How do bacteriophages perforate the bacterial cell wall? – PerfoBac

Bacteriophages are the most abundant microorganisms in the biosphere. At the origin of major discoveries in modern molecular biology, their study is getting increasingly popular in fields as diverse as ecology, genetics, phylogeny and nanophysics. Moreover, medicine and animal health have a reawakened interest in phage therapy, linked to the increased multi-resistance to antibiotics of human, animal and plant pathogens. Bacteriophages show not only a remarkable diversity in host but also in mechanisms regulating their infectious cycle and allowing them to adapt to host constraints. The vast majority of bacteriophages characterised to date are tailed phages that have an icosahedral protein capsid, containing a densely packed double-stranded DNA, and a rigid or flexible tail whose tip proteins serve to recognise the host surface and deliver the genome into the cytoplasm. Binding to the host receptor induces DNA ejection from the capsid and its transfer through the complex host envelope to reach the cytoplasm. In the case of lytic phages, the biosynthetic host machinery is then hijacked to transcribe the viral genome and produce new virions, which are assembled in the cytoplasm following highly regulated assembly pathways. Perforation of the bacterial cell wall is the first step of infection allowing the viral DNA to enter the host. The underlying molecular events are still not elucidated for the Siphoviridae phages, which represent 60% of all phages.

The aim of the PerfoBac project is the elucidation of the perforation mechanism for the Siphoviridae phage T5. PhageT5 appears to be simpler than most other phages in terms of organisation and number of proteins involved. Our strategy is to describe and compare the structure of its tail, before and after ejection of the DNA, which in T5 is the consequence of the sole interaction with the E. coli outer membrane bacterial transporter FhuA. The integration of structural data obtained both in vitro and in vivo, will enable the molecular description of cell wall perforation. This project brings together state-of-the-art technologies in biochemistry of membrane proteins and (near) atomic resolution electron microscopy. The consortium possesses hands-on expertise in handling phage T5 and biochemistry, coupled to that in electron microscopy, enabling to achieve the proposed goal. Positive outcome will be judged by the resolution to quasi atomic resolution of an architecture able to perforate the heavily armoured bacterial cell wall. In addition, original structures and complex molecular mechanisms are expected to be revealed.