Biology subjects

Fortune-Grant, R.

Publications and source records attributed to Fortune-Grant, R..

9 recordsLinked to original sources

bioRxiv2025

Host microbiome determines host specificity of the human whipworm, Trichuris trichiura

Long-term whipworm-host co-evolution has resulted in tropism for the caecum of specific hosts, an organ with the densest microbial population in the body. Here, we demonstrate that the host specificity of human whipworms (Trichuris trichiura) is host microbiome-driven. We successfully establish a T. trichiura infection in a non-primate host using a humanised-microbiota mouse model. We further show, in vitro, that hatching of T. trichiura was triggered by mucosal scrapings of the caecum of human microbiota-associated mice, but not from wild-type mice, which only induced T. muris hatching. Comparative metagenomic analysis of the murine versus humanised microbiomes directly implicated specific bacterial species in T. trichiura egg hatching. Additionally, we demonstrate that host tissue does not directly determine host specificity, as T. trichiura readily infected mouse caecaloids. Our findings indicate that host-microbiome-whipworm co-evolution has resulted in exquisite bacterial-whipworm egg interactions critical for hatching and development of these parasites in their definitive hosts.

microbiology↗

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↗

bioRxiv2023

Airway epithelial cells as a novel intracellular host reservoir for Cryptococcus spores

Human fungal pathogens, including Cryptococcus neoformans, cause 1.5 million annual deaths. Cryptococcus causes disease when it disseminates out of the lung and into the brain which can occur years after initial exposure (latency) via mechanisms that remain unknown. Spores of Cryptococcus display distinct surface epitopes, host-interactions, and disease kinetics to the vegetatively growing yeast morphotype, yet they remain understudied likely contributing to our lack of understanding of pathogenesis. One of the first barriers spores encounter are non-professional phagocytic Airway Epithelial Cells (AECs). Here, we demonstrated that Cryptococcus spores preferentially invade AECs both in vitro and in vivo. Once inside spores can germinate, subsequently replicate, persist and/or escape. This ability to enter AECs correlates with a preferential ability of spore to cross AEC barriers. Together our work indicates that AECs can be invaded by Cryptococcus spores and may serve as a previously ignored intracellular host niche, providing alternative hypothesis for both Cryptococcus dissemination and latency.

microbiology↗

bioRxiv2022

Mutual inhibition of airway epithelial responses supports viral and fungal co-pathogenesis during coinfection

Awareness that fungal coinfection complicates viral respiratory infections causing worse disease outcome has recently emerged. The environmental fungus Aspergillus fumigatus (Af) has been reported as the main driver of fungal coinfection in patients suffering from viral infections caused by Cytomegalovirus, Influenza or more recently SARS-CoV2. The airway epithelium is the first common point of contact between inhaled pathogens and the host. Aberrant airway epithelial cell (AEC) responses against fungal challenge have been described in patients susceptible to aspergillosis. Therefore, it is likely that a dysregulation of AEC responses during fungal-viral coinfection represents a potent driver for the development of fungal disease. Here we used an in vitro model of Af-viral infection of AECs to determine outcomes of spore internalisation, killing and viral replication during coinfection. Our data indicate that viral stimulation, while boosting Af uptake by AECs, limits Af spore killing by those cells, favouring fungal persistence and growth. Type I viral-induced interferon release was significantly decreased in the presence of Af hyphal forms suggesting a possible role of Af secreted factors in modulating viral pathogenicity. We next explored the impact of Af challenge in SARS-CoV2 replication within airway epithelial cells using nano-luciferase as a measure of viral replication. We found that Af increased SARS-CoV2 pathogenicity in a strain-dependent manner. Collectively, our findings demonstrate a mutual inhibition of antifungal and antiviral AEC responses during Af-viral coinfection and also suggest that some fungal factors might be key regulators of co-pathogenicity during in lung infection.

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↗