Biology subjects

Bromley, M.

Publications and source records attributed to Bromley, M..

5 recordsLinked to original sources

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

Aspergillus-mediated allergic airway inflammation is triggered by dendritic cell recognition of a defined spore morphotype, a process that can be targeted via antifungal therapeutics.

BackgroundExposure to fungi, especially Aspergillus fumigatus (A.f.), can elicit potent allergic inflammation that triggers and worsens asthmatic disease. Dendritic cells (DCs), initiate allergic inflammatory responses to allergic stimuli. However, it is unclear if A.f. spores during isotropic growth (early spore swelling) can activate DCs to initiate allergic responses or if germination is required. This lack of basic understanding of how A.f. causes disease is a barrier to the development of new treatments. ObjectiveTo show that a precise A.f. morphotype stage during spore swelling can trigger DCs to mediate allergic inflammatory responses and ascertain if antifungal therapeutics can be effective at suppressing this process. MethodsWe employed an A.f. strain deficient in pyrimidine biosynthesis ({Delta}pyrG) to generate populations of A.f. spores arrested at different stages of isotropic growth (swelling) via temporal removal of uracil and uridine from growth media. These arrested spore stages were cultured with bone marrow derived DCs (BMDCs), and their activation measured via flow cytometry and ELISA to interrogate which growth stage was able to activate BMDCs. These BMDCs were then adoptively transferred into the airways, to assess if they were able to mediate allergic inflammation in naive recipient mice. Allergic airway inflammation in vivo was determined via flow cytometry, ELISA and qPCR. This system was also used to determine if antifungal drug (itraconazole) treatment could alter early stages of spore swelling and therefore BMDC activation and in vivo allergic inflammation upon adoptive transfer. ResultsWe found that A.f. isotropic growth is essential to trigger BMDC activation and mediate allergic airway inflammation. Furthermore, using time arrested A.f. stages, we found that least 3h in growth media enabled spores to swell sufficiently to activate BMDCs to elicit allergic airway inflammation in vivo. Incubation of germinating A.f. with itraconazole reduced spore swelling and partially reduced their ability to activate BMDCs to elicit in vivo allergic airway inflammation. ConclusionIn summary, our results have pinpointed the precise stage of A.f. development when germinating spores are able to activate DCs to mediate downstream allergic airway inflammation. Furthermore, we have identified that antifungal therapeutics can be effective in reducing the potential of A.f. spores to stimulate allergic responses, highlighting a potential mechanism by which antifungal treatment might help to prevent the development of fungal allergy.

immunology↗

bioRxiv2022

Aspergillus fumigatus can exhibit persistence to the fungicidal drug voriconazole

Aspergillus fumigatus is a filamentous fungus that can infect the lungs of patients with immunosuppression and/or underlying lung-diseases. The mortality associated with chronic and invasive aspergillosis infections remain very high, despite availability of antifungal treatments. In the last decade, there has been a worrisome emergence and spread of resistance to the first line antifungals, the azoles. The mortality caused by resistant isolates is even higher, and patient management is complicated as the therapeutic options are reduced. Nevertheless, treatment failure is also common in patients infected with azole-susceptible isolates, which can be due to several non- mutually exclusive reasons, such as poor drug absorption. In addition, the phenomena of tolerance or persistence, where susceptible pathogens can survive the action of an antimicrobial for extended periods, have been associated with treatment failure in bacterial infections, and their occurrence in fungal infections already proposed. Here, we demonstrate that some isolates of A. fumigatus display persistence to voriconazole. A sub-population of the persister isolates can survive for extended periods and even grow at slow rates in the presence of supra-MIC (Minimum Inhibitory Concentration) of voriconazole and seemingly other azoles. Persistence cannot be eradicated with adjuvant drugs or antifungal combinations and seems to reduce the efficacy of treatment for certain individuals in a Galleria mellonella model of infection. Furthermore, persistence implies a distinct transcriptional profile, demonstrating that it is an active response. We propose that azole persistence might be a relevant and underestimated factor that could influence the outcome of infection in human aspergillosis. IMPORTANCEThe phenomena of antibacterial tolerance and persistence, where pathogenic microbes can survive for extended periods in the presence of cidal drug concentrations, have received significant attention in the last decade. Several mechanisms of action have been elucidated, and their relevance for treatment failure in bacterial infections demonstrated. In contrast, our knowledge about antifungal tolerance and, particularly, persistence are still very scarce. In this study, we have characterised the response of the prominent fungal pathogen Aspergillus fumigatus to the first line therapy antifungal voriconazole. We comprehensively show that some isolates display persistence to this fungicidal antifungal and identify various potential mechanisms of action. In addition, using an alternative model of infection, we provide initial evidence to suggest that persistence may cause treatment failure in some individuals. Therefore, we propose that azole persistence is an important factor to consider and further investigate in A. fumigatus.

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↗

bioRxiv2019

Weak Acid Sensitive A, a novel transcription factor required for regulation of weak-acid resistance and spore-spore heterogeneity in Aspergillus niger

Propionic, sorbic and benzoic acids are organic weak acids that are widely used as food preservatives, where they play a critical role in preventing microbial growth. In this study, we uncovered new mechanisms of weak acid resistance in moulds. By screening a library of 401 transcription-factor deletion strains in Aspergillus fumigatus for sorbic acid hypersensitivity, a previously uncharacterised transcription factor was identified, and named as WarA (Weak Acid Resistance A). The orthologous gene in the spoilage mould Aspergillus niger was identified and deleted. WarA was required for resistance to a range of weak acids, including sorbic, propionic and benzoic acids. A transcriptomic analysis was performed to characterise genes regulated by WarA during sorbic acid treatment in A. niger. Several genes were significantly upregulated in the wild type compared with a {Delta}warA mutant, including genes encoding putative weak acid detoxification enzymes and transporter proteins. Among these was An14g03570, a putative ABC-type transporter which we found to be required for weak acid resistance in A. niger. We also show that An14g03570 is a functional homologue of the Saccharomyces cerevisiae protein Pdr12p, and therefore named as PdrA. Lastly, resistance to sorbic acid was found to be highly heterogeneous within genetically-uniform populations of ungerminated A. niger conidia, and we demonstrate that pdrA is a determinant of this heteroresistance. This study has identified novel mechanisms of weak acid resistance in A. niger which could help to inform and improve future food spoilage prevention strategies.\n\nIMPORTANCEWeak acids are widely used as food preservatives, as they are very effective at preventing growth of most species of bacteria and fungi. However, some species of moulds can survive and grow in the concentrations of weak acid employed in food and drink products, thereby causing spoilage with resultant risks for food security and health. Current knowledge of weak acid resistance mechanisms in these fungi is limited, especially in comparison to that in yeasts. We characterised gene functions in the spoilage mould species Aspergillus niger which are important for survival and growth in the presence of weak acid preservatives. Such identification of weak acid resistance mechanisms in spoilage moulds will help to design new strategies to reduce food spoilage in the future.

microbiology↗