Insight into phage Deep-Blue adsorption process : towards the identification of receptor binding proteins

Details

Supervisors

Mahillon Jacques

Faculty

Faculté des bioingénieurs

Degree label

Master [120] : bioingénieur en chimie et bioindustries

Abstract

Bacteriophages, or phages, are viruses that require the bacterial cell machinery for replication. Since their discovery, they have played a key role in bacterial genetics and molecular biology, as well as in biotechnology applications. The infection process of a phage starts with host recognition and adsorption, which involve interactions between a component of the bacterial cell surface, known as the receptor, and a phage receptor binding protein (RBP). Understanding the mechanisms associated with this specificity-determining step may be of great importance to make progress in phage-based technologies. Hence, the aim of this work was to gain insight into the adsorption process of Deep-Blue, a tailed phage of the Myoviridae family that infects members of the B. cereus group. Deep-Blue genome was first compared with related phages using bioinformatic tools and eight putative RBP candidates were selected amongst all tail proteins, namely gp176, gp177, gp178, gp179, gp181, gp185, gp186, and gp188. The choices were made based on detected similarities between the protein of interest and functions that were previously observed in RBPs. Given that RBPs usually consist in a conserved N-terminal part, that anchors the protein to the phage particle, and a variable C-terminal part, responsible for the receptor binding, gp177 and gp181, which display the same pattern, were considered the best candidates. Then, each putative RBP was tagged with a green fluorescent protein (GFP) at its N-terminus. The recombinant proteins were produced using the expression vector pQE-30 and E. coli host cells, except for gp176 that did not get through the molecular cloning step. The recombinant proteins of gp177, gp178, gp179, and gp181 were obtained in soluble form and purified thanks to the N-terminal six-histidine tag. The three other recombinant proteins remained insoluble despite the modification of induction conditions. Finally, cell wall decoration assays were performed on Deep-Blue preferred host, B. weihenstephanensis strain BtB2-4, with the idea that a GFP-tagged RBP that interacts with a component of the bacterial surface will wrap the cells with a fluorescent coat. Insoluble proteins were not purified but cell wall decoration assays were still performed using a slightly modified protocol. Unfortunately, it could not be shown that any of the putative RBP candidates interact with the bacterial cell surface. Every phage must possess at least one RBP, since it is required for host recognition and adsorption. Therefore, it is certain that at least one tail protein of phage Deep-Blue is a RBP. Sadly, this work could not assign the RBP function to any of the selected candidates, since no fluorescent bacteria were observed after cell wall decoration assays. However, it has allowed to prepare the ground for further research, which will undoubtedly lead to the identification of one or more Deep-Blue RBPs.