Characterization of B. thuringiensis mutants resistant to Vp4 infection and identification of their cognate receptors

Details

Supervisors

Mahillon, Jacques

Faculty

Faculté des bioingénieurs

Degree label

Master [120] : bioingénieur en sciences agronomiques, à finalité spécialisée

Abstract

Phages are obligate viruses that rely on their host machinery for their own replication. This infection process begins with the recognition and adsorption of the phage to the host surface through the phage Receptor Binding Protein (RBP) - bacterial receptor interaction. Bacteria have thus developed different defense strategies to modify their receptor, obstruct it or prevent its expression. Hence, the aim of this work was to phenotypically characterize the four spontaneous phage-resistant isolated mutants and identify the mutated receptor that was recognized upon Vp4 adsorption. The mutants phage resistance acquired through the prevention of the adsorption step was confirmed by an adsorption test where Vp4 adsorption decreased of ca. 54% which was corroborated by a cell wall decoration assay where the Baseplate-Wedge (BW) gp119 could weakly decorate the mutants cell surface whereas the RBP gp112 could not recognize it. The presence of a second bacterial receptor was suggested by the lysis from without phenomenon observed on the mutants spot-on-plate. Regarding their phenotype, only slight differences were observed between B. thuringiensis BGSC 4BA1 wild-type and its mutants. The cell treatment of Vp4 host, B. thuringiensis GSX002, with sodium periodate suggested that a sugar moiety could be involved upon Vp4 adsorption, which was also suggested by the presence of carbohydrate-binding module folds in BW-gp119. A sugar competition assay was performed and indicated that the presence of mannose and N-acetylglucosamine interfered with the adsorption of Vp4 to its host. Bioinformatic tools identified the collagen triple helix repeat domain protein (termed collagen 1) as the first candidate gene for receptor function. A B. thuringiensis BGSC 4BA1Δcollagen1 knock-out was thus constructed and an adsorption test confirmed that Vp4 adsorption was decreased but at a lesser extent than with the spontaneous mutants. A cell wall decoration assay reinforced the putative role of collagen 1 in host recognition, either as a receptor or as a stem for decorations serving as receptor, as BW gp119 could not recognize the cell surface of the knock-out mutant. All in one, this work provided a good insight on the phenotypical characterization of the phage-resistant mutants and identified the saccharidic decoration of collagen 1 as a good bacterial receptor candidate recognized by BW gp119 whereas another mutated protein might be recognized by gp112.