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The Division of Structural Biology (STRUBI)
Bacterial pathogen deploys the iminosugar glycosyrin to manipulate plant glycobiology.
The extracellular space (apoplast) in plants is a key battleground during microbial infections. To avoid recognition, the bacterial model phytopathogen Pseudomonas syringae pv. tomato DC3000 produces glycosyrin. Glycosyrin inhibits the plant-secreted β-galactosidase BGAL1, which would otherwise initiate the release of immunogenic peptides from bacterial flagellin. Here, we report the structure, biosynthesis, and multifunctional roles of glycosyrin. High-resolution cryo-electron microscopy and chemical synthesis revealed that glycosyrin is an iminosugar with a five-membered pyrrolidine ring and a hydrated aldehyde that mimics monosaccharides. Glycosyrin biosynthesis was controlled by virulence regulators, and its production is common in bacteria and prevents flagellin recognition and alters the extracellular glycoproteome and metabolome of infected plants. These findings highlight a potentially wider role for glycobiology manipulation by plant pathogens across the plant kingdom.
Permissive central tolerance plus defective peripheral checkpoints license pathogenic memory B cells in CASPR2-antibody encephalitis.
Autoantibody-mediated diseases targeting one autoantigen provide a unique opportunity to comprehensively understand the development of disease-causing B cells and autoantibodies. Convention suggests that such autoreactivities are generated during germinal center reactions. Here, we explore earlier immune checkpoints, focusing on patients with contactin-associated protein-like 2 (CASPR2)-autoantibody encephalitis. In both disease and health, high (~0.5%) frequencies of unmutated CASPR2-reactive naïve B cells were identified. By contrast, CASPR2-reactive memory B cells were exclusive to patients, and their B cell receptors demonstrated affinity-enhancing somatic mutations with pathogenic effects in neuronal cultures and mice. The unmutated, precursor memory B cell receptors showed a distinctive balance between strong CASPR2 reactivity and very limited binding across the remaining human proteome. Our results identify permissive central tolerance, defective peripheral tolerance, and autoantigen-specific tolerance thresholds in humans as sequential steps that license CASPR2-directed pathology. By leveraging the basic immunobiology, we rationally direct tolerance-restoring approaches, with an experimental paradigm applicable across autoimmunity.
The E3 ubiquitin ligase MGRN1 targets melanocortin receptors MC1R and MC4R via interactions with transmembrane adapters.
E3 ubiquitin ligases play a crucial role in modulating receptor stability and signaling at the cell surface, yet the mechanisms governing their substrate specificity remain incompletely understood. Mahogunin Ring Finger 1 (MGRN1) is a membrane-tethered E3 ligase that fine-tunes signaling sensitivity by targeting surface receptors for ubiquitination and degradation. Unlike cytosolic E3 ligases, membrane-tethered E3s require transmembrane adapters to selectively recognize and regulate surface receptors, yet few such ligases have been studied in detail. While MGRN1 is known to regulate the receptor Smoothened (SMO) within the Hedgehog pathway through its interaction with the transmembrane adapter Multiple Epidermal Growth Factor-like 8 (MEGF8), the broader scope of its regulatory network has been speculative. Here, we identify Attractin (ATRN) and Attractin-like 1 (ATRNL1) as additional transmembrane adapters that recruit MGRN1 and regulate cell surface receptor turnover. Through co-immunoprecipitation, we show that ATRN and ATRNL1 likely interact with the RING domain of MGRN1. Functional assays reveal that MGRN1 requires these transmembrane adapters to ubiquitinate and degrade the melanocortin receptors MC1R and MC4R, in a process analogous to its regulation of SMO. Loss of MGRN1 leads to increased surface and ciliary localization of MC4R in fibroblasts and elevated MC1R levels in melanocytes, with the latter resulting in enhanced eumelanin production. These findings expand the repertoire of MGRN1-regulated receptors and provide new insight into a shared mechanism by which membrane-tethered E3 ligases utilize transmembrane adapters to dictate substrate receptor specificity. By elucidating how MGRN1 selectively engages with surface receptors, this work establishes a broader framework for understanding how this unique class of E3 ligases fine-tunes receptor homeostasis and signaling output.
Production of an immunogenic trivalent poliovirus virus-like particle vaccine candidate in yeast using controlled fermentation.
The success of the poliovirus (PV) vaccines has enabled the near-eradication of wild PV, however, their continued use post-eradication poses concerns, due to the potential for virus escape during vaccine manufacture. Recombinant virus-like particles (VLPs) that lack the viral genome remove this risk. Here, we demonstrate the production of PV VLPs for all three serotypes by controlled fermentation using Pichia pastoris. We determined the cryo-EM structure of a new PV2 mutant, termed SC5a, in comparison to PV2-SC6b VLPs described previously and investigated the immunogenicity of PV2-SC5a VLPs. Finally, a trivalent immunogenicity trial using bioreactor-derived VLPs of all three serotypes in the presence of Alhydrogel adjuvant, showed that these VLPs outperform the current IPV vaccine in the standard vaccine potency assay, offering the potential for dose-sparing. Overall, these results provide further evidence that yeast-produced VLPs have the potential to be a next-generation polio vaccine in a post-eradication world.
Molecular basis for the phosphorylation of bacterial tyrosine kinase Wzc.
The regulation of polymerisation and translocation of biomolecules is fundamental. Wzc, an integral cytoplasmic membrane tyrosine autokinase protein serves as the master regulator of the biosynthesis and export of many bacterial capsular polysaccharides and exopolysaccharides. Such polysaccharides play essential roles in infection, defence, and some are important industrial products. Wzc comprises a large periplasmic domain, two transmembrane helices and a C-terminal cytoplasmic kinase domain with a tyrosine-rich tail. Wzc regulates polymerisation functions through cycling the formation and dissociation of an octameric complex, driven by changes in the phosphorylation status of the tyrosine-rich tail. E. coli Wzc serves a model for a wider family of polysaccharide co-polymerases. Here, we determine structures of intermediate states with different extents of phosphorylation. Structural and computational data reveal the pre-ordering of the tyrosine-rich tail, the molecular basis underlying the unidirectionality of phosphorylation events, and the underlying structural dynamics on how phosphorylation status is transmitted.
Human leukocyte antigen alleles associate with COVID-19 vaccine immunogenicity and risk of breakthrough infection
AbstractSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccine immunogenicity varies between individuals, and immune responses correlate with vaccine efficacy. Using data from 1,076 participants enrolled in ChAdOx1 nCov-19 vaccine efficacy trials in the United Kingdom, we found that inter-individual variation in normalized antibody responses against SARS-CoV-2 spike and its receptor-binding domain (RBD) at 28 days after first vaccination shows genome-wide significant association with major histocompatibility complex (MHC) class II alleles. The most statistically significant association with higher levels of anti-RBD antibody was HLA-DQB1*06 (P = 3.2 × 10−9), which we replicated in 1,677 additional vaccinees. Individuals carrying HLA-DQB1*06 alleles were less likely to experience PCR-confirmed breakthrough infection during the ancestral SARS-CoV-2 virus and subsequent Alpha variant waves compared to non-carriers (hazard ratio = 0.63, 0.42–0.93, P = 0.02). We identified a distinct spike-derived peptide that is predicted to bind differentially to HLA-DQB1*06 compared to other similar alleles, and we found evidence of increased spike-specific memory B cell responses in HLA-DQB1*06 carriers at 84 days after first vaccination. Our results demonstrate association of HLA type with Coronavirus Disease 2019 (COVID-19) vaccine antibody response and risk of breakthrough infection, with implications for future vaccine design and implementation.
Detection of mpox and other orthopoxviruses using a lateral flow device as a point-of-care diagnostic.
In 2022, the World Health Organization declared the worldwide outbreak of mpox to be a public health emergency of international concern. The causative monkeypox virus (MPXV) belonged to clade IIb and is transmitted through sexual contact with a low case fatality rate (0.1%), which, together with under-detection, all contributed to a rapid global spread particularly within the MSM (men who have sex with men) community. As MPXV clade II remains circulating worldwide, a new outbreak of the more fatal clade I disease has been declared in Central and East Africa, and remains uncontrolled in part due to the lack of point-of-care (POC) diagnostics for rapid decisions on treatment and self-isolation. To address the lack of POC solutions for mpox, we have designed and evaluated an orthopoxvirus-specific lateral flow device (LFD) that could be used for the diagnosis of mpox. Using an LFD comprising four monoclonal antibodies against the A27 protein, we demonstrate sensitivity to 3 × 105 pfu/mL. This sensitivity is expected to be sufficient for the detection of MPXV from lesion sites and may also be sufficient for other sample types such as saliva and urine. We found that the presence of guanidinium thiocyanate, a common ingredient in inactivating viral transport media, masked the LFD antigen, resulting in false negatives. POC diagnosis of mpox may be possible using an LFD to reduce delays arising from sample shipment to centralized laboratory testing facilities. In order to achieve this, our work demonstrates that an LFD-optimized buffer is required, as the sample collection buffer may have a detrimental impact on sensitivity for clinical material.IMPORTANCEMpox cases have dramatically increased both in traditionally monkeypox virus endemic countries and also worldwide. This increase comes at a time when immunity derived from smallpox vaccination is no longer available. Diagnosis of mpox is complicated due to both disease presentation and the availability of local diagnostic laboratories. The availability of a point-of-care diagnostic tool such as an lateral flow device (LFD) would play an important role to both diagnose and prevent onward transmission. This manuscript provides developers and assessors with key data for defining true sensitivity and specificity of a successful LFD in addition to buffer conditions for sample collection.
A broadly reactive ultralong bovine antibody that can determine the integrity of foot-and-mouth disease virus capsids.
Foot-and-mouth disease vaccination using inactivated virus is suboptimal, as the icosahedral viral capsids often disassemble into antigenically distinct pentameric units during long-term storage, or exposure to elevated temperature or lowered pH, and thus raise a response that is no longer protective. Furthermore, as foot-and-mouth disease virus (FMDV)'s seven serotypes are antigenically diverse, cross-protection from a single serotype vaccine is limited, and most existing mouse and bovine antibodies and camelid single-domain heavy chain-only antibodies are serotype-specific. For quality control purposes, there is a real need for pan-serotype antibodies that clearly distinguish between pentamer (12S) and protective intact FMDV capsid. To date, few cross-serotype bovine-derived antibodies have been reported in the literature. We identify a bovine antibody with an ultralong CDR-H3, Ab117, whose structural analysis reveals that it binds to a deep, hydrophobic pocket on the interior surface of the capsid via the CDR-H3. Main-chain and hydrophobic interactions provide broad serotype specificity. ELISA analysis confirms that Ab117 is a novel pan-serotype and conformational epitope-specific 12S reagent, suitable for assessing capsid integrity.
TomoCPT: a generalizable model for 3D particle detection and localization in cryo-electron tomograms
Cryo-electron tomography is a rapidly developing field for studying macromolecular complexes in their native environments and has the potential to revolutionize our understanding of protein function. However, fast and accurate identification of particles in cryo-tomograms is challenging and represents a significant bottleneck in downstream processes such as subtomogram averaging. Here, we present tomoCPT (Tomogram Centroid Prediction Tool), a transformer-based solution that reformulates particle detection as a centroid-prediction task using Gaussian labels. Our approach, which is built upon the SwinUNETR architecture, demonstrates superior performance compared with both conventional binary labelling strategies and template matching. We show that tomoCPT effectively generalizes to novel particle types through zero-shot inference and can be significantly enhanced through fine-tuning with limited data. The efficacy of tomoCPT is validated using three case studies: apoferritin, achieving a resolution of 3.0 Å compared with 3.3 Å using template matching, SARS-CoV-2 spike proteins on cell surfaces, yielding an 18.3 Å resolution map where template matching proved unsuccessful, and rubisco molecules within carboxysomes, reaching 8.0 Å resolution. These results demonstrate the ability of tomoCPT to handle varied scenarios, including densely packed environments and membrane-bound proteins. The implementation of the tool as a command-line program, coupled with its minimal data requirements for fine-tuning, makes it a practical solution for high-throughput cryo-ET data-processing workflows.
Integrative Approaches to Study Virus Structures.
A virus particle must work as a strongroom to protect its genome, but at the same time it must undergo dramatic conformational changes to infect the cell in order to replicate and assemble progeny. Thus, viruses are miniaturized wonders whose structural complexity requires investigation by a combination of different techniques that can tackle both static and dynamic processes. In this chapter, we will illustrate how major structural techniques such as X-ray crystallography and electron microscopy can be combined with other techniques to determine the structure of complex viruses. The power of these hybrid approaches is discussed through a number of examples.
From Voxels to Viruses: Using Deep Learning and Crowdsourcing to Understand a Virus Factory
Many bioimaging research projects require objects of interest to be identified, located, and then traced to allow quantitative measurement. Depending on the complexity of the system and imaging, instance segmentation is often done manually, and automated approaches still require weeks to months of an individual’s time to acquire the necessary training data for AI models. As such, there is a strong need to develop approaches for instance segmentation that minimize the use of expert annotation while maintaining quality on challenging image analysis problems. Herein, we present our work on a citizen science project we ran called Science Scribbler: Virus Factory on the Zooniverse platform, in which citizen scientists annotated a cryo-electron tomography volume by locating and categorising viruses using point-based annotations instead of manually drawing outlines. One crowdsourcing workflow produced a database of virus locations, and the other workflow produced a set of classifications of those locations. Together, this allowed mask annotation to be generated for training a deep learning–based segmentation model. From this model, segmentations were produced that allowed for measurements such as counts of the viruses by virus class. The application of citizen science–driven crowdsourcing to the generation of instance segmentations of volumetric bioimages is a step towards developing annotation-efficient segmentation workflows for bioimaging data. This approach aligns with the growing interest in citizen science initiatives that combine the collective intelligence of volunteers with AI to tackle complex problems while involving the public with research that is being undertaken in these important areas of science.
Rubisco packaging and stoichiometric composition of the native β-carboxysome in Synechococcus elongatus PCC7942.
Carboxysomes are anabolic bacterial microcompartments that play an essential role in CO2 fixation in cyanobacteria. This self-assembling proteinaceous organelle uses a polyhedral shell constructed by hundreds of shell protein paralogs to encapsulate the key CO2-fixing enzymes Rubisco and carbonic anhydrase. Deciphering the precise arrangement and structural organization of Rubisco enzymes within carboxysomes is crucial for understanding carboxysome formation and overall functionality. Here, we employed cryo-electron tomography and subtomogram averaging to delineate the three-dimensional packaging of Rubiscos within β-carboxysomes in the freshwater cyanobacterium Synechococcus elongatus PCC7942 grown under low light. Our results revealed that Rubiscos are arranged in multiple concentric layers parallel to the shell within the β-carboxysome lumen. We also detected Rubisco binding with the scaffolding protein CcmM in β-carboxysomes, which is instrumental for Rubisco encapsulation and β-carboxysome assembly. Using Quantification conCATamer (QconCAT)-based quantitative mass spectrometry, we determined the absolute stoichiometric composition of the entire β-carboxysome. This study provides insights into the assembly principles and structural variation of β-carboxysomes, which will aid in the rational design and repurposing of carboxysome nanostructures for diverse bioengineering applications.