Structural insights on Siphoviridae bacteriophages infection – SIPHO-PHAGES

1. Scientific background Viruses are designed to protect their genome and to deliver it at high efficiency to a target cell. More than 95 % of the bacterial viruses (bacteriophages or phages) have a double-stranded DNA genome and a tail attached to the portal vertex of the capsid. The tail is responsible for specific recognition of the bacterium surface and for a piercing the bacterial envelope allowing transfer of the genome to the host cell cytoplasm. Most phages have a long (~150 nm) non-contractile tail (Siphoviridae). The tail is composed of an adsorption apparatus connected by a large helïcoidal tube to the capsid. Phage irreversible adsorption to bacterial surface receptors generates a signal transmitted along the tail that triggers DNA release. The first step of Gram+ bacteria infection by Siphoviridae involves interactions between the receptor binding protein, RBP, and a specific receptor distributed on the surface of the cell wall. The RBP is localized at the tip of the phage's tail, in a tail tip (phage SPP1), or in a more complex structure, the baseplate (phages tuc2009 and p2). Our X-ray structures of 3 RBPs from phages infecting Lactococcus lactis revealed their modularity and the saccharidic nature of their receptors. We have also identified the irreversible protein receptor of the Bacillus subtilis phage SPP1 whose isolated ectodomain triggers DNA ejection in vitro. These achievements recommend the synergistic study of phages SPP1, tuc2009 and p2 to investigate the structure of Siphoviridae tails and to characterize the function of their individual components in infection. This overall molecular view is lacking for the non-contractile tail of any phage. Very little is known about infection of Gram+ bacteria despite the abundance and economical impact of their phages. Of major importance is phage infection of L. lactis that impairs fermentation of milk products, a recurrent problem for the dairy industry. 2. Objectives Aunique phage cannot display general features of all Siphoviridae family. We have thus selected three phages (SPP1, tuc2009 and p2) whose tail-tip/baseplate morphologies are sufficiently different to sample the family. The aim of the project is to analyse the structure and function of their tails, and to obtain i) the individual 3D structures of the phage tail-tip or baseplates components at atomic resolution ii) the structures of complexes between interacting components, and iii) reconstructions of tail sub-structures derived from cryoEM. The structural data will provide a framework to interpret and guide studies aiming to characterize (iv) the network of protein-protein interactions necessary for tail assembly, and (v) the molecular rearrangements in the adsorption apparatus and along the tail tube that are triggered by interaction with saccharide/protein receptors. 3. Methodology Thanks to the Structural Genomics platform at AFMB, all the structural proteins of tuc2009 were expressed soluble, while the work with those of SPP1 (VMS and AFMB) and p2 is underway. These proteins will be submitted to crystallization assays using a nano-drop technology developped at AFMB. Interactions between the structural proteins will be detected and analysed using His6/Strep purification coupled to refractometry and DLS in line. They will be further documented by genetic, hydrodynamic and Biacore analyses, while proteins topology in the tail will be established by immuno-electron microscopy. The 3D structures of individual proteins and oligo-complexes will be determined by X-ray diffraction. Larger complexes and tail assembly intermediates from infected cells will be studied by cryo-EM. Crystallographic structures will be fitted into the cryoEM electron density maps to produce structures at pseudo atomic-resolution. Differences between the cryoEM apo-structures and those obtained in complex with the receptor will provide information on the signal transduction pathway for portal vertex opening. 4. Expected results We expect to dissect the assembly pathway of the phage tails into a sequential program of proteins interactions and to obtain insights on the molecular mechanisms controlling assembly of these 4-6 MDa structures. Knowledge of the structure of the tail-tip/baseplate and of its conformational changes upon binding to the receptor will provide answers on the role of each components in bacterial infection. We expect to decipher conformational changes that trigger and communicate the DNA release signal from the RBP to the portal vertex. Besides the three phages analysed in this project, we expect that our results, unique for this family of phages, will also shed light on the infection mechanisms of Siphoviridae in general, and will be useful for the understanding of a wide range of mechanisms in virology. While fundamental, these studies will likely provide the seeds for more applied research concerning the industrial transformation of milk into dairy products.