Structural basis of SPP1 bacteriophage infectivity

Bacterial viruses (bacteriophages or phages) are the most populated biological entity in the Biosphere. Most known bacteriophages have tails that serve as a pipeline at genome delivery into the host cytoplasm during infection. The main structural features of these phages are well known and include an icosahedral head (capsid) that keeps the genome (linear ds DNA) safe from a hazardous environment, a long flexible non-contractile tail attached to the capsid, and a specialised molecular adsorption apparatus located on the free end of the tail. This apparatus is essential for the phage infectivity as it detects a specific receptor in the host cell surface. Once the receptor has been recognised, the phage affixes itself to the bacterial cell wall and forms a channel through the cell membrane. The tail sticks to the cell membrane tightly so that the genome can be delivered straight into the host cell. The interaction of the phage adsorption device with the bacterial cell membrane induces a signal that is transmitted along the tail to the phage head, where the signal stimulates the opening of the connector located between the tail and the head. The connector serves as a valve to keep DNA locked in the capsid. Opening of the connector leads to the DNA release. Biochemical analysis of this process has provided information; however, it is still unclear how the signal propagates through the tail and which phage system components control structural conformational changes. Our study has demonstrated extensive structural rearrangements in the internal wall of the tail tube of SPP1 bacteriophage, however, it remains unknown what sequence of events induces DNA release. We propose that the adsorption device-receptor interaction triggers a conformational switch, which is propagated in a domino-like cascade along the 1600 Å-long helical tail to reach the head-to-tail connector. This leads to opening of the connector culminating in DNA exit from the head into the host cell through the tail tube. To test this hypothesis we need to document the structural changes that occur in the tail structure after receptor binding until the genome is successfully released from the phage particle. In this type of study bacteriophage SPP1 is a unique model since the SPP1 specific receptor has been identified and purified. The process of DNA ejection from phage particles in vitro could be controlled and time dependence can be tested. Since bacteriopaghes are huge asymmetrical macromolecular systems, electron microscopy (EM) in combination with image analysis is the method of choice. Modern methods of sample preparation allow structural conformational changes in phages to be captured and, therefore electron microscopy in combination with biochemical and biophysical methods would allow us to observe the phage in different states. Analysis of two mutant tail structures will clarify a system of interactions between subunits in the tail tube and time resolving experiments will enlighten a basis of the signal propagation. A single particle asymmetrical approach and tomography will be used to localize the connector within the phage capsid before and after DNA ejection. Docking of known or predicted atomic structures of the phage components will allow understanding of structural principles behind signal propagation and function of the capsid gate. The School of Crystallography at Birkbeck College has the EM, computer facilities and software packages required for the project. In 2006 year we have obtained an equipment grant that is providing an FEI 300 keV FEG microscope (Polara), that will be installed in autumn 2007. This microscope will be equipped with the software Leginon that allows automated data collection. Larger data sets are required to improve the reliability of analysis. Statistical approaches developed in the EM groups of Dr. E. Orlova and Prof. H. Saibil allow analysis of heterogeneous data sets.

Much data has been obtained from biochemical analysis of infection in tailed bacteriophages, however the structural basis of the phage tail/cell surface interaction remains unclear. To understand infectivity, we need to find how a signal from the distal area of the tail attached to the host cell is transmitted to the head-to-tail connector to trigger ejection of the genome. The aim of this project is to reveal the sequence and structural nature of the phage infectivity. Bacteriophage SPP1 is unique , as its receptor was identified and purified, thus making an excellent model to study infectivity. Electron microscopy (negative stain and cryo- conditions) and image processing of samples with biochemical and biophysical methods will be used to trap the phage in different stages of infectivity. The purified receptor allows control of the DNA ejection in vitro. Our previous experience and results for SPP1, allow objectives to be formulated: 1. Determine structure of the tail in two phage mutants that carry different forms of the major tail protein: one with gp17.1 that lacks the putative domain exposed to the tail exterior and another formed by gp17.1* that carries this domain.. 2. Determine time-dependent structural changes within the SPP1 tail that accompany signal propagation from the adsorption apparatus to the connector. 3. Structure determination of the connector in two functional states and visualization of DNA inside its channel before release using a single particle asymmetrical approach and tomography 4. Find conformational changes in the connector-tail link area before and after DNA ejection, and possible intermediate states, during DNA ejection. Overall, the project will provide structural information that is necessary to understand the mechanism of infection initiation.