Soutenance de thèse : Study of E. Coli cell wall perforation by bacteriophage T5

Par Alessio d’Acapito (IBS/Groupe Membrane et pathogènes & Groupe Microscopie Electronique et Méthodes)

Bacteriophages are fascinating nanomachines that are highly specific and remarkably efficient in infecting their bacterial hosts. While the cell envelope perforation mechanisms by myophages, such as T4 or of podophages, such as P22 has been well characterized, less is known about the molecular mechanisms of the infection of siphophages, such as T5.

In this work, we used an integrative approach combining cryo-electron tomography (cryo-ET), subtomogram averaging (STA) and single particle analysis (SPA) to obtain snapshots at successive stages of phage T5 infection, from early cell recognition to post DNA injection state. We used E. coli minicells as a simplified cellular model and developed a tailored grid preparation protocol to control the timings of infection and focus on specific timepoints.

First, we solved the structure of T5 L-shaped fibers at 4Å resolution by SPA. Second, we characterized by SPA the structure of the adsorbed but noninfecting phage at 4°C by describing a new intermediate of the tube anchoring mechanism, where the tail tube is brought close to the membrane by the bending of the central fiber but the tube is still closed ; we could conclude that the Cterminal part of the tape measure protein (TMP) pb2-C plays a key structural role.

Lastly, we solved by STA the structure of the infecting phage tip docked on the minicell and of the trans-periplasmic channel that allows the DNA to be translocated into the bacterial cytosol. We propose a model for the channel, formed of three copies of the TMP.

Beyond structural biology, my PhD work was also focused on building tools for the cryo-ET local community. I have developed a tool for “erasing” the membrane signal from sub-volumes extracted with Dynamo as well as a beginner-friendly method for quick tomogram reconstruction from raw data. We made both publicly available on GitHub (https://github.com/alessiodacapito).

Mots clés : cryo-EM, cryo-ET, subtomogram averaging, bacteriophages, bacterial cell wall