Structural basis of bacteriophage T5 infection trigger and E. coli cell wall perforation.

Most bacteriophages present a tail allowing host recognition, cell wall perforation, and viral DNA channeling from the capsid to the infected bacterium cytoplasm. The majority of tailed phages bear a long flexible tail (Siphoviridae) at the tip of which receptor binding proteins (RBPs) specifically interact with their host, triggering infection. In siphophage T5, the unique RBP is located at the extremity of a central fiber. We present the structures of T5 tail tip, determined by cryo-electron microscopy before and after interaction with its E. coli receptor, FhuA, reconstituted into nanodisc. These structures bring out the important conformational changes undergone by T5 tail tip upon infection, which include bending of T5 central fiber on the side of the tail tip, tail anchoring to the membrane, tail tube opening, and formation of a transmembrane channel. The data allow to detail the first steps of an otherwise undescribed infection mechanism.

Deciphering Bacteriophage T5 Host Recognition Mechanism and Infection Trigger.

Bacteriophages, viruses infecting bacteria, recognize their host with high specificity, binding to either saccharide motifs or proteins of the cell wall of their host. In the majority of bacteriophages, this host recognition is performed by receptor binding proteins (RBPs) located at the extremity of a tail. Interaction between the RBPs and the host is the trigger for bacteriophage infection, but the molecular details of the mechanisms are unknown for most bacteriophages. Here, we present the electron cryomicroscopy (cryo-EM) structure of bacteriophage T5 RBPpb5 in complex with its Escherichia coli receptor, the iron ferrichrome transporter FhuA. Monomeric RBPpb5 is located at the extremity of T5's long flexible tail, and its irreversible binding to FhuA commits T5 to infection. Analysis of the structure of RBPpb5 within the complex, comparison with its AlphaFold2-predicted structure, and its fit into a previously determined map of the T5 tail tip in complex with FhuA allow us to propose a mechanism of transmission of the RBPpb5 receptor binding to the straight fiber, initiating the cascade of events that commits T5 to DNA ejection. IMPORTANCE Tailed bacteriophages specifically recognize their bacterial host by interaction of their receptor binding protein(s) (RBPs) with saccharides and/or proteins located at the surface of their prey. This crucial interaction commits the virus to infection, but the molecular details of this mechanism are unknown for the majority of bacteriophages. We determined the structure of bacteriophage T5 RBPpb5 in complex with its E. coli receptor, FhuA, by cryo-EM. This first structure of an RBP bound to its protein receptor allowed us to propose a mechanism of transmission of host recognition to the rest of the phage, ultimately opening the capsid and perforating the cell wall and, thus, allowing safe channeling of the DNA into the host cytoplasm.

Target highlights in CASP14: Analysis of models by structure providers.

The biological and functional significance of selected Critical Assessment of Techniques for Protein Structure Prediction 14 (CASP14) targets are described by the authors of the structures. The authors highlight the most relevant features of the target proteins and discuss how well these features were reproduced in the respective submitted predictions. The overall ability to predict three-dimensional structures of proteins has improved remarkably in CASP14, and many difficult targets were modeled with impressive accuracy. For the first time in the history of CASP, the experimentalists not only highlighted that computational models can accurately reproduce the most critical structural features observed in their targets, but also envisaged that models could serve as a guidance for further studies of biologically-relevant properties of proteins.

Structure, function and assembly of the long, flexible tail of siphophages.

Bacteriophages, viruses that infect bacteria, are the most abundant biological entities on Earth. Siphophages, accounting for ∼60% of known phages, bear a long, flexible tail that allows host recognition and safe delivery of the DNA from the capsid to the cytoplasm of the infected cell. Independently from their host (Gram positive or Gram negative) and the nature of their receptor at its surface (polysaccharide or protein), the core tail architecture of all caudophages and of bacterial phage-derived contractile injection systems share the same structural organisation and are thought to be homologous. Here, we review the recent advances in the structure, function and assembly of the core tail architecture of siphophages.

"French Phage Network" Annual Conference-Fifth Meeting Report.

Attracting about 100 participants, the fifth edition of our French Phages.fr annual conference was once more a success. This year's conference took place at the Institute for Structural Biology on the European Electron and Photon Campus in Grenoble, October 8 and 9, 2019. Similar to previous years, our meeting gathered scientists mainly working in France, from academic labs and hospitals as well as from industry. We also had the pleasure of welcoming attendees from different European countries and even beyond. The conference was divided into four sessions: Ecology and Evolution, Phage Therapy and Biotechnology, Structure and Assembly and Phage-Host Interaction, each opened by a keynote lecture. The talks, selected from abstracts, gave the opportunity for young scientists (especially students and post-docs) to present their project and results in a friendly atmosphere. Poster sessions also favoured interactions and discussions between young researchers and more senior scientists. Here, we provide a summary of the topics developed during the conference.

Extracellular vesicles from activated platelets: a semiquantitative cryo-electron microscopy and immuno-gold labeling study.

Cells release membrane vesicles in their surrounding medium either constitutively or in response to activating signals. Two main types of extracellular vesicles (EVs) are commonly distinguished based on their mechanism of formation, membrane composition and size. According to the current model, EVs shed from the plasma membrane, often called microvesicles, expose phosphatidylserine (PS) and range in size from 100 nm to 1 µm, while EVs originating from endosomal multi-vesicular bodies, called exosomes, contain tetraspanin proteins, including CD63, and range in size from 50 to 100 nm. Heijnen et al. [1] have shown that activated platelets release EVs corresponding to these two types of vesicles, using negative staining electron microscopy (EM) and immuno-gold labeling. Here, we apply cryo-EM and immuno-gold labeling to provide a quantitative analysis of EVs released by platelets activated by thrombin, TRAP and CRP-XL, as well as EVs from serum. We show that EVs activated by these three agonists present a similar size distribution, the majority of them forming a broad peak extending from 50 nm to 1 µm, about 50% of them ranging from 50 to 400 nm. We show also that 60% of the EVs from TRAP or CRP-XL activation expose CD41, a majority of them exposing also PS. To explain the presence of large EVs CD41-negative or PS-negative, several alternative mechanisms of EV formation are proposed. We find also that the majority of EVs in activated platelet samples expose CD63, and distinguish two populations of CD63-positive EVs, namely large EVs with low labeling density and small EVs with high labeling density.

Imaging and Quantification of Extracellular Vesicles by Transmission Electron Microscopy.

Extracellular vesicles (EVs) are cell-derived vesicles that are present in blood and other body fluids. EVs raise major interest for their diverse physiopathological roles and their potential biomedical applications. However, the characterization and quantification of EVs constitute major challenges, mainly due to their small size and the lack of methods adapted for their study. Electron microscopy has made significant contributions to the EV field since their initial discovery. Here, we describe the use of two transmission electron microscopy (TEM) techniques for imaging and quantifying EVs. Cryo-TEM combined with receptor-specific gold labeling is applied to reveal the morphology, size, and phenotype of EVs, while their enumeration is achieved after high-speed sedimentation on EM grids.

High-speed centrifugation induces aggregation of extracellular vesicles.

Plasma and other body fluids contain cell-derived extracellular vesicles (EVs), which participate in physiopathological processes and have potential biomedical applications. In order to isolate, concentrate and purify EVs, high-speed centrifugation is often used. We show here, using electron microscopy, receptor-specific gold labelling and flow cytometry, that high-speed centrifugation induces the formation of EV aggregates composed of a mixture of EVs of various phenotypes and morphologies. The presence of aggregates made of EVs of different phenotypes may lead to erroneous interpretation concerning the existence of EVs harbouring surface antigens from different cell origins.