Biology subjects

Patrick, W. M.

Publications and source records attributed to Patrick, W. M..

8 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↗

bioRxiv2026

Evaluating β-glucanases as cell wall-permeabilising agents against Phytophthora agathidicida oospores

The persistence of specialised survival spores produced by microbial pathogens represents a primary bottleneck in the management of plant diseases. In oomycetes, these spores (known as oospores) are largely impervious to chemical control, allowing them to persist in soil and initiate new infection cycles over many years. A prominent example is the soil-borne pathogen Phytophthora agathidicida, the causal agent of kauri dieback disease, where long-lived oospores hinder conservation efforts in native forests. The resilience of oospores is attributed to their thick wall composed of complex {beta}-glucan layers that render the oospores impermeable to most conventional biocides. Here we have investigated an enzyme-based approach for weakening the oospore cell wall. We searched enzyme databases to select {beta}-glucanases targeting a variety of linkages found in Phytophthora oospore walls. Eight of these {beta}-glucanases were successfully purified and tested for their digestive activity against intact oospores in vitro using a phenol-sulfuric acid assay. We showed that combining these enzymes was crucial to achieve significant digestion through synergies and additive effects. The optimal combination, comprising 1,3-, 1,6-, and 1,3(4)-{beta}-glucanases, was evaluated for its ability to permeabilise oospores to five biocides typically effective only on other, more sensitive lifecycle stages of the pathogen. Using a live/dead fluorescence assay, we observed that the effects of the membrane-targeting biocides were potentiated in oospores that were pre-treated with the {beta}-glucanase mixture. Our results highlight enzymatic cell wall permeabilisation as a promising strategy toward improved management of oospore persistence in kauri forest soils and against broader oomycete threats. KeypointsO_LIOur phenol-sulfuric acid assay can be used to screen for oospore-degrading enzymes. C_LIO_LISynergistic enzyme combinations are essential for effective oospore wall digestion. C_LIO_LIEnzyme pre-treatment sensitises oospores to membrane-targeting biocides. C_LI

biochemistry↗

bioRxiv2026

Engineering antigenic breadth against SARS-CoV-2 by pairing divergent RBDs within a single mRNA immunogen

Vaccines capable of eliciting broadly neutralising antibodies (bnAbs) are a major goal for pandemic preparedness. A persistent challenge across vaccine Wields is how to deliberately recruit the rare B cell clones that recognise conserved epitopes shared across diverse viral variants. BnAbs have been known to frequently emerge through extensive somatic hypermutation during afWinity maturation, here we describe an alternative, structure-driven mechanism for bnAb selection. We designed an mRNA vaccine in which two antigenically distinct SARS-CoV-2 variants (Omicron and Delta; O-{Delta}) receptor binding domains (RBDs) are physically fused on a single polypeptide. This design is predicted to favour B cell antigen receptors capable of engaging conserved epitopes on both RBDs with enhanced avidity. A matched non-divergent tandem RBD (Delta-Delta; {Delta}-{Delta}) served as a control. The divergent (O-{Delta}) immunogen was robustly expressed and retained high-afWinity ACE2 binding. In mice, immunisation elicited potent antibody responses and increased the frequency of antigen-speciWic cross-reactive B cells, recognising Delta, Omicron, and the 2002 pandemic strain SARS-CoV RBDs. Using multicolour RBD tetramers and single-cell B cell receptor sequencing, we show that breadth arises via two distinct pathways. The divergent vaccine preferentially enriches clonally distinct cross-reactive B cells (not present within non-cross-reactive B cell pools) with low levels of somatic hypermutation (SHM), consistent with selection of germline-biased precursors. In contrast, the matched control vaccine yields cross-reactivity primarily within existing clonal lineages (clonal overlap between cross-reactive and non-cross-reactive cells) and at higher mutational burdens, consistent with afWinity-maturation-driven acquisition of breadth. Together, these Windings demonstrate that antigen structure can bias B cell selection towards cross-reactive speciWicities without requiring extensive SHM. This work establishes a simple, modular antigen-design principle in which juxtaposing appropriately divergent antigens on a single scaffold promotes the enrichment of bnAb-prone B cells, providing a scalable strategy for vaccine development against rapidly evolving pathogens.

immunology↗

bioRxiv2021

Assessing the effectiveness of oxathiapiprolin towards Phytophthora agathidicida, the causal agent of kauri dieback disease

Phytophthora species cause disease and devastation of plants in ecological and horticultural settings worldwide. A recently identified species, P. agathidicida, infects and ultimately kills the treasured kauri trees that are endemic to New Zealand. Currently there are few options for controlling or treating P. agathidicida. In this study, we sought to assess the toxicity of the oomycide oxathiapiprolin against several lifecycle stages of two geographically distinct P. agathidicida isolates. Half maximal effective concentration (EC50) values were determined to be approximately 0.1 ng/ml for inhibiting mycelial growth, indicating that P. agathidicida mycelia are more sensitive to oxathiapiprolin than those from most other Phytophthora species that have been studied. Oxathiapiprolin was also highly effective at inhibiting the germination of zoospores (EC50 = 2-9 ng/ml for the two isolates) and oospores (complete inhibition at 100 ng/ml). In addition, oxathiapiprolin delayed the onset of detached kauri leaf infection in a dose-dependent manner. Collectively, the results presented here highlight the significant potential of oxathiapiprolin as a tool to aid in the control of kauri dieback disease.

microbiology↗

bioRxiv2020

A giant leap in sequence space reveals the intracellular complexities of evolving a new function

Selection for a promiscuous enzyme activity provides substantial opportunity for competition between endogenous and new substrates to influence the evolutionary trajectory, an aspect that has generally been overlooked in laboratory directed evolution studies. We evolved the Escherichia coli nitro/quinone reductase NfsA to detoxify chloramphenicol by randomising eight active site residues simultaneously and interrogating [~]250,000,000 reconfigured NfsA variants. Analysis of every possible evolutionary intermediate of the two best chloramphenicol reductases revealed complex epistatic interactions that restrict each hypothetical trajectory. In both cases, improved chloramphenicol detoxification was only possible after one essential substitution had eliminated activity with endogenous quinone substrates. Unlike the predominantly weak trade-offs seen in previous experimental studies, this substrate incompatibility suggests endogenous metabolites have considerable potential to shape evolutionary outcomes. Unselected prodrug-converting activities were mostly unaffected, which emphasises the importance of negative selection to effect enzyme specialisation, and offers an application for the evolved genes as dual-purpose selectable/counter-selectable markers.

biochemistry↗

bioRxiv2019

Assessment of Phenotype Microarray plates for rapid and high-throughput analysis of collateral sensitivity networks

The crisis of antimicrobial resistance is driving research into the phenomenon of collateral sensitivity. Sometimes, when a bacterium evolves resistance to one antimicrobial, it becomes sensitive to others. In this study, we have investigated the utility of Phenotype Microarray (PM) plates for identifying collateral sensitivities with unprecedented throughput. We assessed the relative resistance/sensitivity phenotypes of nine strains of Staphylococcus aureus (two laboratory strains and seven clinical isolates) towards the 72 antimicrobials contained in three PM plates. In general, the PM plates reported on resistance and sensitivity with a high degree of reproducibility. However, a rigorous comparison of PM growth phenotypes with minimum inhibitory concentration (MIC) measurements revealed a trade-off between throughput and accuracy. Small differences in PM growth phenotype did not necessarily correlate with changes in MIC. Thus, we conclude that PM plates are useful for the rapid and high-throughput assessment of large changes in collateral sensitivity phenotypes during the evolution of antimicrobial resistance, but more subtle examples of cross-resistance or collateral sensitivity cannot be reliably identified using this approach.

microbiology↗

bioRxiv2019

A large-scale comparison shows that genetic changes causing antibiotic resistance in experimentally evolved Pseudomonas aeruginosa predict those in naturally evolved bacteria

Pseudomonas aeruginosa is an opportunistic pathogen that causes a wide range of acute and chronic infections. An increasing number of isolates have acquired mutations that make them antibiotic resistant, making treatment more difficult. To identify resistance-associated mutations we experimentally evolved the antibiotic sensitive strain P. aeruginosa PAO1 to become resistant to three widely used anti-pseudomonal antibiotics, ciprofloxacin, meropenem and tobramycin. Mutants were able to tolerate up to 2048-fold higher concentrations of antibiotic than strain PAO1. Genome sequences were determined for thirteen mutants for each antibiotic. Each mutant had between 2 and 8 mutations. There were at least 8 genes mutated in more than one mutant per antibiotic, demonstrating the complexity of the genetic basis of resistance. Additionally, large deletions of up to 479kb arose in multiple meropenem resistant mutants. For all three antibiotics mutations arose in genes known to be associated with resistance, but also in genes not previously associated with resistance. To determine the clinical relevance of mutations uncovered in experimentally-evolved mutants we analysed the corresponding genes in 457 isolates of P. aeruginosa from patients with cystic fibrosis or bronchiectasis as well as 172 isolates from the general environment. Many of the genes identified through experimental evolution had changes predicted to be function-altering in clinical isolates but not in isolates from the general environment, showing that mutated genes in experimentally evolved bacteria can predict those that undergo mutation during infection. These findings expand understanding of the genetic basis of antibiotic resistance in P. aeruginosa as well as demonstrating the validity of experimental evolution in identifying clinically-relevant resistance-associated mutations.\n\nImportanceThe rise in antibiotic resistant bacteria represents an impending global health crisis. As such, understanding the genetic mechanisms underpinning this resistance can be a crucial piece of the puzzle to combatting it. The importance of this research is that by experimentally evolving P. aeruginosa to three clinically relevant antibiotics, we have generated a catalogue of genes that can contribute to resistance in vitro. We show that many (but not all) of these genes are clinically relevant, by identifying variants in clinical isolates of P. aeruginosa. This research furthers our understanding of the genetics leading to resistance in P. aeruginosa and provides tangible evidence that these genes can play a role clinically, potentially leading to new druggable targets or inform therapies.

microbiology↗

bioRxiv2019

Genomic and phenotypic comparison of environmental and patient-derived isolates of Pseudomonas aeruginosa suggest that antimicrobial resistance is rare within the environment

Patient-derived isolates of the opportunistic pathogen Pseudomonas aeruginosa are frequently resistant to antibiotics due to the presence of sequence variants in resistance-associated genes. However, the frequency of antibiotic resistance and of resistance-associated sequence variants in environmental isolates of P. aeruginosa has not been well studied. Antimicrobial susceptibility testing (ciprofloxacin, ceftazidime, meropenem, tobramycin) of environmental (n=50) and cystic fibrosis (n=42) P. aeruginosa isolates was carried out. Following whole genome sequencing of all isolates, twenty-five resistance-associated genes were analysed for the presence of likely function-altering sequence variants. Environmental isolates were susceptible to all antibiotics with one exception, whereas patient-derived isolates had significant frequencies of resistance to each antibiotic and a greater number of likely resistance-associated genetic variants. These findings indicate that the natural environment does not act as a reservoir of antibiotic-resistant P. aeruginosa, supporting a model in which antibiotic susceptible environmental bacteria infect patients and develop resistance during infection.\n\nAuthor Contributions\n\nO_TBL View this table:\norg.highwire.dtl.DTLVardef@19d869eorg.highwire.dtl.DTLVardef@f10c9forg.highwire.dtl.DTLVardef@173a576org.highwire.dtl.DTLVardef@17415e8org.highwire.dtl.DTLVardef@4a5d25_HPS_FORMAT_FIGEXP M_TBL C_TBL

microbiology↗