Investigating Phage- Host Protein Interactions
Dr. Virginia McDonough and Dr. Joseph Stukey
Mycobacteriophages are viruses that infect bacteria in the genus Mycobacterium. The Mycobacterium genus is home to more than 100 identified species including the human pathogens that cause tuberculosis and leprosy. There is evidence that when mycobacteriophages initiate lytic growth – the phage development pathway that results in the immediate production and release of new phage particles concomitant with host cell death – key metabolic machinery of the host cell is strategically seized and redirected for phage replication. Further, the extreme genetic diversity evident in mycobacteriophage genomes suggests this class of viruses use multiple means to carry out this act of cellular piracy. However, the process by which even a single mycobacteriophage hijacks a host cell is unknown. In an effort to understand this process, we have identified multiple mycobacteriophage genes that are cytotoxic when expressed individually in the host M. smegmatis. We hypothesized that at least some of these cytotoxic effects are due to specific phage-host protein-protein interactions. The focus of this work is the cytotoxic VIX80 gene product from mycobacteriophage Vix. Vix gp80 was analyzed using biochemical techniques to find interacting host cell proteins. Vix gp80 was expressed in E. coli and used in a magnetic bead pull-down assay to isolate M. smegmatis proteins that bound to the phage gene product. M. smegmatis Elongation Factor-Tu was isolated using this technique and identified following protein analysis. The interaction of Vix gp80 and EF-Tu will be verified using additional biochemical methods and the yeast two-hybrid system for detecting physical interactions in vivo. A procedure for a two hybrid-system positive control, necessary for functional testing, is being developed and refined.
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