reveals the head domain and receptor binding domain, but the N-terminal assembly region is structurally disordered. In contrast, the assembly region of TSP4 has been purified without the head and receptor domain; pull-down experiments showed that this region binds TSP1. Bacterial halo assays of TSPs 1-3 revealed that all three proteins produced circles of clearing corresponding to polysaccharide depolymerase activity. Site-directed mutagenesis coupled with the halo assay confirmed the identity of TSP3’s catalytic residues as Asp383 and Asp426, which, based on the structure, suggests that the enzyme employs an acid/base mechanism to degrade lipopolysaccharide. The structure-based putative active site of TSP2 suggests that the catalytic machinery comprises Asp506 and Asp571. It was also shown that TSP2, but not TSP1 or TSP3, diminishes CBA120’s ability to infect E. coli O157:H7. Thus, TSP2 is the phage specificity determinant that binds to the lipopolysaccharide of E. coli O157:H7, whereas the bacterial targets of TSP1 and TSP3 remain unknown.en_US
dc.identifierhttps://doi.org/10.13016/2sup-vi4m
dc.identifier.urihttp://hdl.handle.net/1903/22108
dc.language.isoenen_US
dc.subject.pqcontrolledBiochemistryen_US
dc.subject.pquncontrolledPhage CBA120en_US
dc.subject.pquncontrolledtailspike proteinen_US
dc.titleSTRUCTURAL AND FUNCTIONAL STUDIES OF TAILSPIKE PROTEINS FROM ESCHERICHIA COLI O157:H7 PHAGE CBA120en_US
dc.typeDissertationen_US

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