Biology subjects

Foster, A. J.

Publications and source records attributed to Foster, A. J..

7 recordsLinked to original sources

bioRxiv2026

Circular RNA vaccine performance is determined by RNA quality and epitranscriptomic tuning rather than innate activation

Circular RNA (circRNA) is an emerging vaccine modality that is proposed to improve stability, reduce reactogenicity and extend antigen expression compared with linear mRNA. However, the relative contributions to vaccine performance of its covalently closed structure, its purity, and of nucleotide modifications remain poorly described. Here, we systematically dissected these parameters in vivo across two antigen systems. We identified RNA quality as a major determinant of circRNA reactogenicity, with differences in innate immune activation tracking with the presence of residual RNA species in less refined preparations. In contrast, highly purified circRNA exhibited markedly reduced reactogenicity compared with linear mRNA, independent of nucleotide modification. Despite these differences, circRNA and mRNA vaccines elicited comparable antibody titres and T cell responses, indicating that reduced innate activation does not enhance adaptive immune magnitude. Notably, incorporating N6-methyladenosine (m6A) did not affect reactogenicity or antigen expression but selectively enhanced antibody quality, increasing binding affinity and neutralisation capacity. CircRNA vaccination also altered the anatomical distribution of germinal centre responses, reducing splenic antigen-specific germinal centre B cells while preserving lymph node responses. Together, these findings show that circRNA vaccine performance is governed by RNA preparation quality and epitranscriptomic tuning rather than innate activation alone.

immunology↗

bioRxiv2025

Pulcherriminic acid biosynthesis and Transport: Insights from a heterologous system in Saccharomyces cerevisiae

Pulcherriminic acid is an iron chelator produced by some Kluyveromyces and Metschnikowia yeasts. Its biosynthesis is encoded by the four-gene PUL cluster, where PUL1 and PUL2 are the biosynthetic enzymes, PUL3 mediates the uptake of iron-bound pulcherrimin, and PUL4 is a putative regulator. Pulcherriminic acid holds antifungal potential, as the growth of organisms unable to uptake pulcherrimin is inhibited by deficit of essential iron. Thus, a heterologous production system to further characterize and optimize its biosynthesis would be valuable. Using our in-house yeast collection and genomes available in databases, we cloned PUL1 and PUL2 genes from K. lactis and one of our wild Metschnikowia isolates and built an effective production system in S. cerevisiae able to inhibit pathogenic growth. In this context, the K. lactis genes yielded faster pulcherriminic acid production than those from the Metschnikowia isolate and a combinatorial approach showed PUL1 to be the production bottleneck. We further showed that Pul3 is an importer of pulcherrimin, but also mediates the export of pulcherriminic acid and that the growth of pathogens like Candidozyma auris and organisms encoding PUL3 in their genome, previously called "cheaters", is inhibited by pulcherriminic acid, highlighting its potential as an antimicrobial agent.

microbiology↗

bioRxiv2023

Ergosterol mediates aggregation of natamycin in the yeast plasma membrane

Polyene macrolides are antifungal substances, which interact with cells in a sterol-dependent manner. While being widely used, their mode of action is poorly understood. Here, we employ ultraviolet-sensitive (UV) microscopy to show that the antifungal polyene natamycin binds to the yeast plasma membrane (PM) and causes permeation of propidium iodide into cells. Right before membrane permeability becomes compromised, we observed clustering of natamycin in the PM that was independent of PM protein domains. Aggregation of natamycin was paralleled by cell deformation and membrane blebbing as revealed by soft X-ray microscopy. Substituting ergosterol for cholesterol decreased natamycin binding and resulted in reduced clustering of natamycin in the PM. Blocking of ergosterol synthesis necessitates sterol import via the ABC transporters Aus1/Pdr11 to ensure natamycin binding. Quantitative imaging of dehydroergosterol (DHE) and cholestatrienol (CTL), two analogs of ergosterol and cholesterol, respectively, revealed a largely homogeneous lateral sterol distribution in the PM, ruling out that natamycin binds to pre-assembled sterol domains. Depletion of sphingolipids using myriocin increased natamycin binding to yeast cells, likely by increasing the ergosterol fraction in the outer PM leaflet. We conclude that ergosterol-specific aggregation of natamycin in the yeast PM underlies its antifungal activity, which can be synergistically enhanced by inhibitors of sphingolipid synthesis. SignificanceErgosterol is the major sterol in the membranes of fungi and a major target for antifungal treatments. Polyene macrolides, such as natamycin, are known to target ergosterol but the underlying mechanisms for their preference for this yeast sterol compared to mammalian cholesterol is not understood. This study shows that natamycin forms aggregates when associated with yeast S. cerevisiae in an ergosterol-dependent manner. Cholesterol can only partially substitute for ergosterol with respect to natamycin binding and aggregation. Membrane-associated aggregation of natamycin is not the result of pre-formed sterol domains in the cell membrane, as we show by direct visualization of minimally modified ergosterol and cholesterol analogs. Inhibiting sphingolipid synthesis increased membrane association and antifungal activity of natamycin, suggesting that targeting sphingolipids in combination with polyene macrolides could lead to novel drug treatment approaches against fungal infections.

biophysics↗

bioRxiv2022

Rgs1 is a regulator of effector gene expression during plant infection by the rice blast fungus Magnaporthe oryzae

To cause rice blast disease the filamentous fungus Magnaporthe oryzae secretes a battery of effector proteins into host plant tissue to facilitate infection. Effector-encoding genes are expressed only during plant infection and show very low expression during other developmental stages. How effector gene expression is regulated in such a precise manner during invasive growth by M. oryzae is not known. Here, we report a forward-genetic screen to identify regulators of effector gene expression, based on selection of mutants that show constitutive effector gene expression. Using this simple screen, we identify Rgs1, a regulator of G-protein signalling (RGS) protein that is necessary for appressorium development, as a novel transcriptional regulator of effector gene expression, which acts prior to plant infection. We show that an N-terminal domain of Rgs1, possessing transactivation activity, is required for effector gene regulation and acts in an RGS-independent manner. Rgs1 controls expression of at least 60 temporally co-regulated effector genes, preventing their transcription during the pre-penetration stage of development prior to plant infection. A regulator of appressorium morphogenesis is therefore also required for orchestration of pathogen gene expression required for invasive growth by M. oryzae during plant infection.

molecular biology↗

bioRxiv2022

A pandemic clonal lineage of the wheat blast fungus

Wheat, the most important food crop, is threatened by a blast disease pandemic. Here, we show that a clonal lineage of the wheat blast fungus recently spread to Asia and Africa following two independent introductions from South America. Through a combination of genome analyses and laboratory experiments, we show that the decade-old blast pandemic lineage can be controlled by the Rmg8 disease resistance gene and is sensitive to strobilurin fungicides. However, we also highlight the potential of the pandemic clone to evolve fungicide-insensitive variants and sexually recombine with African lineages. This underscores the urgent need for genomic surveillance to track and mitigate the spread of wheat blast outside of South America, and to guide pre-emptive wheat breeding for blast resistance.

evolutionary biology↗

bioRxiv2020

Oxygen-independent chemogenetic protein tags for live-cell fluorescence microscopy

Fluorescent proteins enable targeted visualization of biomolecules in living cells, but their maturation is oxygen-dependent and they are susceptible to aggregation and/or suffer from poor photophysical properties. Organic fluorophores are oxygen-independent with superior photophysical properties, but targeting biomolecules in vivo is challenging. Here, we introduce two oxygen-independent chemogenetic protein (OICP) tags that impart fluorogenicity and fluorescence lifetime enhancement to bound organic dyes. We present a photo- and physicochemical characterization of thirty fluorophores interacting with two OICPs and conclude that aromatic planar structures bind with high specificity to the hydrophobic pockets of the proteins. The binding specificity of the tags and the superior photophysical properties of organic fluorophores enable microscopy of living bacterial and eukaryotic cells. The exchange of photobleached dye for unbleached fluorophore enables prolonged live-cell imaging. Our protein tags provide a general tool for investigating (sub)cellular protein localization and dynamics, protein-protein interactions, and microscopy applications under strictly oxygen-free conditions.

biochemistry↗

bioRxiv2018

CRISPR-Cas9 ribonucleoprotein-mediated co-editing and counterselection in the rice blast fungus

The rice blast fungus Magnaporthe oryzae is the most serious pathogen of cultivated rice and a significant threat to global food security. To accelerate targeted mutation and specific gene editing in this species, we have developed a rapid plasmid-free CRISPR-Cas9-based gene editing method. It has previously been reported in M. oryzae that transformation with plasmids expressing Cas9 can generate specific mutations using sgRNAs, directing the endonuclease to specific genes. We show, however, that expression of Cas9 is highly toxic to M. oryzae, rendering this approach impractical. We demonstrate that using purified Cas9 pre-complexed to RNA guides to form ribonucleoproteins (RNPs), provides an alternative and very effective gene editing procedure. When used in combination with oligonucleotide or PCR-generated donor DNAs, generation of strains with specific base pair edits, in-locus gene replacements, or multiple gene edits, is very rapid and straightforward. Additionally, we report a novel counterselection strategy which allows creation of precisely edited fungal strains that contain no foreign DNA and are completely isogenic to the wild type. Together, these developments represent a scalable improvement in the precision and speed of genetic manipulation in M. oryzae and are likely to be broadly applicable to other fungal species.

microbiology↗