déroulement complet du processus infectieux. De plus, la synthèse de A1 commande la dégradation de
l'ADN bactérien et le détournement de la machinerie de transcription. Nous disposons de phages mutés
dans les gènes A1 et A2, ce qui permet d'étudier la fonction des gènes précoces et leurs interactions avec
les protéines de l'hôte alors que le transfert de l'ADN est bloqué à la première étape. Ces propriétés de
T5 permettent d'étudier le détournement des fonctions cellulaires indépendamment des autres étapes de
l'infection (réplication du génome et assemblage de la particule virale).
Ce projet de thèse s'inscrit dans l'étude globale des mécanismes moléculaires qui contrôlent le
transfert de l'ADN en deux étapes et la neutralisation des ressources de l'hôte. Le premier objectif est de
caractériser la fonction des protéine A1 et A2. Ces protéines peuvent être surproduites et purifiées et
nous avons débuté leur caractérisation biochimique. Des mutants de T5 seront construits, codant pour
A1 ou A2 fusionnées avec des étiquettes de type "TAP-tag" afin d'identifier les protéines virales et
bactériennes partenaires de A1 et A2 au début de l'infection. Les interactions ainsi établies seront
analysées in vitro en reconstituant les complexes à partir des protéines purifiées. Si ces complexes
peuvent être obtenus de manière stable, leur caractérisation structurale sera effectuée.
Ce projet intégrera également l'identification d'autres gènes précoces impliqués dans la neutralisation
des fonctions bactériennes. Différentes données génétiques indiquent que seulement 5 gènes, en plus de
A1 et A2, sont essentiels pour l'arrêt immédiat de la croissance de l'hôte et le détournement de son
métabolisme. La fonction et les partenaires de ces protéines précoces seront explorés par des approches
génétiques et biochimiques.
Project description:
Bacteriophages, the natural predators of bacteria, can kill their host with high efficiency. In the early
stages of infection phages defeat bacterial defenses and exploit specific cellular functions to serve their
needs and propagate in their host. The strategies by which phages take over the host cell resources
remain obscure and have been studied in a very limited number of phages. An up to date investigation
of these mechanisms is essential for the main following reasons: i) The early-expressed genes are highly
diverse from one phage to another and most of them have no assigned function. They thus represent a
library of novel genes whose functions are crucial for host neutralization. ii) By deciphering the
mechanisms of host takeover, we expect to identify host factors targeted by phage, which constitute
attractive targets for new antimicrobial drugs.
We use the Escherichia coli phage T5 as a model system to investigate the early stages of phage
infection. T5 uses a unique two-step DNA delivery strategy that controls the timing of host function
diversion. After binding of T5 to its host receptor FhuA, only 8% of the genome enters the bacterial cell
before injection temporarily stops. Early proteins encoded by this region of the genome are then
expressed and affect vital host functions: the bacterial DNA is degraded, the RNA polymerase is
redirected towards the transcription of viral genes, the systems of restriction/modification and of DNA
repair are inactivated. Two of early proteins, A1 and A2, are required for resuming the DNA transfer,
which takes place after a few minutes, allowing the completion of the infectious process. Moreover the
synthesis of A1 controls the onset of bacterial DNA degradation and transcription machinery hijacking.
As we have T5 mutants of the A1 or A2 proteins, we can “freeze” infection just after the first step
transfer of the viral genome. This gives us the opportunity to investigate the mechanism of host takeover
independently of the further steps of phage replication and assembly.
This PhD project is part of a global study of the molecular mechanisms, which control the two-step
DNA transfer and the neutralization of host resources. First, it aims at characterizing the function of A1
and A2 proteins. These proteins can be overproduced and purified and we have undertaken their
biochemical characterization. New mutants of T5 will be constructed, encoding tag fusion proteins A1
or A2 (type “TAP-tag”) in order to identify the viral and bacterial partners of A1 and A2 at the onset of
infection. The interactions established in this way will be analyzed in vitro by reconstituting the
complexes starting from purified proteins. If stable complexes can be obtained, their structural
characterization will be carried out.
This project will also include the identification of other early genes involved in the neutralization of
bacterial functions. Genetic data indicate that only 5 genes, besides A1 and A2, are essential for the
immediate diversion of the bacterial metabolism. The functions and partners of these early proteins will
be investigated by genetic and biochemical approaches.