Phages express receptor-binding protein (RBP) that interact with receptors on the bacterial surface, as the first step in the infection process. We have previously investigated the host recognition of Kuttervirus phage S117 that express four TSPs. Each TSP recognize different O-antigens on E. coli and Salmonella. However, S117 are also able to infect the E. coli strains EcoR4 and K12 strains that lack the O-antigen with low efficiency. Furthermore, when S117 was spotted on a S. Typhimurium strain where the O-antigen was knocked out the phage was still able to infect the mutant, but to a comparable efficiency to EcoR4 and K12 strains. Taken together, these observations could suggest that S117 uses the LPS as a primary receptor where the TSP degrades the O-antigens in order to get to an unknown secondary receptor present on both E. coli and Salmonella.
We have experimental evidences that FepA and TonB involved in transport of enterobactin, a siderophore that binds to ferric ions, could be the secondary host receptor. In addition, we have identified a putative tail needle / knob protein (orf175) in S117 with structural similarity to the knob of Shigella phage Sf6. The aim of the project is to investigate further S117 host secondary receptor and its associated receptor binding protein.
You will be in charge of creating mutants in Salmonella enterica LT2C for FepA and TonB using the lambda red or CRISPR system, to test if the O-antigen alone could be enough for S117 to infect its host. A FeENT inhibition assay, which will block FepA receptor, will be carried out in parallel to validate the receptor recognition.
Furthermore, you will express the protein encoded by phage S117, orf175, in fusion with a GFP or Nanoluciferase to check its binding capacities to its Salmonella LT2C host strain.
Cloning, mutant generation using Lambda red or CRISPR, protein expression and purification, fluorescent microscopy (if enough time)
PhD student Anders Nørgaard (protein expression/purification) & PhD Cedric Woudstra (Cloning / mutant generation)