Přehled o publikaci

Bacteriophages and phage therapy are getting more attention as the numbers of the infections caused by multidrug resistant bacteria increase. However, the usage of phages and phage derived products is limited due to the lack of knowledge about phage-bacterium interactions. In this thesis, the virion structure and the replication mechanism of Casadabanvirus siphophage JBD30 in a single P. aeruginosa cell, as well as in the context of P. aeruginosa biofilm was studied. The structure of JBD30 virion, empty particle, procapsid and a virion attached to its receptor, P. aeruginosa type IV pilus, were resolved using the cryo-electron microscopy single particle analysis approach. The combination of cryo-electron tomography and super-resolution fluorescence imaging enabled description of key stages of JBD30 replication cycle starting with the recognition of the host cell and genome delivery, and ending with a release of new phage progeny. Light-sheet fluorescence microscope equipped with a unique flow-cell system was used for P. aeruginosa biofilm cultivation and time-lapse monitoring of JBD30 dissemination through the grown biofilm. For the purpose of the fluorescence microscopy a mutant of phage JBD30 with cyan fluorescent protein fused to the C-terminus of major capsid protein was prepared using a yeast-based phage-engineering platform. P. aeruginosa cells were labelled using plasmids ensuring stable expression of green and red fluorescent proteins. Results of the thesis enabled me to propose a detailed time-resolved structural model of phage JBD30 – P. aeruginosa interaction. It is likely that the structural features and replication mechanisms described here for bacteriophage JBD30 are conserved among siphophages that utilize type IV pilus as an initial receptor. The understanding of phage replication mechanism, identification of key proteins involved in the host cell recognition and genome delivery, and their structural characterization will enable rational design of phage mutants and derivatives efficient in targeting specific pathogenic bacteria.