Antigen self-anchoring onto bacteriophage T5 capsid-like particles for vaccine design.

The promises of vaccines based on virus-like particles stimulate demand for universal non-infectious virus-like platforms that can be efficiently grafted with large antigens. Here, we harnessed the modularity and extreme affinity of the decoration protein pb10 for the capsid of bacteriophage T5. SPR experiments demonstrated that pb10 fused to mCherry or to the model antigen ovalbumin (Ova) retained picomolar affinity for DNA-free T5 capsid-like particles (T5-CLPs), while cryo-EM studies attested to the full occupancy of the 120 capsid binding sites. Mice immunization with CLP-bound pb10-Ova chimeras elicited strong long-lasting anti-Ova humoral responses involving a large panel of isotypes, as well as CD8+ T cell responses, without any extrinsic adjuvant. Therefore, T5-CLP constitutes a unique DNA-free bacteriophage capsid able to display a regular array of large antigens through highly efficient chemical-free anchoring. Its ability to elicit robust immune responses paves the way for further development of this novel vaccination platform.

Real-time detection of virus antibody interaction by label-free common-path interferometry.

Viruses have a profound influence on all forms of life, motivating the development of rapid and minimally invasive methods for virus detection. In this study, we present a novel methodology that enables quantitative measurement of the interaction between individual biotic nanoparticles and antibodies in solution. Our approach employs a label-free, full-field common-path interferometric technique to detect and track biotic nanoparticles and their interactions with antibodies. It is based on the interferometric detection of light scattered by viruses in aqueous samples for the detection of individual viruses. We employ single-particle tracking analysis to characterize the size and properties of the detected nanoparticles, and to monitor the changes in their diffusive mobility resulting from interactions. To validate the sensitivity of our detection approach, we distinguish between particles having identical diffusion coefficients but different scattering signals, using DNA-loaded and DNA-devoid capsids of the Escherichia coli T5 virus phage. In addition, we have been able to monitor, in real time, the interaction between the bacteriophage T5 and purified antibodies targeting its major capsid protein pb8, as well as between the phage SPP1 and nonpurified anti-SPP1 antibodies present in rabbit serum. Interestingly, these virus-antibody interactions are observed within minutes. Finally, by estimating the number of viral particles interacting with antibodies at different concentrations, we successfully quantify the dissociation constant Kd of the virus-antibody reaction using single-particle tracking analysis.

Strategies for Bacteriophage T5 Mutagenesis: Expanding the Toolbox for Phage Genome Engineering.

Phage genome editing is crucial to uncover the molecular mechanisms of virus infection and to engineer bacteriophages with enhanced antibacterial properties. Phage genetic engineering relies mostly on homologous recombination (HR) assisted by the targeted elimination of wild-type phages by CRISPR-Cas nucleases. These strategies are often less effective in virulent bacteriophages with large genomes. T5 is a virulent phage that infects Escherichia coli. We found that CRISPR-Cas9 system (type II-A) had ununiform efficacies against T5, which impairs a reliable use of CRISPR-Cas-assisted counterselection in the gene editing of T5. Here, we present alternative strategies for the construction of mutants in T5. Bacterial retroelements (retrons) proved to be efficient for T5 gene editing by introducing point mutations in the essential gene A1. We set up a protocol based on dilution-amplification-screening (DAS) of phage pools for mutant enrichment that was used to introduce a conditional mutation in another essential gene (A2), insert a new gene (lacZα), and construct a translational fusion of a late phage gene with a fluorescent protein coding gene (pb10-mCherry). The method should be applicable to other virulent phages that are naturally resistant to CRISPR/Cas nucleases.

[Antibacterial applications of bacteriophages]. / Les applications antibactériennes des bactériophages.

In the 1917 article in which Félix d'Hérelle describes his first observations and proposes the name of bacteriophage, he also reports the first use of these viruses to treat bacterial infections, thus giving birth to phage therapy. Soon after antibiotics supplanted bacteriophages. Today, bacteria resistant to multiple antibiotics become a growing public health issue worldwide. This situation has revived research aiming at developing the antibacterial activity of bacteriophages to treat patients as well as diseases in animals and plants. In fact, the areas of applications of bacteriophages as antibacterial are widening as current solutions of chemical nature are questioned. This review summarizes the basic principles of therapeutic applications of bacteriophages and presents recent data in areas where commercial exploitation is occurring or about to emerge.

[A century of research on bacteriophages]. / Un siècle de recherche sur les bactériophages.

Bacteriophages have a prominent place in the living world. They participate to our understanding of the living world through three main aspects : (i) the dissection of the most intimist aspects of viral infection molecular mechanisms (molecular biology), (ii) the description and functioning mechanisms of ecosystems (ecology), and (iii) the adaptive dynamics of integrated viral and host-cell populations (evolution). This review looks back at the genesis of these fundamental findings and draws a picture of the most active fields of current research.

Capsid expansion of bacteriophage T5 revealed by high resolution cryoelectron microscopy.

The large (90-nm) icosahedral capsid of bacteriophage T5 is composed of 775 copies of the major capsid protein (mcp) together with portal, protease, and decoration proteins. Its assembly is a regulated process that involves several intermediates, including a thick-walled round precursor prohead that expands as the viral DNA is packaged to yield a thin-walled and angular mature capsid. We investigated capsid maturation by comparing cryoelectron microscopy (cryo-EM) structures of the prohead, the empty expanded capsid both with and without decoration protein, and the virion capsid at a resolution of 3.8 Å for the latter. We detail the molecular structure of the mcp, its complex pattern of interactions, and their evolution during maturation. The bacteriophage T5 mcp is a variant of the canonical HK97-fold with a high level of plasticity that allows for the precise assembly of a giant macromolecule and the adaptability needed to interact with other proteins and the packaged DNA.

Virus-like particle size and molecular weight/mass determination applying gas-phase electrophoresis (native nES GEMMA).

(Bio-)nanoparticle analysis employing a nano-electrospray gas-phase electrophoretic mobility molecular analyzer (native nES GEMMA) also known as nES differential mobility analyzer (nES DMA) is based on surface-dry analyte separation at ambient pressure. Based on electrophoretic principles, single-charged nanoparticles are separated according to their electrophoretic mobility diameter (EMD) corresponding to the particle size for spherical analytes. Subsequently, it is possible to correlate the (bio-)nanoparticle EMDs to their molecular weight (MW) yielding a corresponding fitted curve for an investigated analyte class. Based on such a correlation, (bio-)nanoparticle MW determination via its EMD within one analyte class is possible. Turning our attention to icosahedral, non-enveloped virus-like particles (VLPs), proteinaceous shells, we set up an EMD/MW correlation. We employed native electrospray ionization mass spectrometry (native ESI MS) to obtain MW values of investigated analytes, where possible, after extensive purification. We experienced difficulties in native ESI MS with time-of-flight (ToF) detection to determine MW due to sample inherent characteristics, which was not the case for charge detection (CDMS). nES GEMMA exceeds CDMS in speed of analysis and is likewise less dependent on sample purity and homogeneity. Hence, gas-phase electrophoresis yields calculated MW values in good approximation even when charge resolution was not obtained in native ESI ToF MS. Therefore, both methods-native nES GEMMA-based MW determination via an analyte class inherent EMD/MW correlation and native ESI MS-in the end relate (bio-)nanoparticle MW values. However, they differ significantly in, e.g., ease of instrument operation, sample and analyte handling, or costs of instrumentation. Graphical abstract.

Neutral mass spectrometry of virus capsids above 100 megadaltons with nanomechanical resonators.

Measurement of the mass of particles in the mega- to gigadalton range is challenging with conventional mass spectrometry. Although this mass range appears optimal for nanomechanical resonators, nanomechanical mass spectrometers often suffer from prohibitive sample loss, extended analysis time, or inadequate resolution. We report on a system architecture combining nebulization of the analytes from solution, their efficient transfer and focusing without relying on electromagnetic fields, and the mass measurements of individual particles using nanomechanical resonator arrays. This system determined the mass distribution of ~30-megadalton polystyrene nanoparticles with high detection efficiency and effectively performed molecular mass measurements of empty or DNA-filled bacteriophage T5 capsids with masses up to 105 megadaltons using less than 1 picomole of sample and with an instrument resolution above 100.

"French Phage Network"-Third Meeting Report.

In its third year of existence, the French Phage Network (Phages.fr) is pursuing its expansion. With more than 25 groups, mostly based in France, working on the various aspects of phage research, the network has increased its visibility, interactivity, and activity. The third meeting of the Phages.fr network, held on November 2017 at the Gif-sur-Yvette Centre National de la Recherche Scientifique (CNRS) campus, was a great opportunity for many young scientists to present their work and interact with more senior scientists, amongst which several were invited from abroad. Here we provide a summary of the work presented at this occasion during the oral presentations and poster sessions.

Bacteriophage T5 tail tube structure suggests a trigger mechanism for Siphoviridae DNA ejection.

The vast majority of phages, bacterial viruses, possess a tail ensuring host recognition, cell wall perforation and safe viral DNA transfer from the capsid to the host cytoplasm. Long flexible tails are formed from the tail tube protein (TTP) polymerised as hexameric rings around and stacked along the tape measure protein (TMP). Here, we report the crystal structure of T5 TTP pb6 at 2.2 Å resolution. Pb6 is unusual in forming a trimeric ring, although structure analysis reveals homology with all classical TTPs and related tube proteins of bacterial puncturing devices (type VI secretion system and R-pyocin). Structures of T5 tail tubes before and after interaction with the host receptor were determined by cryo-electron microscopy at 6 Å resolution. Comparison of these two structures reveals that host-binding information is not propagated to the capsid through conformational changes in the tail tube, suggesting a role of the TMP in this information transduction process.

High affinity anchoring of the decoration protein pb10 onto the bacteriophage T5 capsid.

Bacteriophage capsids constitute icosahedral shells of exceptional stability that protect the viral genome. Many capsids display on their surface decoration proteins whose structure and function remain largely unknown. The decoration protein pb10 of phage T5 binds at the centre of the 120 hexamers formed by the major capsid protein. Here we determined the 3D structure of pb10 and investigated its capsid-binding properties using NMR, SAXS, cryoEM and SPR. Pb10 consists of an α-helical capsid-binding domain and an Ig-like domain exposed to the solvent. It binds to the T5 capsid with a remarkably high affinity and its binding kinetics is characterized by a very slow dissociation rate. We propose that the conformational exchange events observed in the capsid-binding domain enable rearrangements upon binding that contribute to the quasi-irreversibility of the pb10-capsid interaction. Moreover we show that pb10 binding is a highly cooperative process, which favours immediate rebinding of newly dissociated pb10 to the 120 hexamers of the capsid protein. In extreme conditions, pb10 protects the phage from releasing its genome. We conclude that pb10 may function to reinforce the capsid thus favouring phage survival in harsh environments.

Can Changes in Temperature or Ionic Conditions Modify the DNA Organization in the Full Bacteriophage Capsid?

We compared four bacteriophage species, T5, λ, T7, and Φ29, to explore the possibilities of DNA reorganization in the capsid where the chain is highly concentrated and confined. First, we did not detect any change in DNA organization as a function of temperature between 20 to 40 °C. Second, the presence of spermine (4+) induces a significant enlargement of the typical size of the hexagonal domains in all phages. We interpret these changes as a reorganization of DNA by slight movements of defects in the structure, triggered by a partial screening of repulsive interactions. We did not detect any signal characteristic of a long-range chiral organization of the encapsidated DNA in the presence and in the absence of spermine.

Correct Assembly of the Bacteriophage T5 Procapsid Requires Both the Maturation Protease and the Portal Complex.

The 90-nm-diameter capsid of coliphage T5 is organized with T=13 icosahedral geometry and encloses a double-stranded DNA genome that measures 121kbp. Its assembly follows a path similar to that of phage HK97 but yielding a larger structure that includes 775 subunits of the major head protein, 12 subunits of the portal protein and 120 subunits of the decoration protein. As for phage HK97, T5 encodes the scaffold function as an N-terminal extension (∆-domain) to the major head protein that is cleaved by the maturation protease after assembly of the initial prohead I form and prior to DNA packaging and capsid expansion. Although the major head protein alone is sufficient to assemble capsid-like particles, the yield is poor and includes many deformed structures. Here we explore the role of both the portal and the protease in capsid assembly by generating constructs that include the major head protein and a combination of protease (wild type or an inactive mutant) and portal proteins and overexpressing them in Escherichia coli. Our results show that the inactive protease mutant acts to trigger assembly of the major head protein, probably through binding to the ∆-domain, while the portal protein regulates assembly into the correct T=13 geometry. A cryo-electron microscopy reconstruction of prohead I including inactivated protease reveals density projecting from the prohead interior surface toward its center that is compatible with the ∆-domain, as well as additional internal density that we assign as the inactivated protease. These results reveal complexity in T5 beyond that of the HK97 system.

Structure of the Receptor-Binding Carboxy-Terminal Domain of the Bacteriophage T5 L-Shaped Tail Fibre with and without Its Intra-Molecular Chaperone.

Bacteriophage T5, a Siphovirus belonging to the order Caudovirales, has a flexible, three-fold symmetric tail, to which three L-shaped fibres are attached. These fibres recognize oligo-mannose units on the bacterial cell surface prior to infection and are composed of homotrimers of the pb1 protein. Pb1 has 1396 amino acids, of which the carboxy-terminal 133 residues form a trimeric intra-molecular chaperone that is auto-proteolyzed after correct folding. The structure of a trimer of residues 970-1263 was determined by single anomalous dispersion phasing using incorporated selenomethionine residues and refined at 2.3 Å resolution using crystals grown from native, methionine-containing, protein. The protein inhibits phage infection by competition. The phage-distal receptor-binding domain resembles a bullet, with the walls formed by partially intertwined beta-sheets, conferring stability to the structure. The fold of the domain is novel and the topology unique to the pb1 structure. A site-directed mutant (Ser1264 to Ala), in which auto-proteolysis is impeded, was also produced, crystallized and its 2.5 Å structure solved by molecular replacement. The additional chaperone domain (residues 1263-1396) consists of a central trimeric alpha-helical coiled-coil flanked by a mixed alpha-beta domain. Three long beta-hairpin tentacles, one from each chaperone monomer, extend into long curved grooves of the bullet-shaped domain. The chaperone-containing mutant did not inhibit infection by competition.

Insights into bacteriophage T5 structure from analysis of its morphogenesis genes and protein components.

Bacteriophage T5 represents a large family of lytic Siphoviridae infecting Gram-negative bacteria. The low-resolution structure of T5 showed the T=13 geometry of the capsid and the unusual trimeric organization of the tail tube, and the assembly pathway of the capsid was established. Although major structural proteins of T5 have been identified in these studies, most of the genes encoding the morphogenesis proteins remained to be identified. Here, we combine a proteomic analysis of T5 particles with a bioinformatic study and electron microscopic immunolocalization to assign function to the genes encoding the structural proteins, the packaging proteins, and other nonstructural components required for T5 assembly. A head maturation protease that likely accounts for the cleavage of the different capsid proteins is identified. Two other proteins involved in capsid maturation add originality to the T5 capsid assembly mechanism: the single head-to-tail joining protein, which closes the T5 capsid after DNA packaging, and the nicking endonuclease responsible for the single-strand interruptions in the T5 genome. We localize most of the tail proteins that were hitherto uncharacterized and provide a detailed description of the tail tip composition. Our findings highlight novel variations of viral assembly strategies and of virion particle architecture. They further recommend T5 for exploring phage structure and assembly and for deciphering conformational rearrangements that accompany DNA transfer from the capsid to the host cytoplasm.

Crystal structure of pb9, the distal tail protein of bacteriophage T5: a conserved structural motif among all siphophages.

The tail of Caudovirales bacteriophages serves as an adsorption device, a host cell wall-perforating machine, and a genome delivery pathway. In Siphoviridae, the assembly of the long and flexible tail is a highly cooperative and regulated process that is initiated from the proteins forming the distal tail tip complex. In Gram-positive-bacterium-infecting siphophages, the distal tail (Dit) protein has been structurally characterized and is proposed to represent a baseplate hub docking structure. It is organized as a hexameric ring that connects the tail tube and the adsorption device. In this study, we report the characterization of pb9, a tail tip protein of Escherichia coli bacteriophage T5. By immunolocalization, we show that pb9 is located in the upper part of the cone of the T5 tail tip, at the end of the tail tube. The crystal structure of pb9 reveals a two-domain protein. Domain A exhibits remarkable structural similarity with the N-terminal domain of known Dit proteins, while domain B adopts an oligosaccharide/oligonucleotide-binding fold (OB-fold) that is not shared by these proteins. We thus propose that pb9 is the Dit protein of T5, making it the first Dit protein described for a Gram-negative-bacterium-infecting siphophage. Multiple sequence alignments suggest that pb9 is a paradigm for a large family of Dit proteins of siphophages infecting mostly Gram-negative hosts. The modular structure of the Dit protein maintains the basic building block that would be conserved among all siphophages, combining it with a more divergent domain that might serve specific host adhesion properties.

Crystallization of the C-terminal domain of the bacteriophage T5 L-shaped fibre.

Tails of bacteriophage T5 (a member of the Siphoviridae family) were studied by electron microscopy. For the distal parts of the L-shaped tail fibres, which are involved in host cell receptor binding, a low-resolution volume was calculated. Several C-terminal fragments of the fibre were expressed and purified. Crystals of two of them were obtained that belonged to space groups P63 and R32 and diffracted synchrotron radiation to 2.3 and 2.9 Å resolution, respectively. A single-wavelength anomalous dispersion data set to 2.5 Å resolution was also collected from a selenomethionine-derivatized crystal of one of the fragments, which belonged to space group C2.

Assessing the conformational changes of pb5, the receptor-binding protein of phage T5, upon binding to its Escherichia coli receptor FhuA.

Within tailed bacteriophages, interaction of the receptor-binding protein (RBP) with the target cell triggers viral DNA ejection into the host cytoplasm. In the case of phage T5, the RBP pb5 and the receptor FhuA, an outer membrane protein of Escherichia coli, have been identified. Here, we use small angle neutron scattering and electron microscopy to investigate the FhuA-pb5 complex. Specific deuteration of one of the partners allows the complete masking in small angle neutron scattering of the surfactant and unlabeled proteins when the complex is solubilized in the fluorinated surfactant F6-DigluM. Thus, individual structures within a membrane protein complex can be described. The solution structure of FhuA agrees with its crystal structure; that of pb5 shows an elongated shape. Neither displays significant conformational changes upon interaction. The mechanism of signal transduction within phage T5 thus appears different from that of phages binding cell wall saccharides, for which structural information is available.

A two-state cooperative expansion converts the procapsid shell of bacteriophage T5 into a highly stable capsid isomorphous to the final virion head.

Capsids of double-stranded DNA (dsDNA) bacteriophages initially assemble into compact procapsids, which undergo expansion upon the genome packaging. This shell remodeling results from a structural rearrangement of head protein subunits. It is a critical step in the capsid maturation pathway that yields final particles capable to withstand the huge internal pressure generated by the packed DNA. Here, we report on the expansion process of the large capsid (T=13) of bacteriophage T5. We purified the intermediate prohead II form, which is competent for packaging the 121-kbp dsDNA genome, and we investigated its morphology and dimensions using cryo-electron microscopy and small-angle X-ray scattering. Decreasing the pH or the ionic strength triggers expansion of prohead II, converting them into thinner and more faceted capsids isomorphous to the mature virion particles. At low pH, prohead II expansion is a highly cooperative process lacking any detectable intermediate. This two-state reorganization of the capsid lattice per se leads to a remarkable stabilization of the particle. The melting temperature of expanded T5 capsid is virtually identical with that of more complex shells that are reinforced by inter-subunit cross-linking (HK97) or by additional cementing proteins (T4). The T5 capsid with its "simple" two-state conversion thus appears to be a very attractive model for investigating the mechanism of the large-scale allosteric transition that takes place upon the genome packaging of dsDNA bacteriophages.