Investigating the cytotoxic effects of mycobacteriophage Vix gene 80 (611.3)

Abstract

A bacteriophage 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 modified by the infecting phage and redirected toward making new phage particles. Protein‐protein interactions are likely involved in this process. In this work, gene 80 of mycobacteriophage Vix, 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 caused growth inhibition. The Vix80 protein shares 68% amino acid identity with the product of gene 77 of mycobacteriophage L5. The L5_77 protein has been previously shown to exhibit cytotoxic properties, and interacts with MSMEG_3532, a L‐serine dehydratase. The Vix80 and MSMEG_3532 proteins were expressed in Escherichia coli and purified, but attempts to show a physical interaction in vitro have not succeeded. In addition, no interaction between of Vix80 and MSMEG_3532 was observed through two‐hybrid analysis. HHMI‐Pred analysis found that Vix80 contains a conserved domain of unknown function (DUF) near the N‐terminus. The Vix80 gene was dissected, and the N‐terminal 66 residues, encompassing the entire DUF, was found to be cytotoxic to M. smegmatis. DUF was found to be homologous to a region of three M.smegmatis ORFs, two of which are related by alternate initiation points of the same sequence. Efforts to test the interaction of Vix80 and the VIX80 DUF with themselves and with the three host proteins are described. 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.Grant Funding Source: Supported by a Richard Decker Biology Summer Research Award