Biology subjects

Bromley, M. J.

Publications and source records attributed to Bromley, M. J..

8 recordsLinked to original sources

bioRxiv2022

COVID-19 Associated Pulmonary Aspergillosis isolates are genomically diverse but similar to each other in their responses to infection-relevant stresses

Secondary infections caused by the pulmonary fungal pathogen Aspergillus fumigatus are a significant cause of mortality in patients with severe Coronavirus Disease 19 (COVID-19). Even though epithelial cell damage and aberrant cytokine responses have been linked with susceptibility to COVID-19 associated pulmonary aspergillosis (CAPA), little is known about the mechanisms underpinning co-pathogenicity. Here, we analysed the genomes of 11 A. fumigatus isolates from patients with CAPA in three centres from different European countries. CAPA isolates did not cluster based on geographic origin in a genome-scale phylogeny of representative A. fumigatus isolates. Phenotypically, CAPA isolates were more similar to the A. fumigatus A1160 reference strain than to the Af293 strain when grown in infection-relevant stresses; except for interactions with human immune cells wherein macrophage responses were similar to those induced by the Af293 reference strain. Collectively, our data indicates that CAPA isolates are genomically diverse but are more similar to each other in their responses to infection-relevant stresses. A larger number of isolates from CAPA patients should be studied to identify genetic drivers of co-pathogenicity in patients with COVID-19. ImportanceCoronavirus disease 2019 (COVID-19)-associated pulmonary aspergillosis (CAPA) has been globally reported as a life-threatening complication in some patients with severe COVID-19. Most of these infections are caused by the environmental mould Aspergillus fumigatus which ranks third in the fungal pathogen priority list of the WHO. However, little is known about the molecular epidemiology of Aspergillus fumigatus CAPA strains. Here, we analysed the genomes of 11 A. fumigatus isolates from patients with CAPA in three centres from different European countries and, carried out phenotypic analyses with a view to understand the pathophysiology of the disease. Our data indicates that A. fumigatus CAPA isolates are genomically diverse but are more similar to each other in their responses to infection-relevant stresses.

cell biology↗

bioRxiv2022

Functional characterization of the RNA interference pathway in A. fumigatus reveals its potential for antifungal therapy

The RNA interference (RNAi) pathway has evolved numerous functionalities in eukaryotes, with many on display in Kingdom Fungi. RNAi can regulate gene expression, facilitate drug resistance, or even be altogether lost to improve growth potential in some fungal pathogens. In the WHO fungal priority pathogen, Aspergillus fumigatus, the RNAi system is known to be intact and functional. To extend our limited understanding of A. fumigatus RNAi, we first investigated the genetic variation in RNAi-associated genes in a collection of 217 environmental and 83 clinical genomes, where we found that RNAi components are conserved even in clinical strains. Using endogenously expressed inverted-repeat transgenes complementary to a conditionally essential gene (pabA) or a nonessential gene (pksP), we determined that a subset of the RNAi componentry is active in inverted-repeat transgene silencing in conidia and mycelium. Analysis of mRNA-seq data from RNAi double-knockout strains linked the A. fumigatus dicer-like enzymes (DclA/B) and RNA-dependent RNA polymerases (RrpA/B) to regulation of conidial ribosome biogenesis genes; however, surprisingly few endogenous small RNAs were identified in conidia that could explain this broad change. Although RNAi was not clearly linked to growth or stress response defects in the RNAi knockouts, serial passaging of RNAi knockout strains for six generations resulted in lineages with diminished spore production over time, indicating that loss of RNAi can exert a fitness cost on the fungus. Cumulatively, A. fumigatus RNAi appears to play an active role in defense against double-stranded RNA species alongside a previously unappreciated housekeeping function in regulation of conidial ribosomal biogenesis genes.

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

Every single conidium in Aspergillus fumigatus caspofungin tolerant strains are intrinsically caspofungin tolerant

Aspergillus fumigatus is a human fungal pathogen that causes a disease named aspergillosis. Echinocandins, such as the fungistatic drug caspofungin (CAS) are used as second-line therapy. Some A. fumigatus clinical isolates can survive and grow in higher CAS concentrations, a phenomenon known as "caspofungin paradoxical effect" (CPE). Here we investigate if CPE is due to a subpopulation of conidia produced by a CAS tolerant strain, indicative of a persistence phenotype or is caused by all the conidia which would be consistent with a tolerance phenotype. We evaluated 67 A. fumigatus clinical isolates for CPE growth and used a novel CPE Index (CPEI) classified them as CPE+ (CPEI [≥] 0.40) or CPE- (CPEI [≤] 0.20). Conidia produced by three CPE+ clinical isolates, CEA17 (CPEI=0.52), Af293 (CPEI=0.64), CM7555 (CPEI=0.58) all showed the ability to grow in high levels of CAS while all conidia produced by the CPE- isolate IFM61407 (CPEI=0.12) strain showed no evidence of tolerance. Given the importance of calcium/calcineurin/transcription factor CrzA pathway in CPE regulation, we also evaluated {Delta}crzAAf293 (CPE-) and {Delta}crzACEA17 (CPE+) conidia tolerance to CAS. All {Delta}crzACEA17 conidia showed CPE+ while 100 % of {Delta}crzAAf293 spores are CPE-. As all spores derived from an individual strain are phenotypically indistinct with respect to CPE it is likely that CPE is a genetically encoded adaptive trait that should be considered an antifungal tolerant phenotype. As the CPEI shows that the strength of the CPE is not uniform between strains we propose that the mechanisms that govern this phenomenon are multi-factorial.

microbiology↗

bioRxiv2022

Azole resistance associated regulatory motifs within the promoter of cyp51A in Aspergillus fumigatus

Aspergillus fumigatus is one of the deadliest fungal species causing hundreds of thousands of deaths each year. As azoles provide the preferred first-line option for treatment of Aspergillosis, the increase in rates of resistance and the poor therapeutic outcomes for those infected with a resistant isolate constitutes a serious global health threat. Azole resistance is frequently associated with specific tandem repeat duplications of a promoter element upstream of cyp51A, the gene which encodes the target for this drug class in A. fumigatus. This promoter element is recognized by the activating transcription factors SrbA and AtrR. This region also provides a docking platform for the CCAAT-binding-complex (CBC) and HapX that cooperate in the regulation of genes involved in iron-consuming pathways including cyp51A. Here, we studied the regulatory contribution of SrbA, AtrR, CBC and HapX binding sites on cyp51A expression and azole resistance during different iron availability employing promoter mutational analysis and protein/DNA interaction analysis. This strategy revealed iron status-dependent and -independent roles of these regulatory elements. We show that promoter occupation by both AtrR and SrbA is required for iron-independent steady-state transcriptional activation of cyp51A and its induction during short-term iron exposure relies on HapX binding. We further uncover the HapX binding site as repressor element the disruption of which elevates cyp51A expression and azole resistance regardless of iron availability.

molecular biology↗

bioRxiv2021

Aspergillus fumigatus ffmA encodes a C2H2-containing transcriptional regulator that modulates azole resistance and is required for normal growth

The production of a collection of disruption mutant strains corresponding to a large number of transcription factors from the filamentous fungal pathogen Aspergillus fumigatus has permitted rapid identification of transcriptional regulators involved in a range of different processes. Here we characterize a gene designated ffmA (favors fermentative metabolism) as an C2H2-containing transcription factor that is required for azole drug resistance and normal growth. Loss of ffmA caused cells to exhibit significant defects in growth, either under untreated or azole-challenged conditions. Loss of FfmA caused a reduction in expression of the AbcG1 ATP-binding cassette transporter, previousy shown to contribute to azole resistance. Strikingly, overproduction of the AtrR transcription factor gene restored a wild-type growth phenotype to a ffmA{Delta} strain. Overexpression of AtrR also suppressed the defect in AbcG1 expression caused by loss of FfmA. Replacement of the ffmA promoter with a doxycycline-repressible promoter restored near normal growth in the absence of doxycycline. Finally, chromatin immunoprecipitation experiments indicated that FfmA bound to its own promoter as well as to the abcG1 promoter. These data imply that FfmA and AtrR interact both with respect to abcG1 expression and also more broadly to regulate hyphal growth. ImportanceInfections associated with azole-resistant forms of the primary human pathogen, Aspergillus fumigatus, are associated with poor outcomes in patient populations. This makes analysis of the mechanisms underlying azole resistance of A. fumigatus a high priority. In this work, we describe characterization of a gene designated ffmA that encodes a sequence-specific transcriptional regulator. We identified ffmA in a screen of a collection of gene disruption mutant strains made in A. fumigatus. Loss of ffmA caused sensitivity to azole drugs and also a large reduction in normal growth. We found that overproduction of the AtrR transcription factor was able to restore growth to ffmA null cells. We provide evidence that FfmA can recognize promoters of genes involved in azole resistance as well as the ffmA promoter itself. Our data indicate that FfmA and AtrR interact to support azole resistance and normal growth.

microbiology↗

bioRxiv2021

Olorofim and the azoles are antagonistic in Aspergillus fumigatus and functional genomic screens reveal mechanisms of cross resistance.

Aspergillosis, in its various manifestations, is a major cause of morbidity and mortality. Very few classes of antifungal drugs have been approved for clinical use to treat these diseases and resistance to the first line therapeutic class, the triazoles, is increasing. A new class of antifungals that target pyrimidine biosynthesis, the orotomides, are currently in development with the first compound in this class, olorofim in late-stage clinical trials. In this study, we identify an antagonistic action of the triazoles on the action of olorofim. We show that this antagonism is the result of an azole induced upregulation of the pyrimidine biosynthesis pathway and regulation. Intriguingly, we show that loss of function in the higher order transcription factor, HapB a member of the heterotrimeric HapB/C/E (CBC) complex or the regulator of nitrogen metabolic genes AreA, leads to cross resistance to both the azoles and olorofim indicating that factors that govern resistance are under common regulatory control. However loss of azole induced antagonism requires decoupling of the pyrimidine biosynthetic pathway in a manner independent of the action of a single transcription factor. Our study provides a first insight into antagonism between the azoles and olorofim through dysregulation of the pyrimidine and ergosterol pathway, showing complex crosstalk between these two pathways.

microbiology↗

bioRxiv2021

Tracing patterns of evolution and acquisition of drug resistant Aspergillus fumigatus infection from the environment using population genomics

Infections caused by opportunistic fungal pathogens are increasingly resistant to first-line azole antifungal drugs. However, despite its clinical importance, little is known about the extent to which susceptible patients acquire infection from drug resistant genotypes in the environment. Here, we present a population genomic analysis of the mould Aspergillus fumigatus from across the United Kingdom and Republic of Ireland. First, we show occurrences where azole resistant isolates of near identical genotypes were obtained from both environmental and clinical sources, indicating with high confidence the infection of patients with resistant isolates transmitted from the environment. Second, we find that the fungus is structured into two clades ( A and B) with little interclade recombination and the majority of environmental azole resistance genetically clustered inside Clade A. Genome-scans show the impact of selective sweeps across multiple regions of the genome. These signatures of positive selection are seen in regions containing canonical genes encoding fungicide resistance in the ergosterol biosynthetic pathway, whilst other regions under selection have no defined function. Phenotyping identified genes in these regions that could act as modifiers of resistance showing the utility of reverse genetic approaches to dissect the complex genomic architecture of fungal drug resistance. Understanding the environmental drivers and genetic basis of evolving fungal drug resistance needs urgent attention, especially in light of increasing numbers of patients with severe viral respiratory tract infections who are susceptible to opportunistic fungal superinfections.

genomics↗