Title

Weapons of Cellular Destruction: Investigating the Cytotoxic Effects of Mycobacteriophage Vix Gene 80

Student Author(s)

Danielle Goodman

Faculty Mentor(s)

Dr. Joseph Stukey; Dr. Virginia McDonough

Document Type

Poster

Event Date

4-12-2013

Abstract

A bacteriophage, or phage, is a virus that infects and reproduces in bacteria. During productive infections – those that result in construction and release of infectious phage particles – key host cell metabolic processes are often modified by the infecting phage and redirected toward making new phage particles. Protein-protein interactions are likely involved in this process. Identifying the relevant phage and host gene products and understanding how phage exploit their host’s weaknesses could lead to new therapeutic options for many bacterial illnesses. In this work, mycobacteriophage Vix, gene 80, a gene cytotoxic to host strain Mycobacterium smegmatis, was studied. Our hypothesis was that an interaction between the Vix80 gene product and a host cell protein, possibly MSMEG_3532, affects host cell metabolism and causes growth inhibition. Vix80 protein shares 68% amino acid identity with the product of gene 77 of mycobacteriophage L5, a gene that has been previously shown to exhibit cytotoxic properties and interacts with MSMEG_3532, a L-serine dehydratase. Both Vix80 and L5_77 gene products contain a conserved domain of unknown function near the N-terminus. The Vix80 gene was dissected and the N-terminal conserved domain was tested separately for cytotoxic activity. The N-terminal 66 residues, encompassing the entire conserved domain of unknown function, was found to be cytotoxic to M. smegmatis. Efforts to express and purify both the Vix80 and MSMEG_3532 proteins in Escherichia coli and show a physical interaction in vitro have not succeeded due to extremely low solubility of our T7-antigen tagged MSMEG_3532 protein. We continue to explore this approach with modifications, including the removal of the T7-antigen tag on MSMEG_3532. Expression of the Vix80 protein in E.coli was also cytotoxic indicating existence of a conserved host target in this species, or, that Vix80 cytotoxicity does not require an interaction with a specific host protein. Finally, to examine whether the Vix80 gene is required for phage Vix infection of M. smegmatis, we are in the process of removing the genomic copy of Vix80 in phage Vix.

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