Biology subjects

Bignell, E.

Publications and source records attributed to Bignell, E..

12 recordsLinked to original sources

bioRxiv2026

Inferring antifungal drug synergy from Candidozyma auris optical density data using Bayesian mechanistic modelling

Antifungal drug-resistant Candidozyma auris (C. auris) is a threat to human health worldwide. Combination antifungal drug therapy has emerged as a promising approach to combat drug-resistant C. auris because some drugs interact synergistically to increase fungal clearance when co-administered. Moreover, combination regimens that either rapidly act or completely kill C. auris could mitigate development of on-treatment resistance. However, traditional checkerboard methods to identify synergistic drug combinations only inspect fungal growth at a single timepoint. As a result, they cannot be used to estimate the rate of drug-action or to hypothesise on fungicidal or fungistatic drug-action. Mechanistic modelling would allow us to quantify time-dependent drug-action and infer killing or inhibitory action, but these models are usually fit to direct measurements of fungal growth whose collection is currently not scalable to many time-points and drug combinations. In this paper, we propose a Bayesian mechanistic modelling approach that could detect drug-synergy, estimate drug-action over time and investigate fungicidal or fungistatic drug-activity from optical density (OD600) data alone. OD600 is quicker and easier to collect than direct measurements of fungal growth and therefore more amenable to high-throughput susceptibility testing. By fitting our model to time-course OD600 data of a multi-drug-resistant C. auris isolate growing in mono- and combination drug regimens, we successfully inferred synergy between previously confirmed synergistic antifungal drugs (anidulafungin with manogepix or with 5-flucytosine) and linked our models inferred kinetic parameters to fungicidal and fungistatic action on C. auris growth, which matched drug-activity reported in literature where known. We validated that our model outperformed baseline logistic and Gompertz models using cross validation stratified by OD600 replicates. Our results represent the much-needed groundwork for identifying drug combinations for subsequent experimental testing for use in clinics based on their synergy, temporal drug-action and fungicidal or fungistatic activities inferred from OD600 data alone. Author SummaryThere is an urgent need to locate novel treatments to better treat antifungal drug-resistant Candidozyma auris infections. Combination therapy is a promising approach where two or more antifungal drugs are administered and interact synergistically to enhance fungal clearance. If these combinations are fast acting or eradicate fungi through killing, then they could also reduce the chance of resistance developing during treatment. The synergy of antifungal drug combinations is currently assessed by checkerboard methodologies that compare fungal growth under drug combinations to that under a single drug. However, checkerboard methodologies record only one time-point. Hence, they cannot evaluate drug combinations timeframe of action and follow-up studies are required to determine which combinations could optimally enhance killing. We developed a Bayesian mechanistic model that could detect synergy between drugs, estimate rates of drug-action and investigate killing and inhibition drug-action using only optical density (OD600) data of C. auris. OD600-based measurement of fungal growth is more amenable to large-scale drug testing than data typically used for mechanistic modelling, such as microscopy data. This work serves as a foundation for more targeted drug testing that identifies promising drug combinations based on their inferred drug-synergy and hypothesised killing (or inhibition) rates.

systems biology↗

bioRxiv2025

Clinical Candida Lactobacillus Isolate Pairs Reveal Distinct Host-Pathogen Interactions in a Vaginal Epithelial Model

Vulvovaginal candidiasis (VVC) is a highly prevalent condition affecting up to 75% of women, yet the mechanisms underpinning the transition from asymptomatic colonisation by Candida albicans to symptomatic infection remain incompletely understood. VVC is hormonally driven and rare in young girls and postmenopausal women. A central challenge lies in dissecting the complex interplay between microbial communities, hormonal factors, and host epithelial responses. While C. albicans is part of the normal vaginal microbiota at low abundance, its morphogenic switch to the hyphal form is closely linked with pathogenicity. Concomitantly, the dominance of specific Lactobacillus species is known to modulate vaginal health, but their precise role in shaping host-pathogen interactions during VVC is unresolved. We hypothesised that clinical isolate pairs of C. albicans and Lactobacillus spp. may differ in their ability to drive host epithelial damage, and that hormonal signals such as estrogen may exacerbate these interactions. To test this, we developed an in vitro VVC model using human vaginal epithelial cell (VEC) monolayers co-cultured with clinical isolate pairs. Using LDH release as a marker of cytotoxicity, extracellular pH monitoring, and immunoassays of mitogen-activated protein kinase (MAPK) phosphorylation, we interrogated host responses across microbial conditions in the presence or absence of estrogen. Our findings demonstrate that two clinical isolate pairs exhibit divergent behaviours. Pair C (C. albicans 203 and L. gasseri 167) suppressed epithelial cytotoxicity and maintained acidic pH, while Pair D (C. albicans 205 and L. jensenii 172) enhanced host damage and did not acidify the environment. Morphological differences in C. albicans isolates correlated with MAPK activation profiles, supporting a phenotype-specific host response. These results reveal that strain-level interactions within the vaginal microbiota significantly modulate host-pathogen dynamics.

microbiology↗

bioRxiv2025

Mgl2+ cDC2 triggering of fungal allergic inflammation depends on a spore induced glycolytic shift fuelled by local availability of glucose

Fungal spores are a major cause of severe asthmatic disease. However, the precise events that cause individuals to become sensitised to spores are poorly understood. Mgl2+ type 2 conventional dendritic cells (Mgl2+ cDC2s) are critical in coordinating allergic airway inflammation in mice following repeated exposure to inhaled spores. Yet, whether these DCs are directly acquiring spores from the airway, and the downstream mechanism(s) upon fungal uptake causing DCs to trigger allergic inflammation are unknown. Here we find that spores are acquired by lung DCs after inhalation although these events are rare ([~] 0.5% of the cDC2 population). Transcriptomics on isolated spore+ Mgl2+ cDC2s, compared to spore- Mgl2+ cDC2s from the same environment, revealed that a major consequence of fungal uptake was a boost in metabolic activity. Single-cell metabolic profiling revealed this increase in Mgl2+ cDC2 metabolism upon spore acquisition was fuelled by a glycolytic shift. To pinpoint if nutrient availability and acquisition is an important determinant of this response, mass spectrometry-based metabolomics revealed that, during fungal allergic inflammation, the local airway nutrient environment is altered. To ascertain which of these could be fuelling DC responses, we identified which substrates that feed into glycolysis were crucial. Of these, we found that glucose availability acts as a key rheostat in shaping cDC2 responses to spores. These data highlight a crucial role for glycolytic metabolism in driving cDC2 responses to spores, which is governed by glucose availability, defining novel targets for future therapeutic development of fungal allergic inflammation.

immunology↗

bioRxiv2024

Live-cell imaging of pathogenic fungal hyphae reveal dynamic cellular responses to clinical antifungals

Antifungal susceptibility testing quantifies end-point fungal biomass in liquid cultures initiated from non-invasive yeast or spore morphologies. However, end-point analyses obscure informative spatio-temporal responses to drug exposures. In the major fungal pathogens Aspergillus fumigatus and Candida albicans we used microfluidic-coupled, fluorescence-mediated live-cell imaging to capture the real-time responses of fungal hyphae to clinical concentrations of AmBisome or Caspofungin. In both fungi, AmBisome exposure caused rapid growth arrest, extensive hyphal vacuolation and membrane blebbing. Responses to Caspofungin exposure were slower with initial lytic effects occurring after 1.5 or 4 hours in A.fumigatus and C.albicans, respectively. Whilst C.albicans hyphae undergo unsalvageable hyphal lysis in response to Caspofungin, A.fumigatus exhibit several compensatory growth behaviours, including a novel resuscitative growth form, that circumvent lytic events to maintain apical and sub-apical hyphal growth. This study reveals how the differing biologies of the two pathogens affected outcomes and contributes to the highly disparate rates of antifungal efficacy amongst commonly used drugs, where spore/yeast-derived inhibitory doses may underestimate the dose required to arrest/kill the invasive hyphal morphotypes of fungal pathogens in vitro.

cell biology↗

bioRxiv2024

The sulfur-related metabolic status of Aspergillus fumigatus during infection reveals cytosolic serine hydroxymethyltransferase as a promising antifungal target.

Sulfur metabolism is an essential aspect of fungal physiology and is known to be crucial for pathogenicity. Fungal sulfur metabolism comprises anabolic and catabolic routes that are not well- conserved in mammals, and therefore can be considered a promising source of prospective novel antifungal targets. To gain insight into the status of the Aspergillus fumigatus sulfur-related metabolism during infection we used a NanoString custom nCounter TagSet and compared the expression of 68 key metabolic genes in different murine models of invasive pulmonary aspergillosis, at three different time-points, and a variety of in vitro conditions. We identified a set of 15 genes that are consistently expressed at higher levels in vivo than in vitro, suggesting that they may be particularly relevant for intrapulmonary growth and therefore constitute promising drug targets. Indeed, the role of five of the fifteen genes had previously been empirically validated, supporting the likelihood that the remaining candidates are relevant. In addition, the analysis of the dynamics of gene expression at the early (16h), mid (24h-1) and late (72h) time-points uncovered potential disease initiation and progression factors. We further characterised one of the identified genes, encoding the cytosolic serine hydroxymethyltransferase ShmB, and demonstrated that it is an essential gene of A. fumigatus and that it is also required for virulence in a murine model of established pulmonary infection. We further show that the structure of the ligand binding pocket of the fungal enzyme differs significantly from its human counterpart, suggesting that specific inhibitors can be designed. Therefore, in vivo transcriptomics is a powerful tool to identify genes crucial for fungal pathogenicity that might encode promising antifungal target candidates. AUTHOR SUMMARYAspergillus fumigatus is an opportunistic human fungal pathogen that causes devastating chronic and invasive infections in immunocompromised patients. Our arsenal of antifungal drugs to fight this and other fungal pathogens is very limited, partly because of the high similarity between eukaryotic fungal and human cells makes the identification of suitable drug targets a challenging task. Furthermore, targets identified in vitro are often not effective in vivo, as their action is not relevant for fungal virulence. To address this challenge, we compared the expression profiles of a set of genes involved in sulfur metabolism, a promising source of potential drug targets, in numerous in vitro and in vivo conditions to identify favourable antifungal candidates. Subsequently, we validated one of the highlighted genes, demonstrating that it is essential for A. fumigatus viability and virulence, and that it can likely be targeted by specific inhibitors. Hence, we show the potential of using in vivo transcriptomics to identify targets that contribute to virulence, propose various candidates for future studies and present a novel target validated for further antifungal drug development.

microbiology↗

bioRxiv2024

Inferring fungal growth rates from optical density data

Quantifying fungal growth underpins our ability to effectively treat severe fungal infections. Current methods quantify fungal growth rates from time-course morphology-specific data, such as hyphal length data. However, automated large-scale collection of such data lies beyond the scope of most clinical microbiology laboratories. In this paper, we propose a mathematical model of fungal growth to estimate morphology-specific growth rates from easy-to-collect, but indirect, optical density (OD600) data of Aspergillus fumigatus growth (filamentous fungus). Our method accounts for OD600 being an indirect measure by explicitly including the relationship between the indirect OD600 measurements and the calibrating true fungal growth in the model. Therefore, the method does not require de novo generation of calibration data. Our model outperformed reference models at fitting to and predicting OD600 growth curves and overcame observed discrepancies between morphology-specific rates inferred from OD600 versus directly measured data in reference models that did not include calibration. Author summaryQuantifying fungal growth is essential for antifungal drug discovery and monitoring antifungal resistance. As fungal growth is complex, with fungal morphology (shape) dynamically changing over time, previous studies have quantified fungal growth by estimating growth rates during specific fungal morphologies (morphology-specific growth rates) or by mathematically modelling fungal growth. However, collecting time-series data that captures the morphological information required for mathematical model fitting or estimating morphology-specific growth rates is prohibitively time consuming for large-scale drug testing in most microbiology laboratories. Alternatively, fungal growth can be quickly, although indirectly, quantified by measuring the optical density (OD) of a broth culture. However, changes in OD are not always reflective of true changes in fungal growth because OD is an indirect measure. This paper proposes a method to model fungal growth and estimate a morphology-specific growth rate from indirect OD600 measurements of the major mould pathogen, Aspergillus fumigatus. We explicitly model the relationship between measured indirect OD600 data and true fungal growth (calibration). The presented work serves as the much-needed foundation for estimating and comparing morphology-specific fungal growth rates in varying antifungal drug concentrations using only OD600 data.

systems biology↗

bioRxiv2022

Heightened efficacy of anidulafungin when used in combination with manogepix or 5-flucytosine against Candida auris in vitro

Candida auris is an emerging, multi-drug resistant fungal pathogen that causes refractory colonisation and life-threatening invasive nosocomial infections. The high proportion of C. auris isolates that display antifungal resistance severely limits treatment options. Combination therapies provide a possible strategy to enhance antifungal efficacy and prevent the emergence of further resistance. Therefore, we examined drug combinations using antifungals that are already in clinical use or undergoing clinical trials. Using checkerboard assays we screened combinations of 5-flucytosine and manogepix (the active form of the novel antifungal drug fosmanogepix) with anidulafungin, amphotericin B or voriconazole against drug resistant and susceptible C. auris isolates from clades I and III. Fractional inhibitory concentration indices (FICI values) of 0.28-0.75 and 0.36-1.02 were observed for combinations of anidulafungin with manogepix or 5-flucytosine, respectively, indicating synergistic activity. The high potency of these anidulafungin combinations was confirmed using live-cell microfluidics-assisted imaging of fungal growth. In summary, combinations of anidulafungin with manogepix or 5-flucytosine show great potential against both resistant and susceptible C. auris isolates.

microbiology↗

bioRxiv2022

Distinct cohorts of Aspergillus fumigatus transcription factors are required for epithelial damage occurring via contact- or soluble effector-mediated mechanisms

Damage to the lung epithelium is a unifying feature of disease caused by the saprophytic fungus Aspergillus fumigatus. However, the mechanistic basis and the regulatory control of such damage is poorly characterized. Previous studies have identified A. fumigatus mediated pathogenesis as occurring at early ([≤] 16 hours) or late (>16 hours) phases of the fungal interaction with epithelial cells, and respectively involve direct contact with the host cell or the action of soluble factors produced by mature fungal hyphae. Both early and late phases of epithelial damage have been shown to be subject to genetic regulation by the pH-responsive transcription factor PacC. This study sought to determine whether other transcriptional regulators play a role in modulating epithelial damage. In particular, whether the early and late phases of epithelial damage are governed by same or distinct regulators. Furthermore, whether processes such as spore uptake and hyphal adhesion, that have previously been documented to promote epithelial damage, are governed by the same cohorts of epithelial regulators. Using 479 strains from the recently constructed library of A. fumigatus transcription factor null mutants, two high-throughput screens assessing epithelial cell detachment and epithelial cell lysis were conducted. A total of 17 transcription factor mutants were found to exhibit reproducible deficits in epithelial damage causation. Of these, 10 mutants were defective in causing early phase damage via epithelial detachment and 8 mutants were defective in causing late phase damage via epithelial lysis. Remarkably only one transcription factor, PacC, was required for causation of both phases of epithelial damage. The 17 mutants exhibited varied and often unique phenotypic profiles with respect to fitness, epithelial adhesion, cell wall defects, and rates of spore uptake by epithelial cells. Strikingly, 9 out of 10 mutants deficient in causing early phase damage also exhibited reduced rates of hyphal extension, and culture supernatants of 7 out of 8 mutants deficient in late phase damage were significantly less cytotoxic. Our study delivers the first high-level overview of A. fumigatus regulatory genes governing lung epithelial damage, suggesting highly coordinated genetic orchestration of host-damaging activities that govern epithelial damage in both space and time. Contribution to the Field StatementAspergillus fumigatus is a soil dwelling fungus that can cause lethal lung infections in individuals with a compromised immune system. Disease initiates with inhalation of the fungal spores, followed by growth of the fungus leading to destruction of the lung. Our understanding of the A. fumigatus genes and mechanisms driving lung damage leading to establishment of disease is limited. This study has identified the genes moderating lung damage by assessing 479 regulatory mutants of A. fumigatus for their ability to cause epithelial damage using a lung cell line model. We observed that distinct cohorts of transcriptional regulators are required for driving early, and late phases of epithelial damage and that early- and late-occurring damage are associated respectively with hyphal growth rates and secreted fungal products. This study is the first to reveal that mechanistically distinct programs of host damage elicited during early and late stage of fungal interaction with epithelial cells are genetically regulated via distinct cohorts of A. fumigatus transcription factors.

microbiology↗

bioRxiv2022

Exploring a novel genomic safe-haven site in the human pathogenic mould Aspergillus fumigatus.

Aspergillus fumigatus is the most important airborne fungal pathogen and allergen of humans causing high morbidity and mortality worldwide. The factors that govern pathogenicity of this organism are multi-factorial and are poorly understood. Molecular tools to dissect the mechanisms of pathogenicity in A. fumigatus have improved significantly over the last 20 years however many procedures have not been standardised for A. fumigatus. Here, we present a new genomic safe-haven locus at the site of an inactivated transposon, named SH-aft4, which can be used to insert DNA sequences in the genome of this fungus without impacting its phenotype. We show that we are able to effectively express a transgene construct from the SH-aft4 and that natural regulation of promoter function is conserved at this site. Furthermore, the SH-aft4 locus is highly conserved in the genome of a wide range of clinical and environmental isolates including the isolates commonly used by many laboratories CEA10, Af293 and ATCC46645, allowing a wide range of isolates to be manipulated. Our results show that the aft4 locus can serve as a site for integration of a wide range of genetic constructs to aid functional genomics studies of this important human fungal pathogen.

microbiology↗

bioRxiv2022

Epithelial uptake of Aspergillus fumigatus drives efficient fungal clearance in vivo and is aberrant in Chronic Obstructive Pulmonary Disease (COPD)

Hundreds of spores of the common mould Aspergillus fumigatus (Af) are inhaled daily by human beings, representing a constant, often fatal, threat to our respiratory health. The small size of Af spores suggest that interactions with Airway Epithelial Cells (AECs) are frequent and we and others have previously demonstrated that AECs are able to internalise Af spores. We thus hypothesised that Af spore uptake and killing by AECs is important for driving efficient fungal clearance in vivo and that defective spore uptake and killing would represent major risk factors for Aspergillus-related diseases. In order to test this, we utilised single-cell approaches based on Imaging Flow Cytometry (IFC) and live-cell microfluidic imaging to measure spore uptake and outcomes in vitro, in vivo and using primary human AECs. In vitro, viability of immortalised AECs was largely unaffected by Af uptake and AECs were able to significantly curtail the growth of internalised spores. Applying our approach directly to infected mouse lungs we demonstrated, for the first time, that Af spores are internalised and killed by AECs during whole animal infection, whereby only ~3% of internalised spores remained viable after 8 hours of co-incubation with murine AECs. Finally, in vitro analysis of primary human AECs from healthy and at-risk donors revealed significant alterations in the uptake and consequent outcomes in Chronic Obstructive Pulmonary Disease (COPD), whereby gorging COPD-derived AECs were unable to quell intracellular Af as efficiently as healthy primary AECs. We have thus demonstrated that AECs efficiently kill Af spores upon uptake in vivo and that this process is altered in COPD, a well-known risk factor for debilitating fungal lung disease, thereby suggesting that AECs critically contribute to the efficient clearance of inhaled Af spores and that dysregulation of curative AEC responses represents a potent driver of Aspergillus-related diseases.

microbiology↗

bioRxiv2021

Aspergillus fumigatus drives tissue damage via iterative assaults upon mucosal integrity and immune homeostasis

The human lung is constantly exposed to Aspergillus fumigatus spores, the most prevalent worldwide cause of fungal respiratory disease. Pulmonary tissue damage is a unifying feature of Aspergillus-related diseases; however, the mechanistic basis of damage is not understood. In the lungs of susceptible hosts A. fumigatus undergoes an obligatory morphological switch involving spore germination and hyphal growth. We modelled A. fumigatus infection in cultured A549 human pneumocytes, capturing phosphoactivation status of five host signalling pathways, nuclear translocation & DNA binding of eight host transcription factors, and expression of nine host response proteins over six time points encompassing exposures to live fungus and the secretome thereof. The resulting dataset, comprised of more than 1000 data points, reveals that pneumocytes mount differential responses to A. fumigatus spores, hyphae and soluble secreted products via the NF-kB, JNK, and JNK + p38 pathways respectively. Importantly, via selective degradation of host pro-inflammatory (IL-6 and IL-8) cytokines and growth factors (FGF-2), fungal secreted products reorchestrate the host response to fungal challenge as well as driving multiparametric epithelial damage, culminating in cytolysis. Dysregulation of NF-kB signalling, involving iterative stimulation of canonical and non-canonical signalling, was identified as a significant feature of host damage both in vitro and in a mouse model of invasive aspergillosis. Our data demonstrate that composite tissue damage results from iterative exposures to different fungal morphotypes and secreted products and suggest that modulation of host responses to fungal challenge might represent a unified strategy for therapeutic control of pathologically distinct types of Aspergillus-related disease. IMPORTANCEPulmonary aspergillosis is a spectrum of diseases caused primarily by Aspergillus fumigatus. This fungus is ubiquitous in the environment and grows as a mold, which harbors and disperses spores into the environment. Like other airborne pathogens, the lung mucosa is the first point of contact with the fungus post inhalation. The outcome and severity of disease depends on the host-fungal interaction at the lung interface. We studied how the human lung interacts with spore, germ tube and hyphae growth forms to understand the sequence and dynamics of the early events, which are critical drivers of disease development and progression. Our work is significant in identifying, in response to fungal secreted products, non-canonical NF-kB activation via RelB as being a driving factor in fungus-mediated lung damage. This process could be modulated therapeutically to protect the integrity of infected lung mucosae.

microbiology↗

bioRxiv2020

Targeting methionine synthase in a fungal pathogen causes a metabolic imbalance that impacts cell energetics, growth and virulence

There is an urgent need to develop novel antifungals to tackle the threat fungal pathogens pose to human health. In this work, we have performed a comprehensive characterisation and validation of the promising target methionine synthase (MetH). We uncover that in Aspergillus fumigatus the absence of this enzymatic activity triggers a metabolic imbalance that causes a reduction in intracellular ATP, which prevents fungal growth even in the presence of methionine. Interestingly, growth can be recovered in the presence of certain metabolites, which evidences that metH is a conditionally essential gene. As this implies that for a correct validation MetH should be targeted in established infections, we have validated the use of the tetOFF genetic model for fungal research and optimised its performance to mimic treatment of established infections. We show that repression of metH in growing hyphae halts growth in vitro, which translates into a beneficial effect when targeting established infections using this model in vivo. Finally, a structural-based virtual screening of methionine synthases reveals key differences between the human and fungal structures and unravels features in the fungal enzyme that can guide the design of novel specific inhibitors. Therefore, methionine synthase is a valuable target for the development of new antifungals. IMPORTANCEFungal pathogens are responsible for millions of life-threatening infections on an annual basis worldwide. The current repertoire of antifungal drugs is very limited and, worryingly, resistance has emerged and already become a serious threat to our capacity to treat fungal diseases. The first step to develop new drugs often is to identify molecular targets which inhibition during infection can prevent pathogen growth. However, the current models are not suitable to validate targets in established infections. Here we have characterised the promising antifungal target methionine synthase in great detail, using the prominent fungal pathogen Aspergillus fumigatus as a model. We have uncovered the underlying reason for its essentiality and confirmed its druggability. Furthermore, we have optimised the use of a genetic system to show a beneficial effect of targeting methionine synthase in established infections. Therefore, we believe that antifungal drugs to target methionine synthase should be pursued and additionally, we propose that antifungal targets should be validated in a model of established infection.

microbiology↗