CE11 - Caractérisation des structures et relations structure-fonctions des macromolécules biologiques

Molecular mechanisms of phage T5 induced immunity – ImmunoPhage

Submission summary

Bacteriophages or phages, i.e. bacterial viruses, are the most abundant living entities on Earth. They have a profound impact on global biogeochemical cycles and on climate, exerted through effects on marine microbial ecology. They are however present in all biotopes, from the atmosphere to the deep biosphere and our intestine. Long used just as a tool to develop modern genetics and molecular biology, the scientific community is now realising the importance of phages on our environment. Phages are now starting to be used in a vast variety of applications such as drug delivery, highly specific bio-sensors, viral-based electronics, and bio- and nano-technologies, without forgetting their reborn use as anti-bacterial agents in the treatment of human and animal infections, particularly promising against the increasing amount of multi-drug-resistant pathogens. The trigger for infection is the recognition of the host, which induces phage perforation of the heavily armoured bacterial cell wall, allowing the injection of its DNA into the host cytoplasm. In the case of lytic phages, the biosynthetic host machinery is then highjacked to allow viral replication, transcription and translation, and eventually liberation of the new virions, killing the host cell. During this vulnerable time, phages protect the new viral factory from over-infection by modifying the bacterial receptors at the host cell surface, preventing recognition by other phages. This will also prevent the newly released virions to bind to cell wall fragments of the lysed host, increasing phage fitness greatly. Because inhibition of host recognition by other phages protects the infected bacterium, we term this phenomenon “phage immunity”.
The ImmunoPhage proposal aims at deciphering the mechanisms of host recognition and inhibition in the case of bacteriophage T5, a phage infecting E. coli that bears a long, non-contractile flexible tail (60% of all phages). At the tip of T5 straight fibre at the distal end of the tail, pb5, the T5 Receptor Binding Protein, irreversibly binds FhuA, an outermembrane iron-ferrichrome transporter. This interaction commits the phage to infection. Next to the pb5 gene in the T5 genome is encoded a small lipoprotein, Llp, that has been shown to be targeted to the inner leaflet of the outer-membrane and to interact with FhuA. Its production prevents infection of the producing cells by free or progeny T5 but also by any other phages which receptor is FhuA. It also inhibits Fe-Ferrichrome transport. How does pb5 interact with FhuA? What are the conformational changes induced in pb5 that allow the transmission of the host recognition information to the rest of the phage? How does the binding of a protein to the periplamic surface of FhuA inhibit the binding of all other ligands on the extracellular surface of the protein? We will answer these questions by determining the structures of pb5, Llp and of the FhuA-pb5 and FhuA-Llp complexes, either by crystallography, NMR or cryo-electron microscopy (cryo-EM), and by studying at the biochemical and biophysical level the FhuA-Llp interactions. We have been working on the FhuA-pb5 (150 kDa) complex for some time, trying to obtain good quality crystals without success. With the resolution revolution of cryo-EM, the time is ripe to tackle smaller complexes. Results should give insights into the early stage of the T5 life cycle but maybe also into the Fe-ferrichrome transport.
This project brings together state-of-the-art technologies: biochemistry, biophysics and structural analysis of phage and membrane proteins and (near) atomic resolution EM. The consortium possesses hands-on expertise in handling phage T5 and membrane proteins, coupled to that in cryo-EM and other structural techniques, enabling to achieve the proposed goal. Positive outcome will be judged by the resolution to atomic resolution of FhuA in complex with two very different partners.

Project coordination


The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.



Help of the ANR 357,084 euros
Beginning and duration of the scientific project: November 2020 - 36 Months

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