Structural basis of SPP1 bacteriophage infectivity

Lead Research Organisation: Birkbeck College
Department Name: Biological Sciences

Abstract

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 th 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.

Technical Summary

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.

Publications

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Chaban Y (2015) Structural rearrangements in the phage head-to-tail interface during assembly and infection. in Proceedings of the National Academy of Sciences of the United States of America

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Cuniasse P (2017) Structures of biomolecular complexes by combination of NMR and cryoEM methods. in Current opinion in structural biology

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Javed A (2017) The ribosome and its role in protein folding: looking through a magnifying glass. in Acta crystallographica. Section D, Structural biology

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Langlois C (2015) Bacteriophage SPP1 tail tube protein self-assembles into ß-structure-rich tubes. in The Journal of biological chemistry

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Lhuillier S (2009) Structure of bacteriophage SPP1 head-to-tail connection reveals mechanism for viral DNA gating. in Proceedings of the National Academy of Sciences of the United States of America

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Orlova E (2012) Bacteriophages

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Orlova EV (2015) A molecular syringe that kills cells. in Nature structural & molecular biology

 
Description The structure of the bacteriophage SPP1 capsid was determined at subnanometer resolution by cryo-electron microscopy and single-particle analysis (White et al, 2012). The icosahedral capsid is composed of the major capsid protein gp13 and the auxiliary protein gp12, which are organized in a T7 lattice. DNA is arranged in layers with a distance of 24.5 Å. gp12 forms spikes that are anchored at the center of gp13 hexamers. In a gp12-deficient mutant, the centers of hexamers are closed by loops of gp13 coming together to protect the SPP1 genome from the outside environment. The HK97-like fold was used to build a pseudoatomic model of gp13. Its structural organization remains unchanged upon tail binding and following DNA release. gp13 exhibits enhanced thermostability in the DNA-filled capsid. A remarkable convergence between the thermostability of the capsid and those of the other virion components was found, revealing that the overall architecture of the SPP1 infectious particle coevolved toward high robustness.
Our results obtained in the following years demonstrate that assembly of the head-to-tail interface and DNA release from the SPP1 virion is accomplished by subtle structural rearrangements in the head-to-tail interface proteins. A crucial player is the gp16 protein that forms the stopper by an allosteric mechanism to retain the viral genome and that opens for DNA ejection during infection. Hindrance with DNA flow by the gp16 stopper can reduce the rate of DNA exit from the virion. We propose that the closed conformation of SPP1 gp16 is the system-stable state whose opening is imposed by signaling for genome release by the tail end when it attaches the cell. The coordinated action of the different head-to-tail interface components thus is essential to ensure the timing of stopper opening for free flow of DNA. This sophisticated part of mechanics combines features of a flexible joint that can act as a camera aperture with robust architecture that can withstand the strong forces produced by the internal pressure of packed DNA at the beginning of the DNA ejection process [47 ± 6 atm for wild-type SPP1]. The ability of the head-to-tail interface to serve as gatekeeper for the viral genome is a key requirement for building viruses with a capsid container carrying tightly packed DNA combined with a long tail tube device for delivery of genetic information to the host cell, the most successful virion design for infecting bacteria.
Exploitation Route Tailed bacteriophages are divided into three families according to their tail morphology. The family Siphoviridae have long and flexible non-contractile tails and represents ~60% of all known tailed species. Siphophages infect most bacteria including pathogenic species such as Staphylococcus aureus a major human and animal pathogen that causes both hospital-acquired and community-acquired infections. The SPP1 bacteriophage is a model system for siphophages of Gram-positive bacteria that include a large number of species that are human and animal pathogens. Analysis of SPP1 infection is of great importance for understanding and usage of phage infection for eradicating bacterial diseases or for industrial applications such as the dairy industry.
Beneficiaries and interested parties:
(1) The Immediate beneficiaries include researchers in academia (national and international) and in the private commercial sector (phage therapy treatment, phage-mediated biocontrol companies).
Interested academics are:
(i) Those who seek methodological advances in single particle analysis by electron microscopy.
(ii) Structural biologists employing diverse biophysical techniques to relate structure to function.
(iii) Microbiologists who are involved in studies of infectivity of Gram-positive bacteria by phages.
(2) Long-term direct beneficiaries would include:
(i) Medical scientists. Pharmaceutical sector of UK.
(ii) Those who use phages in the food industry.
(iii) The wider population who will benefit from improved health and wealth that would accompany a reduction in infectious diseases targeting the pathogenic bacteria and improvement of food quality distributed via supermarkets and all together UK economy.
Sectors Education,Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology

 
Description The funds were used to perform the fundamental structural studies of the SPP1 bacteriophage, to the structure of its tail and conformational changes after DNA ejection. Using. CryoEM and image processing combined with atomic strictures allowed us to validate structures obtained and suggests the mechanism of signal transduction to open the channel of the virus to begin release of genom into the host cell. Cryo EM analysis and the structure of the SPP1 bacteriophage capsid obtained helped us to identify the fold of the capsid protein.
First Year Of Impact 2010
Sector Environment,Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology
 
Description Collaborative Computational Project for Electron cryo-Microscopy (CCP-EM): Supporting the software infrastructure for cryoEM techniques. 2015- 2020.
Amount £1,177,000 (GBP)
Funding ID MR/N009614/1 
Organisation Medical Research Council (MRC) 
Department MRC Partnership Grant
Sector Public
Country United Kingdom
Start  
 
Title Analysis of heterogeneity of samples 
Description Statistical methods for separation of molecular images according to their sizes or conformations 
Type Of Material Biological samples 
Year Produced 2009 
Provided To Others? Yes  
Impact Usage of the new tool by researches in structural biology using EM 
 
Title EM structural data 
Description Structural densitiy maps of portal proteins 
Type Of Material Biological samples 
Provided To Others? Yes  
Impact Densty maps for the core complexes 
 
Title Electron microscopy 
Description Analysis of the T4S systems in different organisms. The T4S systems are involved into active pathogenesis of many bacteria. We study the structural bases of the complexes to reveal a mechanism of thier activity. 
Type Of Material Biological samples 
Provided To Others? No  
Impact Possible development of drugs, that would prevent pathogenic activity of bacteria and preventing of the horizontal transfer of genomic material between bacteria, a reason for their high resistance to antibiotics. 
 
Title Image processing of EM micrographs 
Description Structural analysis of biological samples (proteins and macro biological complexes using digital approaches for alignment of images, their classification, determination of space orientation of the images and finally using methods of structural reconstructions of bio complexes in space. 
Type Of Material Biological samples 
Provided To Others? Yes  
Impact Approach has been implementsed in the package IMAGIC and broadly used in electron microscopy/ 
 
Title EMDB 
Description This is a database (EMDB) that archives all structures obtained by electron microscopy (in negative stain and in cryo). All our results are deposited to this data base became available to other scientists 
Type Of Material Database/Collection of data 
Provided To Others? Yes  
Impact All our deposited data became available to the broad EM community and helps to analyse similarity and differences between bio complexes and investigate their functional activity. 
 
Title PDB 
Description The Protein Data Bank archive (PDB) serves as the single repository of information about the 3D structures of proteins, nucleic acids, and complex assemblies. It allows to validate structures and assess their quality using widely accepted standards and criteria. 
Type Of Material Database/Collection of data 
Year Produced 2008 
Provided To Others? Yes  
Impact That helps to us and other groups to make reliable interpretations of complexes and understand their functionality 
 
Description Barley Stripe Mosaic Virus 
Organisation Moscow State University
Country Russian Federation 
Sector Academic/University 
PI Contribution My lab in Birkbeck College has obtained the first high resolution structure of BSMV obtained by cryo-electron microscopy. We discovered that BSMV forms two types of virions, which differ in the number of coat protein (CP) subunits per turn and interactions between the CP subunits. BSMV CP subunits lack substantial axial contacts and are held together by a previously unobserved lateral contact formed at the virion surface via an interacting loop, which protrudes from the CP hydrophobic core to the adjacent CP subunit. These data provide an insight into diversity in structural organisation of helical plant viruses.
Collaborator Contribution Our partners have purified samples and performed mutagenesis to verify structural finding.
Impact The manuscript is published in Structure, a Phd student E. Pechnikova has got her PhD degree in Moscow. Dr. E. Pechnikova has got her job on FEI (Industry)
Start Year 2010
 
Description Structural analysis of bacteriophages 
Organisation Institute for Integrative Biology of the Cell (I2BC)
Country France 
Sector Academic/University 
PI Contribution During the last decade, we determined and published several EM structures of different components of the bacteriophage system: a portal protein, an isolated head-to-tail interface, a tail, and a capsid. We now report structures of the supramolecular complex forming the complete portal-tail interface extracted from functional phages in a form still able to react efficiently with the host receptor like in the infectious phage system . These nearly in vivo experiments combined with mutagenesis, structural EM analysis and modeling provide major novel functional and mechanistic insights on this structure.
Collaborator Contribution my collaborator Prof P. Tavares has very close interaction with my group and provides samples of purified complexes and performs mutagenesis to reveal a mechanism of DNA deliver to host cells, in another words a mechanism of infection by bacteriophages. At the present moment
Impact PMID: 22514336, 19433794, 17611601, 17363899, 12628918, 11501993, 10467096, 8890151, 26278173, 25991862 Viral Molecular Machines, Chapter 25, , ISBN 978-1-4614-0980-9 Orlova, Elena V. (2012) Bacteriophages and their structural organisation. In: Kurtboke, I. (ed.) Bacteriophages. Rijeka, Croatia: InTech, pp. 3-30. ISBN 9789535102724.
 
Description X-ray Crystallography 
Organisation University of York
Country United Kingdom 
Sector Academic/University 
PI Contribution EM visualisation of protein/DNA complexes, discussions on running projects, Collaboration on structural conformations in E1 heleicase Structural analysis of capsid proteins in SPP1 bacteriophage
Collaborator Contribution The lab by prof A. Antson has provedid as with a number of atomic models of proteins involved into intraction with DNA During the last year (2015) we had discussions on onformation of the capsid proteins. Prof. F. Antson is working now on the X-ray structure of the mutant gp11 (capsid protein from the SPP1phage). The lab by prof A. Antson has analysed structure of the helicase domain of E1 helcase for Bovine Papiloma virus. This structure has been used in analysis of EM sturcture of E1.
Impact This is multi-disciplinary collaboration: fusion of electron microscopy, crystallography and biochemistry. We have the paper published on the portal protein structure and a new publiscation is anticipated in the nearest future on the E1 helicase. At the present moment we are working on atomic structure os the capsid protein gp11 (Spp1 bacteriophage). That will revial how the mature capsid is able to hold genome inside at such high pressure. Our presliminraty analysis indicated that the capsid protein in the SPP1bacteriophage has a mechanism of interaction that differ ftom HK97 and Lambda phages.
Start Year 2006
 
Description Open days in university 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Type Of Presentation Poster Presentation
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact 400 people attended the event. During the open days I have been engaged into inter5sting conversation related to explanation of what is it structural biology, its role in medicine and education, improvement of environment.

Increasieng a number of undegraduates and graduates in BIrkbeck College
Year(s) Of Engagement Activity Pre-2006,2006,2007,2010,
 
Description Participation on Open days of Birkbeck college. 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Explanation of general ideas biolcogical studies, importance of structural studies and how it can be achieved. Making the links between microbiology, structural studies and development of means against diseases. Explanations of how the mutations in biological complexes can cause cancers and what we have to understand to be able to i=restore the functions of these molecules
Year(s) Of Engagement Activity 2010,2011,2012,2013,2014