Factors of prescribing phage therapy among UK healthcare professionals: Evidence from conjoint experiment and interviews.

With the global challenge of antimicrobial resistance (AMR), interest in the development of antibiotic alternatives has surged worldwide. While phage therapy is not a new phenomenon, technological and socio-economic factors have limited its implementation in the Western world. There is now a resurged effort, especially in the UK, to address these challenges. In this study, we collect survey data on UK general practitioners (n = 131) and other healthcare professionals (n = 103), as well as interviews with medical professionals (n = 4) and a focus group with medical students (n = 6) to explore factors associated with their willingness to prescribe phage therapy to patients. The interviews with medical professionals show support for the expansion of bacteriophage clinical trials and highlight their role as a viable alternative to antibiotics. A conjoint experiment reveals that success rate, side effect rate, and patient attitude to treatment are the decisive factors when it comes to phage therapy prescription; in contrast, the effects of administration route, type of treatment, and severity of infection were not statistically significant. Moreover, we show that general practitioners overall are more likely to recommend phage treatment to patients, compared to other healthcare professionals. The results of the study suggest that phage therapy has a potential to be widely accepted and used by healthcare workers in the UK.

Expression, Purification, and Cryo-EM Structural Analysis of an Outer Membrane Secretin Channel.

Secretin proteins form pores in the outer membranes of Gram-negative bacteria, and as such provide a means of transporting a wide variety of molecules out of or in to the cell. They are important components of several different bacterial secretion systems, surface filament assembly machineries, and virus assembly complexes. Despite accommodating a diverse assortment of molecules, including virulence factors, folded proteins, and whole viruses, the secretin family of proteins is highly conserved, particularly in their membrane-embedded ß-barrel domain. We describe here a protocol for the expression, purification and cryo-EM structural determination of the pIV secretin from the Ff family of filamentous bacteriophages.

The consequence of ATP synthase dimer angle on mitochondrial morphology studied by cryo-electron tomography.

Mitochondrial ATP synthases form rows of dimers, which induce membrane curvature to give cristae their characteristic lamellar or tubular morphology. The angle formed between the central stalks of ATP synthase dimers varies between species. Using cryo-electron tomography and sub-tomogram averaging, we determined the structure of the ATP synthase dimer from the nematode worm C. elegans and show that the dimer angle differs from previously determined structures. The consequences of this species-specific difference at the dimer interface were investigated by comparing C. elegans and S. cerevisiae mitochondrial morphology. We reveal that C. elegans has a larger ATP synthase dimer angle with more lamellar (flatter) cristae when compared to yeast. The underlying cause of this difference was investigated by generating an atomic model of the C. elegans ATP synthase dimer by homology modelling. A comparison of our C. elegans model to an existing S. cerevisiae structure reveals the presence of extensions and rearrangements in C. elegans subunits associated with maintaining the dimer interface. We speculate that increasing dimer angles could provide an advantage for species that inhabit variable-oxygen environments by forming flatter more energetically efficient cristae.

Structure of the two-component S-layer of the archaeon Sulfolobus acidocaldarius.

Surface layers (S-layers) are resilient two-dimensional protein lattices that encapsulate many bacteria and most archaea. In archaea, S-layers usually form the only structural component of the cell wall and thus act as the final frontier between the cell and its environment. Therefore, S-layers are crucial for supporting microbial life. Notwithstanding their importance, little is known about archaeal S-layers at the atomic level. Here, we combined single-particle cryo electron microscopy, cryo electron tomography, and Alphafold2 predictions to generate an atomic model of the two-component S-layer of Sulfolobus acidocaldarius. The outer component of this S-layer (SlaA) is a flexible, highly glycosylated, and stable protein. Together with the inner and membrane-bound component (SlaB), they assemble into a porous and interwoven lattice. We hypothesise that jackknife-like conformational changes in SlaA play important roles in S-layer assembly.

CryoEM reveals that ribosomes in microsporidian spores are locked in a dimeric hibernating state.

Translational control is an essential process for the cell to adapt to varying physiological or environmental conditions. To survive adverse conditions such as low nutrient levels, translation can be shut down almost entirely by inhibiting ribosomal function. Here we investigated eukaryotic hibernating ribosomes from the microsporidian parasite Spraguea lophii in situ by a combination of electron cryo-tomography and single-particle electron cryo-microscopy. We show that microsporidian spores contain hibernating ribosomes that are locked in a dimeric (100S) state, which is formed by a unique dimerization mechanism involving the beak region. The ribosomes within the dimer are fully assembled, suggesting that they are ready to be activated once the host cell is invaded. This study provides structural evidence for dimerization acting as a mechanism for ribosomal hibernation in microsporidia, and therefore demonstrates that eukaryotes utilize this mechanism in translational control.

Structure, Biology, and Applications of Filamentous Bacteriophages.

The closely related Escherichia coli Ff filamentous phages (f1, fd, and M13) have taken a fantastic journey over the past 60 years, from the urban sewerage from which they were first isolated, to their use in high-end technologies in multiple fields. Their relatively small genome size, high titers, and the virions that tolerate fusion proteins make the Ffs an ideal system for phage display. Folding of the fusions in the oxidizing environment of the E. coli periplasm makes the Ff phages a platform that allows display of eukaryotic surface and secreted proteins, including antibodies. Resistance of the Ffs to a broad range of pH and detergents facilitates affinity screening in phage display, whereas the stability of the virions at ambient temperature makes them suitable for applications in material science and nanotechnology. Among filamentous phages, only the Ffs have been used in phage display technology, because of the most advanced state of knowledge about their biology and the various tools developed for E. coli as a cloning host for them. Filamentous phages have been thought to be a rather small group, infecting mostly Gram-negative bacteria. A recent discovery of more than 10 thousand diverse filamentous phages in bacteria and archaea, however, opens a fascinating prospect for novel applications. The main aim of this review is to give detailed biological and structural information to researchers embarking on phage display projects. The secondary aim is to discuss the yet-unresolved puzzles, as well as recent developments in filamentous phage biology, from a viewpoint of their impact on current and future applications.

A restrictor complex of ZC3H4, WDR82, and ARS2 integrates with PNUTS to control unproductive transcription.

The transcriptional termination of unstable non-coding RNAs (ncRNAs) is poorly understood compared to coding transcripts. We recently identified ZC3H4-WDR82 ("restrictor") as restricting human ncRNA transcription, but how it does this is unknown. Here, we show that ZC3H4 additionally associates with ARS2 and the nuclear exosome targeting complex. The domains of ZC3H4 that contact ARS2 and WDR82 are required for ncRNA restriction, suggesting their presence in a functional complex. Consistently, ZC3H4, WDR82, and ARS2 co-transcriptionally control an overlapping population of ncRNAs. ZC3H4 is proximal to the negative elongation factor, PNUTS, which we show enables restrictor function and is required to terminate the transcription of all major RNA polymerase II transcript classes. In contrast to short ncRNAs, longer protein-coding transcription is supported by U1 snRNA, which shields transcripts from restrictor and PNUTS at hundreds of genes. These data provide important insights into the mechanism and control of transcription by restrictor and PNUTS.

Phage therapy and the public: Increasing awareness essential to widespread use.

Today, the antimicrobial resistance (AMR) crisis is shaping a world where previously treatable infections can kill. This has revitalised the development of antibiotic alternatives, such as phage therapy. The therapeutic use of phages, viruses that infect and kill bacteria, was first explored over a century ago. However, most of the Western world abandoned phage therapy in favour of antibiotics. While the technical feasibility of phage therapy has been increasingly investigated in recent years, there has been minimal effort to understand and tackle the social challenges that may hinder its development and implementation. In this study, we assess the UK public's awareness, acceptance, preferences and opinions regarding phage therapy using a survey, fielded on the Prolific online research platform. The survey contained two embedded experiments: a conjoint and framing experiment (N = 787). We demonstrate that acceptance of phage therapy among the lay public is already moderate, with a mean likelihood of acceptance of 4.71 on a scale of 1 (not at all likely to accept phage therapy) to 7 (very likely to accept phage therapy). However, priming participants to think about novel medicines and antibiotic resistance significantly increases their likelihood of using phage therapy. Moreover, the conjoint experiment reveals that success and side effect rate, treatment duration, and where the medicine has been approved for use has a statistically significant effect on participants' treatment preferences. Investigations altering the framing of phage therapy, to highlight positive and negative aspects, reveal a higher acceptance of the treatment when described without using perceived harsh words, such as "kill" and "virus". Combined, this information provides an initial insight into how phage therapy could be developed and introduced in the UK to maximise acceptance rate.

Cryo-electron microscopy of the f1 filamentous phage reveals insights into viral infection and assembly.

Phages are viruses that infect bacteria and dominate every ecosystem on our planet. As well as impacting microbial ecology, physiology and evolution, phages are exploited as tools in molecular biology and biotechnology. This is particularly true for the Ff (f1, fd or M13) phages, which represent a widely distributed group of filamentous viruses. Over nearly five decades, Ffs have seen an extraordinary range of applications, yet the complete structure of the phage capsid and consequently the mechanisms of infection and assembly remain largely mysterious. In this work, we use cryo-electron microscopy and a highly efficient system for production of short Ff-derived nanorods to determine a structure of a filamentous virus including the tips. We show that structure combined with mutagenesis can identify phage domains that are important in bacterial attack and for release of new progeny, allowing new models to be proposed for the phage lifecycle.

Interaction of the periplasmic chaperone SurA with the inner membrane protein secretion (SEC) machinery.

Gram-negative bacteria are surrounded by two protein-rich membranes with a peptidoglycan layer sandwiched between them. Together they form the envelope (or cell wall), crucial for energy production, lipid biosynthesis, structural integrity, and for protection against physical and chemical environmental challenges. To achieve envelope biogenesis, periplasmic and outer-membrane proteins (OMPs) must be transported from the cytosol and through the inner-membrane, via the ubiquitous SecYEG protein-channel. Emergent proteins either fold in the periplasm or cross the peptidoglycan (PG) layer towards the outer-membrane for insertion through the ß-barrel assembly machinery (BAM). Trafficking of hydrophobic proteins through the periplasm is particularly treacherous given the high protein density and the absence of energy (ATP or chemiosmotic potential). Numerous molecular chaperones assist in the prevention and recovery from aggregation, and of these SurA is known to interact with BAM, facilitating delivery to the outer-membrane. However, it is unclear how proteins emerging from the Sec-machinery are received and protected from aggregation and proteolysis prior to an interaction with SurA. Through biochemical analysis and electron microscopy we demonstrate the binding capabilities of the unoccupied and substrate-engaged SurA to the inner-membrane translocation machinery complex of SecYEG-SecDF-YidC - aka the holo-translocon (HTL). Supported by AlphaFold predictions, we suggest a role for periplasmic domains of SecDF in chaperone recruitment to the protein translocation exit site in SecYEG. We propose that this immediate interaction with the enlisted chaperone helps to prevent aggregation and degradation of nascent envelope proteins, facilitating their safe passage to the periplasm and outer-membrane.

Electron cryo-microscopy reveals the structure of the archaeal thread filament.

Pili are filamentous surface extensions that play roles in bacterial and archaeal cellular processes such as adhesion, biofilm formation, motility, cell-cell communication, DNA uptake and horizontal gene transfer. The model archaeaon Sulfolobus acidocaldarius assembles three filaments of the type-IV pilus superfamily (archaella, archaeal adhesion pili and UV-inducible pili), as well as a so-far uncharacterised fourth filament, named "thread". Here, we report on the cryo-EM structure of the archaeal thread. The filament is highly glycosylated and consists of subunits of the protein Saci_0406, arranged in a head-to-tail manner. Saci_0406 displays structural similarity, but low sequence homology, to bacterial type-I pilins. Thread subunits are interconnected via donor strand complementation, a feature reminiscent of bacterial chaperone-usher pili. However, despite these similarities in overall architecture, archaeal threads appear to have evolved independently and are likely assembled by a distinct mechanism.

An archaellum filament composed of two alternating subunits.

Archaea use a molecular machine, called the archaellum, to swim. The archaellum consists of an ATP-powered intracellular motor that drives the rotation of an extracellular filament composed of multiple copies of proteins named archaellins. In many species, several archaellin homologs are encoded in the same operon; however, previous structural studies indicated that archaellum filaments mainly consist of only one protein species. Here, we use electron cryo-microscopy to elucidate the structure of the archaellum from Methanocaldococcus villosus at 3.08 Å resolution. The filament is composed of two alternating archaellins, suggesting that the architecture and assembly of archaella is more complex than previously thought. Moreover, we identify structural elements that may contribute to the filament's flexibility.

CryoEM structure of the outer membrane secretin channel pIV from the f1 filamentous bacteriophage.

The Ff family of filamentous bacteriophages infect gram-negative bacteria, but do not cause lysis of their host cell. Instead, new virions are extruded via the phage-encoded pIV protein, which has homology with bacterial secretins. Here, we determine the structure of pIV from the f1 filamentous bacteriophage at 2.7 Å resolution by cryo-electron microscopy, the first near-atomic structure of a phage secretin. Fifteen f1 pIV subunits assemble to form a gated channel in the bacterial outer membrane, with associated soluble domains projecting into the periplasm. We model channel opening and propose a mechanism for phage egress. By single-cell microfluidics experiments, we demonstrate the potential for secretins such as pIV to be used as adjuvants to increase the uptake and efficacy of antibiotics in bacteria. Finally, we compare the f1 pIV structure to its homologues to reveal similarities and differences between phage and bacterial secretins.

Maintenance of complex I and its supercomplexes by NDUF-11 is essential for mitochondrial structure, function and health.

Mitochondrial supercomplexes form around a conserved core of monomeric complex I and dimeric complex III; wherein a subunit of the former, NDUFA11, is conspicuously situated at the interface. We identified nduf-11 (B0491.5) as encoding the Caenorhabditis elegans homologue of NDUFA11. Animals homozygous for a CRISPR-Cas9-generated knockout allele of nduf-11 arrested at the second larval (L2) development stage. Reducing (but not eliminating) expression using RNAi allowed development to adulthood, enabling characterisation of the consequences: destabilisation of complex I and its supercomplexes and perturbation of respiratory function. The loss of NADH dehydrogenase activity was compensated by enhanced complex II activity, with the potential for detrimental reactive oxygen species (ROS) production. Cryo-electron tomography highlighted aberrant morphology of cristae and widening of both cristae junctions and the intermembrane space. The requirement of NDUF-11 for balanced respiration, mitochondrial morphology and development presumably arises due to its involvement in complex I and supercomplex maintenance. This highlights the importance of respiratory complex integrity for health and the potential for its perturbation to cause mitochondrial disease. This article has an associated First Person interview with Amber Knapp-Wilson, joint first author of the paper.

Inter-membrane association of the Sec and BAM translocons for bacterial outer-membrane biogenesis.

The outer-membrane of Gram-negative bacteria is critical for surface adhesion, pathogenicity, antibiotic resistance and survival. The major constituent - hydrophobic ß-barrel Outer-Membrane Proteins (OMPs) - are first secreted across the inner-membrane through the Sec-translocon for delivery to periplasmic chaperones, for example SurA, which prevent aggregation. OMPs are then offloaded to the ß-Barrel Assembly Machinery (BAM) in the outer-membrane for insertion and folding. We show the Holo-TransLocon (HTL) - an assembly of the protein-channel core-complex SecYEG, the ancillary sub-complex SecDF, and the membrane 'insertase' YidC - contacts BAM through periplasmic domains of SecDF and YidC, ensuring efficient OMP maturation. Furthermore, the proton-motive force (PMF) across the inner-membrane acts at distinct stages of protein secretion: (1) SecA-driven translocation through SecYEG and (2) communication of conformational changes via SecDF across the periplasm to BAM. The latter presumably drives efficient passage of OMPs. These interactions provide insights of inter-membrane organisation and communication, the importance of which is becoming increasingly apparent.

Cryo-electron microscopy reveals two distinct type IV pili assembled by the same bacterium.

Type IV pili are flexible filaments on the surface of bacteria, consisting of a helical assembly of pilin proteins. They are involved in bacterial motility (twitching), surface adhesion, biofilm formation and DNA uptake (natural transformation). Here, we use cryo-electron microscopy and mass spectrometry to show that the bacterium Thermus thermophilus produces two forms of type IV pilus ('wide' and 'narrow'), differing in structure and protein composition. Wide pili are composed of the major pilin PilA4, while narrow pili are composed of a so-far uncharacterized pilin which we name PilA5. Functional experiments indicate that PilA4 is required for natural transformation, while PilA5 is important for twitching motility.

Architecture and modular assembly of Sulfolobus S-layers revealed by electron cryotomography.

Surface protein layers (S-layers) often form the only structural component of the archaeal cell wall and are therefore important for cell survival. S-layers have a plethora of cellular functions including maintenance of cell shape, osmotic, and mechanical stability, the formation of a semipermeable protective barrier around the cell, and cell-cell interaction, as well as surface adhesion. Despite the central importance of S-layers for archaeal life, their 3-dimensional (3D) architecture is still poorly understood. Here we present detailed 3D electron cryomicroscopy maps of archaeal S-layers from 3 different Sulfolobus strains. We were able to pinpoint the positions and determine the structure of the 2 subunits SlaA and SlaB. We also present a model describing the assembly of the mature S-layer.

The ER morphology-regulating lunapark protein induces the formation of stacked bilayer discs.

Lunapark (Lnp) is a conserved membrane protein that localizes to and stabilizes three-way junctions of the tubular ER network. In higher eukaryotes, phosphorylation of Lnp may contribute to the conversion of the ER from tubules to sheets during mitosis. Here, we report on the reconstitution of purified Lnp with phospholipids. Surprisingly, Lnp induces the formation of stacked membrane discs. Each disc is a bicelle, with Lnp sitting in the bilayer facing both directions. The interaction between bicelles is mediated by the cytosolic domains of Lnp, resulting in a constant distance between the discs. A phosphomimetic Lnp mutant shows reduced bicelle stacking. Based on these results, we propose that Lnp tethers ER membranes in vivo in a cell cycle-dependent manner. Lnp appears to be the first membrane protein that induces the formation of stacked bicelles.