Phages Deep-Blue and Deep-Purple holins : identification and characterization

Details

Supervisors

Mahillon, Jacques

Faculty

Faculté des bioingénieurs

Degree label

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

Abstract

Bacteriophages or phages are viruses that can specifically infect bacteria and utilize the cell machinery for multiplication. Within the host, phages enter either in a lysogenic (dormant) life cycle, or in a lytic cycle, at the end of which most phages kill the host to allow the release of their virion progeny. Phages have been used in therapy since early 20th, before the advent of antibiotics, and later in molecular biology, where they have been crucial in setting the bases of genetics. Today, a better understanding of their lytic activity is essential, since their bactericidal and transducing properties display enormous potential for phage-based technologies in the agro-food and medical sectors. In this Master thesis, we investigated the lysis actors of Deep-Blue and Deep- Purple, two phages infecting members of the Bacillus cereus group. The focus was set on holins, small proteins that oligomerize in the inner cell membrane at the end of the lytic cycle, creating pores that allow other lytic proteins, such as endolysins, to complete the lysis process. To this end, a bioinformatic analysis based on the putative holins topology, gene localization and similarity to other holins, identified two candidates : Deep-Blue HolB and Deep-Purple HolP. Experiments were then performed to determine their lytic activity (OD monitoring), effect on cell viability (CFU counts), the size of the lesions they caused (𝛽-galactosidase assay), and their localization in the cells (confocal microscopy, using GFP fusions), first in Escherichia coli, then in Bacillus thuringiensis. Truncated versions of the proteins were also designed and tested to assess the specific role(s) of their trans-membrane domains, and N-ter and C-ter extremities. Results in E. coli confirmed that HolB and HolP are holins, given that they caused cell lysis, reduced cell viability, and formed lesions in the hosts membrane. Moreover, we identified the minimal protein fragments lethal for the cells. In HolB, the C-ter domain appears to be dispensable for lysis. We further found that the N-ter extremity and the two transmembrane domains are implicated in toxicity. Regarding HolP, we observed that the N-ter extremity is essential for HolP activity, as well as the ten last amino acids of the C-ter extremity. Unfortunately, so far, we were unable to observe lytic activity of the various holin constructions in B. thuringiensis. Different strains, culture and induction conditions were assayed. Co-expression of holins with their cognate endolysins was also attempted without success. Finally, we studied the possibility of HolB and HolP working with other lytic proteins to achieve lysis in their natural host, as it has been observed for other phages. We found a second holin-like protein for Deep- Purple, Gp33, which was assayed for lytic activity in E. coli. The preliminary results are promising and Gp33 and HolP co-expression should be attempted. No such protein was found for Deep-Blue, which may rely on a novel lysis system. In conclusion, this Master thesis offers a new perspective on phages Deep-Blue and Deep-Purple holins, HolB and HolP, and provides the first information on holins encoded by phages targeting members of the B. cereus group.