Biology subjects

von Itzstein, M.

Publications and source records attributed to von Itzstein, M..

9 recordsLinked to original sources

bioRxiv2026

SARS-CoV-2 spike protein-associated sialoglycoconjugates induce nanoscale filipodia to facilitate micro-size platelet clotting

COVID-19 disease is associated with thrombosis, but the pathogenic mechanism remains unclear. Here, we investigate how SARS-CoV-2 spike protein causes platelet activation and aggregation. Our three-dimensional ultrastructural analyses showed that invaginated platelet structures, open canalicular system (OCS), expanded upon activation, trapping viral particles in the process. Binding with platelet OCS concealed SAR-CoV-2 spike-coated particles from virion detection in platelet-depleted blood plasma. Both SARS-CoV-2 spike coated-particles and recombinant spikes specifically induced platelet aggregation with nanoscale filipodia extensions, with the terminal sialic acids of the SARS-CoV-2 spike protein-associated sialoglycoconjugates being the key determinant in platelet activation. Our work illustrates that virus-associated sialic acids, not proteins, are functionally responsible for SARS-CoV-2 induced thrombotic events, providing a mechanistic insight on how glycosylation contributes to disease severity in COVID-19. This study lays the foundation for the development of glycan-modified vaccines with reduced risks of thrombosis.

molecular biology↗

bioRxiv2026

Filament formation is a conserved mechanism of Thoeris SIR2 effector activation

Thoeris defence systems protect bacteria from phages via abortive infection. In type I Thoeris systems, ThsA effectors containing silent information regulator 2 (SIR2) and SMF/DprA-LOG (SLOG) domains are activated by the cyclic ADP-ribose (ADPR) isomer 3'cADPR, triggering abortive infection via nicotinamide adenine dinucleotide (NAD+) depletion. 3'cADPR activates the NADase activity of Bacillus subtilis ThsA tetramers via filament formation of its SIR2 domains, but the molecular details of how 3'cADPR triggers this process remain incompletely understood. Here, we demonstrate that ThsA activation by 3'cADPR-induced SIR2 filament formation is conserved in type I Thoeris systems from Streptococcus equi and Entercoccus facium. We present cryo-electron microscopy structures of the S. equi ThsA filament bound to 3'cADPR and the non-cleavable NAD+ analog carba-NAD+, and of the S. equi ThsA tetramer bound to 3'cADPR. These structures reveal that SIR2 filament formation is required to stabilise an active site conformation that can bind and hydrolyse NAD+. The structures also show that 3'cADPR induces quaternary alterations in the SLOG dimers and consequently the SIR2 tetramer to enable ThsA filament formation. Collectively, our study provides a comprehensive understanding of 3'cADPR-induced activation of type I Thoeris effectors.

microbiology↗

bioRxiv2025

Human parainfluenza virus infection remodels the host cell glycome

Human parainfluenza virus type 3 (HPIV-3) remains a major cause of respiratory illness particularly among young children, the elderly and immunocompromised individuals. Despite significant efforts in therapeutic discovery research, there is neither an effective antiviral nor a vaccine available against HPIV-3. Host cell glycosylation is known to play a pivotal role in virus entry and replication. While some host glycan-based cellular receptors for HPIV-3 have been identified, the dynamics of the host glycome upon HPIV-3 infection has never been studied. Herein, we report the first mass spectrometry-based study that provides direct insight into the remodelling of the human lung adenocarcinoma cell (A549) glycome upon HPIV-3 infection. In this study we observed that HPIV-3 infection led to significant host-cell glycome changes in both oligomannose and sialylated complex-type N-glycans. Moreover, notable changes were also observed in both core 1 and core 2 type O-glycans, along with distinct glycosphingolipid remodelling in infected cells compared to their mock-infected counterparts. Our study presents the first evidence that hPIV-3 infection alters host-cell glycome, offering new insights into the viruss impact on host cellular processes.

microbiology↗

bioRxiv2025

Ultra-fast and highly sensitive protein structure alignment with segment-level representations and block-sparse optimization

Deep learning models for protein structure prediction have given rise to extreme growth in 3D structure data. As a result, traditional methods for geometric structure alignment are too slow to effectively search modern structure libraries. In this study we introduce SPfast - a fully geometric method for structure-based alignment which accelerates search by more than 2 orders of magnitude while increasing sensitivity by 21% and 5% compared with foldseek and TMalign respectively. Using the significant speed of SPfast to conduct more than 100B pairwise comparisons between bona fide uncharacterized proteins and a large-scale, annotated structure library uncovers new biological insights relating to type III secretion in pathogenic bacteria and identifies novel toxin-antitoxin systems. Putative SPfast-based functional assignments are supported by orthogonal evidence including shared genomic context and high-confidence AlphaFold3 complex modelling.

bioinformatics↗

bioRxiv2024

Remodelling of the glycome of B-cell precursor acute lymphoblastic leukemia cells developing drug-tolerance

Reduced responsiveness of precursor B-acute lymphoblastic leukemia (BCP-ALL) to chemotherapy can be first detected in the form of minimal residual disease leukemia cells that persist after 28 days of initial treatment. The ability of these cells to resist chemotherapy is partly due to the microenvironment of the bone marrow, which promotes leukemia cell growth and provides protection, particularly under these conditions of stress. It is unknown if and how the glycocalyx of such cells is remodelled during the development of tolerance to drug treatment, even though glycosylation is the most abundant cell surface post-translational modification present on the plasma membrane. To investigate this, we performed omics analysis of BCP-ALL cells that survived a 30-day vincristine chemotherapy treatment while in co-culture with bone marrow stromal cells. Proteomics showed decreased levels of some metabolic enzymes. Overall glycocalyx changes included a shift from Core-2 to less complex Core-1 O-glycans, and reduced overall sialylation, with a shift from 2-6 to 2-3 linked Neu5Ac. Interestingly, there was a clear increase in bisecting complex N-glycans with a concomitant increased mRNA expression of MGAT3, the only enzyme known to form bisecting N-glycans. These small but reproducible quantitative differences suggest that individual glycoproteins become differentially glycosylated. Glycoproteomics confirmed glycosite-specific modulation of cell surface and lysosomal proteins in drug-tolerant BCP-ALL cells, including HLA-DRA, CD38, LAMP1 and PPT1. We conclude that drug-tolerant persister leukemia cells that grow under continuous chemotherapy stress have characteristic glycotraits that correlate with and perhaps contribute to their ability to survive and could be tested as neoantigens in drug-resistant leukemia.

cancer biology↗

bioRxiv2022

Drug repurposing for therapeutic discovery against human metapneumovirus infection

Human metapneumovirus (HMPV) is recognised as an important cause of pneumonia in infants, elderly, and immunocompromised individuals worldwide. The absence of an antiviral treatment or vaccine strategy against HMPV infection creates a high burden on the global health care system. Drug repurposing has become increasingly attractive for the treatment of emerging and endemic diseases as it requires less research and development costs than traditional drug discovery. In this study, we developed an in vitro medium-throughput screening assay that allows for the identification of novel anti-HMPV drugs candidates. Out of [~]2400 compounds, we identified eleven candidates with a dose-dependent inhibitory activity against HMPV infection. Additionally, we further described the mode of action of five anti-HMPV candidates with low in vitro cytotoxicity. Two entry inhibitors, Evans Blue and aurintricarboxylic acid, and three post-entry inhibitors, mycophenolic acid, mycophenolate mofetil, and 2,3,4-trihydroxybenzaldehyde, were identified. Among them, the mycophenolic acid series displayed the highest levels of inhibition, due to the blockade of intracellular guanosine synthesis. Importantly, MPA has significant potential for drug repurposing as inhibitory levels are achieved below the approved human oral dose. Our drug-repurposing strategy proved to be useful for the rapid discovery of novel hit candidates to treat HMPV infection and provide promising novel templates for drug design. HighlightsO_LIThere is currently no treatment against acute HMPV infection. C_LIO_LIWe developed a medium-throughput screening for drug repurposing against HMPV infection. C_LIO_LIEvaluation of a large drug library identified 5 candidates with low M to nM IC50 values against HMPV growth. C_LIO_LIThe approved drug mycophenolic acid (MPA) is a nM inhibitor of HMPV in vitro infection C_LIO_LIHuman MPA plasma levels upon oral dosing are 10x higher than MPA IC50 against HMPV in vitro infection. C_LI

pharmacology and toxicology↗

bioRxiv2022

A phasevarion controls multiple virulence traits, including expression of vaccine candidates, in Streptococcus pneumoniae

Streptococcus pneumoniae is the most common cause of bacterial illness worldwide. Current vaccines based on the polysaccharide capsule (PCV-13 and PPSV-23) are only effective against a limited number of the >100 capsular serotypes. A universal vaccine based on conserved protein antigens requires a thorough understanding of gene expression in S. pneumoniae. Restriction-Modification (R-M) systems, classically described as a defence against bacteriophage, are almost ubiquitous in the bacterial domain, and roles other than phage defence. All S. pneumoniae strains encode the SpnIII R-M system. This system contains a phase-variable methyltransferase that randomly switches specificity, and controls expression of multiple genes; a phasevarion. We aimed to determine the role of the SpnIII phasevarion during pneumococcal pathobiology and determine if phase-variation resulted in differences in expression of protein antigens that are being investigated as vaccine candidates. Using locked S. pneumoniae strains that express a single SpnIII methyltransferase specificity, we found significant differences in clinically relevant traits, including survival in blood, and adherence to and invasion of human cells. Crucially, we also observed differences in expression of numerous proteinaceous vaccine candidates, which complicates selection of protein antigens for inclusion in a universal protein-based pneumococcal vaccine. This study will inform future vaccine design against S. pneumoniae by ensuring only stably expressed candidates are included in a rationally designed vaccine. Significance StatementS. pneumoniae is the worlds foremost bacterial pathogen. S. pneumoniae encodes a randomly expressed epigenetic regulator, a phasevarion (phase-variable regulon), that results in random expression of multiple genes. Previous work demonstrated that the pneumococcal SpnIII phasevarion switches between six different expression states, generating six unique phenotypic variants in a pneumococcal population. Here, we show that this phasevarion generates multiple phenotypic differences relevant to pathobiology. Importantly, expression of conserved protein antigens varies with phasevarion switching. As capsule expression, a major pneumococcal virulence factor, is also controlled by the phasevarion, our work will inform the selection of the best candidates to include in a rationally designed, universal pneumococcal vaccine.

microbiology↗

bioRxiv2021

Overcoming microenvironment-mediated chemoprotection through stromal Galectin-3 inhibition in acute lymphoblastic leukemia

Environmentally-mediated drug resistance in B-cell precursor acute lymphoblastic leukemia (BCP-ALL) significantly contributes to relapse. Stromal cells in the bone marrow environment protect leukemia cells by secretion of chemokines as cues for BCP-ALL migration towards, and adhesion to, stroma. Stromal cells and BCP-ALL cells communicate through stromal galectin-3. Here, we investigated the significance of stromal galectin-3 to BCP-ALL cells. We used CRISPR/Cas9 genome editing to ablate galectin-3 in stromal cells and found that galectin-3 is dispensable for steady-state BCP-ALL proliferation and viability. However, efficient leukemia migration and adhesion to stromal cells are significantly dependent on stromal galectin-3. Importantly, loss of stromal galectin-3 production sensitized BCP-ALL cells to conventional chemotherapy. We therefore tested novel carbohydrate-based small molecule compounds (Cpd14 and Cpd17) with high specificity for galectin-3. Consistent with results obtained using galectin-3-knockout stromal cells, treatment of stromal-BCP-ALL co-cultures inhibited BCP-ALL migration and adhesion. Moreover, these compounds induced anti-leukemic responses in BCP-ALL cells including a dose-dependent reduction of viability and proliferation, induction of apoptosis and, importantly, inhibition of drug resistance. Collectively, these findings indicate galectin-3 regulates BCP-ALL cell responses to chemotherapy through the interactions between leukemia cells and the stroma, and show that a combination of galectin-3 inhibition with conventional drugs can sensitize the leukemia cells to chemotherapy.

cancer biology↗

bioRxiv2021

Better together - Relative retention time plus spectral matching improves automated glycan characterization using PGC-nLC-IT-ESI-MS/MS

Porous Graphitized Carbon nano-liquid chromatography tandem mass spectrometry (PGC-nLC-MS/MS) is a glycomics technique with the unique capacity to differentiate isobaric glycans. The lack of suitable software tools integrating chromatography and MS-information delivered by PGC-nLC-MS/MS has been limiting fast and robust glycan identification and quantitation. We report a LC-system-independent strategy called GlycoRRT that combines relative retention time (RRT) and negative ion fragment spectra analyses for isobaric structure-specific glycomics of PGC-nLC-MS/MS data. The GlycoRRT toolset is fully customizable and easily adaptable enabling semi-automated high-throughput structural assignments. The current library contains over 200 entries and their individual meta-data (MS instrumentation, experimental conditions, retention times, fragmentation profiles and glycan structural diagnostic ion features) relevant for reliable data analyses. The GlycoRRT workflow was employed to map the N- and O-glycome in blood group matched human plasma and urine as well as decipher Immunoglobulin (IgG) glycosylation features from 13 different animal species. We have also developed visualization tools to enable a consistent, reliable, and reproducible analysis of large sets of multidimensional PGC-nLC-MS/MS glycomics data. This comprehensive glycan resource provides the glycan map of human and animal species, will serve as a reference in dissecting the role of glycans in host pathogen interaction and zoonotic disease transmission.

biochemistry↗