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A multifaceted approach to gain insight into the mechanisms underlying novel phage-host interactions

Binsabaan, Saeed (2023) A multifaceted approach to gain insight into the mechanisms underlying novel phage-host interactions. Doctoral Dissertation, University of Pittsburgh. (Unpublished)

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Several billions of years of bacteriophage evolution have given rise to different mechanisms by which phages interact with their bacterial hosts. Many of these mechanisms have been characterized; however, metagenomics studies suggest that phage-host interactions are potentially regulated by a wide variety of mechanisms still waiting to be discovered.
Mycobacteriophages are a group of bacteriophages that infect mycobacterial species. Metagenomic studies suggest that mycobacteriophage genomes are remarkably diverse. As of March 2023, 12,381 phages have been isolated from a single mycobacterial species, Mycobacterium smegmatis mc2 155, and 2184 genomes have been completely sequenced. These genomes harbor an extensive reservoir of genes coding for proteins of unknown function, many of which have unique sequences that do not match any protein sequence in the available protein database. This vast array of unexplored proteins represents a great source to gain insight into the potential novel pathways by which phage proteins mediate the interaction with the host cell. This thesis explores new mechanisms regulating phage-mycobacterial host interactions by investigating selected novel mycobacteriophage proteins. We selected Phaedrus gp82, encoded by the Cluster B mycobacteriophage Phaedrus, as a candidate protein with no known function or structural homolog but is predicted to crystallize. We showed that Phaedrus gp82 is a toxic protein that severely reduces colony size when overexpressed in Mycobacterium smegmatis. This effect arises from the interaction of Phaedrus gp82 with an essential M. smegmatis protein, MoxR, a multifunctional ATPase known to have chaperone function. The structure of Phaedrus gp82 was solved using x-ray crystallography at 1.4 Å resolution revealing that the protein consists of two domains, the base and wing domain. The electron density map revealed that Phaedrus gp82 contains a disordered loop, which was critical for MoxR binding. A mutation in this disordered loop disrupts the interaction with MoxR and abolishes the small colony phenotype. We also revealed that the wing domain and acidic residues D38, E43, and D45 are critical for the resulting phenotype and likely play a secondary role in MoxR interactions.
We propose that Phaedrus gp82 functions by reducing the ATPase activity of MoxR, thereby reducing the levels of properly folded MoxR clients. The complete collection of proteins that require MoxR activity to achieve their folded state is unknown, but one example, the essential protein RipA is known. Therefore, it is reasonable to speculate that overexpression of Phaedrus gp82 may be cytotoxic because of the resulting decrease in properly folded MoxR clients. The findings presented here provide new insight into potential undiscovered pathways governing phage-host interactions; and underline the importance of mycobacteriophage genomics as a promising tool to identify new drug targets for the treatment of mycobacterial pathogens.


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Item Type: University of Pittsburgh ETD
Status: Unpublished
CreatorsEmailPitt UsernameORCID
Binsabaan, Saeedsab297@pitt.edusab2970009-0009-6590-0564
ETD Committee:
TitleMemberEmail AddressPitt UsernameORCID
Committee ChairVanDemark, Andrewandyv@pitt.eduandyv
Committee MemberRobert, Dudaduda@pitt.edududa
Committee MemberHatfull, Grahamgfh@pitt.edugfh
Committee MemberBerman, Andreaajb190@pitt.eduajb190
Committee MemberVan Tyne, Dariavantyne@pitt.eduvantyne
Date: 5 September 2023
Date Type: Publication
Defense Date: 25 May 2023
Approval Date: 5 September 2023
Submission Date: 24 July 2023
Access Restriction: No restriction; Release the ETD for access worldwide immediately.
Number of Pages: 127
Institution: University of Pittsburgh
Schools and Programs: Dietrich School of Arts and Sciences > Biological Sciences
Degree: PhD - Doctor of Philosophy
Thesis Type: Doctoral Dissertation
Refereed: Yes
Uncontrolled Keywords: Phaedrus gp82, MoxR ATPase, Adephagia gp73, Mycobacteriophage, Phage-host interactions
Date Deposited: 05 Sep 2023 16:15
Last Modified: 05 Sep 2023 16:15


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