Enzymes and mechanisms used by phage Vp4 to enter and exit its bacterial host

Details

Supervisors

Mahillon, Jacques

Faculty

Faculté des sciences

Degree label

Master [120] en biochimie et biologie moléculaire et cellulaire, à finalité approfondie

Abstract

Phages are natural bacterial viruses that have evolved various enzymes and mechanisms targeting the bacterial cell envelope (BCE) in order to enter and exit their bacterial host. Every phage infection begins with the adsorption in which virions interact with the BCE to eventually deliver their DNA. Since this process can be altered by polysaccharide (PS) barriers and the peptidoglycan (PG) mesh, many phages produce depolymerases and ectolysins as PS- and PG-degrading enzymes, respectively. At the end of the lifecycle, the host cell lysis releasing phage progeny involves at least two proteins: the holin, a hole-forming membrane protein, and the endolysin, a PG-degrading enzyme relying on holin function to reach its substrate. As understanding these critical processes is paramount for fundamental phage research and phage technologies development, this Master thesis aimed at investigating and characterizing the proteins involved in the adsorption and lysis processes of the phage Vp4 infecting members of the Bacillus cereus group. Bioinformatic analyses enabled to select one ectolysin candidate, the tail protein gp110 predicted to harbor a C-terminal enzymatically active domain (EAD) potentially involved in PG degradation, and two depolymerase candidates, gp77 and gp104 displaying homologies with beta-helix structures, common in depolymerases, and PS-degrading activities. The bacteriolytic activity of purified gp110 was confirmed by conducting turbidity reduction assays (TRAs) with bacterial cells. When testing several strains, gp110 lytic activity was almost only restricted to members of the B. cereus group, including emetic strains and strains insensitive to Vp4 infection. Unfortunately, so far, no depolymerase activity could be detected for both gp77 and gp104 as no degradation zones could be observed when performing spotting tests. PlyV76 was investigated as the putative Vp4 endolysin because of its predicted modular structure with a N-terminal EAD and a C-terminal cell wall binding domain (CBD). Its lytic activity, presumably due to PG-degradation, was confirmed by performing TRAs with both bacterial cells and extracted cell wall (CW). Its lytic activity was mostly specific to B. cereus group members, including emetic strains and strains insensitive to Vp4. By conducting TRAs in different conditions, PlyV76 turned out to be stable in a broad range of pH, but sensitive to temperature and salt concentration. Then, evaluating the activity of PlyV76 potential EAD alone in TRAs validated its catalytic function. Also, by fusing the putative CBD to a GFP and assessing its interaction with the bacterial CW by fluorescence microscopy, its binding function was confirmed and its binding spectrum, which mirrored the lytic spectrum, was evaluated. Next, HolV99, the holin candidate, was predicted to be a membrane protein with two transmembrane domains (TMDs) and an N-in C-in topology when inserted into the membrane. HolV99 was first expressed in an E. coli strain producing the T7 lysozyme, a PG-degrading enzyme. The candidate appeared to exert a hole-forming function releasing the T7 lysozyme to the PG since a cell lysis was validated by optical density monitoring, colony forming unit reduction assessment and beta-galactosidase assay. Also, confocal microscopy imaging of GFP fusions indicated a membrane localization in both E. coli and B. thuringiensis cells. In addition, by assessing the remaining hole-forming activity of TMD deleted and truncated versions of HolV99, the first TMD appeared to be essential for the hole-forming function, while the N- and C-terminal regions seemed to be involved in lysis timing regulation. Finally, HolV99-PlyV76 co-expression was attempted in B. thuringiensis to reconstitute Vp4 lysis event in a homologous context, without any success yet.