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Aspergillus fumigatus is a saprophytic fungus that can cause a variety of human diseases known as aspergillosis. Mycotoxin gliotoxin (GT) production is important for its virulence and must be tightly regulated to avoid excess production and toxicity to the fungus. GT self-protection by GliT oxidoreductase and GtmA methyltransferase activities is related to the subcellular localization of these enzymes and how GT can be sequestered from the cytoplasm to avoid increased cell damage. Here, we show that GliT:GFP and GtmA:GFP are localized in the cytoplasm and in vacuoles during GT production. Peroxisomes are also required for proper GT production and self-defense. The Mitogen-Activated Protein (MAP) kinase MpkA is essential for GT production and self-protection, interacts physically with GliT and GtmA and it is necessary for their regulation and subsequent presence in the vacuoles. Our work emphasizes the importance of dynamic compartmentalization of cellular events for GT production and self-defense.

More than 10 million people suffer from lung diseases caused by the pathogenic fungus Aspergillus fumigatus. The azole class of antifungals represent first line therapeutics for most of these infections however resistance is rising. Identification of novel antifungal targets that, when inhibited, synergise with the azoles will aid the development of agents that can improve therapeutic outcomes and supress the emergence of resistance. As part of the A. fumigatus genome-wide knockout program (COFUN), we have completed the generation of a library that consists of 120 genetically barcoded null mutants in genes that encode the protein kinase cohort of A. fumigatus. We have employed a competitive fitness profiling approach (Bar-Seq), to identify targets which when deleted result in hypersensitivity to the azoles and fitness defects in a murine host. The most promising candidate from our screen is a previously uncharacterised DYRK kinase orthologous to Yak1 of Candida albicans, a TOR signalling pathway kinase involved in modulation of stress responsive transcriptional regulators. Here we show that the orthologue YakA has been repurposed in A. fumigatus to regulate blocking of the septal pore upon exposure to stress via phosphorylation of the Woronin body tethering protein Lah. Loss of YakA function reduces the ability of A. fumigatus to penetrate solid media and impacts growth in murine lung tissue. We also show that 1-ethoxycarbonyl-beta-carboline (1-ECBC), a compound previously shown to inhibit Yak1 in C. albicans prevents stress mediated septal spore blocking and synergises with the azoles to inhibit A. fumigatus growth.

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 subpopulation of the persister isolates can survive for extended periods and even grow at low rates in the presence of supra-MIC of voriconazole and seemingly other azoles. Persistence cannot be eradicated with adjuvant drugs or antifungal combinations and seemed 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. Importance: The 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 of antifungal tolerance and, in particular, persistence is still very limited. In this study, we have characterized 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 propose 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. J.A. is funded by an Atracción de Talento Modalidad 1 (020-T1/BMD-200) contract of the Madrid Regional Government. J.S. has been funded by a BSAC Scholarship (bsac-2016-0049). C.V. was funded by FAPESP (2108/00715-3 and 2020/01131-5). G.H.G. hasbeen funded by FAPESP (2016/07870-9 and 2021/04977-5), CNPq (301058/2019-9 and404735/2018-5) and by the NIH/NIAID (grant R01AI153356). S.G. was cofunded by the NIHR Manchester Research Centre and the Fungal Infection Trust. Sí

Aspergillus fumigatus is the main etiological agent of aspergillosis. The antifungal drug caspofungin (CSP) can be used against A. fumigatus, and CSP tolerance is observed. We have previously shown that the transcription factor FhdA is important for mitochondrial activity. Here, we show that FhdA regulates genes transcribed by RNA polymerase II and III. FhdA influences the expression of tRNAs that are important for mitochondrial function upon CSP. Our results show a completely novel mechanism that is impacted by CSP.

Aspergillus fumigatusis 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 ofA. fumigatusdisplay 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 aGalleria mellonellamodel 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 pathogenAspergillus fumigatusto 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 inA. 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.

Damage to the lung epithelium is a unifying feature of disease caused by the saprophytic fungus

Aspergillosis, in its various manifestations, is a major cause of morbidity and mortality. Very few classes of antifungal have been approved for clinical use to treat these diseases and resistance to the first line therapeutics is increasing. A new class of antifungals, the orotomides, are currently in development with the first compound in this class olorofim in late-stage clinical trials. In this study, we characterise a network of genes that govern olorofim response in A. fumigatus. We reveal that the number of transcription factors that regulate olorofim susceptibility are far fewer than we have previously observed for the azoles and the change in sensitivity observed in these isolates is less extreme. Intriguingly, loss of function in two higher order transcriptional regulators, 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. However, a clinical azole resistant isolate with a point mutation in HapE (hapEP88L) retains sensitivity to olorofim. Our transcriptomic analysis suggests that altered sensitivity to olorofim may emerge via modification of genes involved in the production of pyrimidine biosynthetic precursors. Finally, we also show that the action of the azoles are antagonistic to olorofim in vitro.

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Infections caused by the fungal pathogen Aspergillus fumigatus are increasingly resistant to first-line azole antifungal drugs. However, despite its clinical importance, little is known about how susceptible patients acquire infection from drug-resistant genotypes in the environment. Here, we present a population genomic analysis of 218 A. fumigatus isolates from across the UK and Ireland (comprising 153 clinical isolates from 143 patients and 65 environmental isolates). First, phylogenomic analysis shows strong genetic structuring into two clades (A and B) with little interclade recombination and the majority of environmental azole resistance found within clade A. Second, 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. Third, genome-wide scans identified selective sweeps across multiple regions indicating a polygenic basis to the trait in some genetic backgrounds. These signatures of positive selection are seen for loci containing the canonical genes encoding fungicide resistance in the ergosterol biosynthetic pathway, while other regions under selection have no defined function. Lastly, pan-genome analysis identified genes linked to azole resistance and previously unknown resistance mechanisms. 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. Published version This study was partially supported by an unrestricted education grant from Gilead Sciences through their investigator sponsored research programme. J. Rhodes, T.S., A.P.B., P.S.D., D.A.J. and M.C.F. were supported by grants from the NERC (nos. NE/P001165/1 and NE/P000916/1), the UK Medical Research Council (MRC) (no. MR/R015600/1) and Wellcome Trust (no. 219551/Z/19/Z). D.A.J. is also funded by the Medical Research Council (grant no. MR/V037315/1) and Cystic Fibrosis Trust (grant no. SRC015). D.A.J. is funded by the Department of Health and Social Care (DHSC) Centre for Antimicrobial Optimisation (CAMO), Imperial College London. The views expressed in this publication are those of the authors and not necessarily those of the DHSC, National Health Service or National Institute for Health Research (NIHR). M.C.F. is a CIFAR Fellow in the Fungal Kingdom programme. K.D. was supported by a PhD studentship awarded by the School of Medicine, Trinity College Dublin. P.G.M. and J. Renwick (Dublin) received a project grant from the National Children’s Hospital, Tallaght University Hospital, which in part supported this work. A.W. and E.B. are supported by the Wellcome Trust Strategic Award (grant no. 097377), MRC Centre for Medical Mycology (grant no. MR/N006364/2) at the University of Exeter and a Biotechnology and Biological Sciences Research Council EASTBIO grant (no. BB/M010996/1). The authors also acknowledge the Imperial College London Cystic Fibrosis Strategic Research Centre and NIHR CAMO.

The origin of isolates routinely used by the community of Aspergillus fumigatus researchers is periodically a matter of intense discussion at our centre, as the construction of recombinant isolates have sometimes followed convoluted routes, the documentation describing their lineages is fragmented, and the nomenclature is confusing. As an aide memoir, not least for our own benefit, we submit the following account and tabulated list of strains (Table 1) in an effort to collate all of the relevant information in a single, easily accessible document. To maximise the accuracy of this record we have consulted widely amongst the community of Medical Mycologists using these strains. All the strains described are currently available from one of these organisations, namely the Fungal Genetics Stock Centre (FGSC), FungiDB, Ensembl Fungi and The National Collection of Pathogenic Fungi (NCPF) at Public Health England. Display items from this manuscript are also featured on FungiDB. Lay abstract We present a concise overview on the definition, origin and unique genetic makeup of the Aspergillus fumigatus isolates routinely in use by the fungal research community, to aid researchers to describe past and new strains and the experimental differences observed more accurately.

Highlights • In vitro assembled CRISPR/Cas9-gRNA complexes can mediate selection free transformation. • Single gRNAs can be used to target mutagenesis effectively. • Targeted point mutations can be achieved utilising single strand DNA oligonucleotides as repair template., Aspergillus fumigatus is a saprophytic fungal pathogen that is the cause of more than 300,000 life-threatening infections annually. Our understanding of pathogenesis and factors contributing to disease progression are limited. Development of rapid and versatile gene editing methodologies for A. fumigatus is essential. CRISPR-Cas9 mediated transformation has been widely used as a novel genome editing tool and has been used for a variety of editing techniques, such as protein tagging, gene deletions and site-directed mutagenesis in A. fumigatus. However, successful genome editing relies on time consuming, multi-step cloning procedures paired with the use of selection markers, which can result in a metabolic burden for the host and/or unintended transcriptional modifications at the site of integration. We have used an in vitro CRISPR-Cas9 assembly methodology to perform selection-free genome editing, including epitope tagging of proteins and site-directed mutagenesis. The repair template used during this transformation use 50 bp micro-homology arms and can be generated with a single PCR reaction or by purchasing synthesised single stranded oligonucleotides, decreasing the time required for complex construct synthesis.

Generally, growth assays for filamentous fungi have been performed on solid media, either as dilution series or spot tests. However, the solid media environment does not accurately mimic the environment encountered during infection (ie the mammalian lung). Previously, we have developed a methodology to perform liquid growth assays in time for A. fumigatus and other filamentous fungi including analysis via mathematical modelling. This protocol is designed for Aspergillus species (and other filamentous fungi) to generate growth curves in liquid media in a 96-well plate. This can be done in high-throughput to generate 96 growth curves per run.

Generally, growth assays for filamentous fungi have been performed on solid media, either as dilution series or spot tests. However, the solid media environment does not accurately mimic the environment encountered during infection (ie the mammalian lung). Previously, we have developed a methodology to perform liquid growth assays in time for A. fumigatus and other filamentous fungi including analysis via mathematical modelling. This protocol is designed for Aspergillus species (and other filamentous fungi) to generate growth curves in liquid media in a 96-well plate. This can be done in high-throughput to generate 96 growth curves per run.

Generally, growth assays for filamentous fungi have been performed on solid media, either as dilution series or spot tests. However, the solid media environment does not accurately mimic the environment encountered during infection (ie the mammalian lung). Previously, we have developed a methodology to perform liquid growth assays in time for A. fumigatus and other filamentous fungi including analysis via mathematical modelling. This protocol is designed for Aspergillus species (and other filamentous fungi) to generate growth curves in liquid media in a 96-well plate. This can be done in high-throughput to generate 96 growth curves per run.

We consider the situation where a temporal process is composed of contiguous segments with differing slopes and replicated noise-corrupted time series measurements are observed. The unknown mean of the data generating process is modelled as a piecewise linear function of time with an unknown number of change-points. We develop a Bayesian approach to infer the joint posterior distribution of the number and position of change-points as well as the unknown mean parameters. A-priori, the proposed model uses an overfitting number of mean parameters but, conditionally on a set of change-points, only a subset of them influences the likelihood. An exponentially decreasing prior distribution on the number of change-points gives rise to a posterior distribution concentrating on sparse representations of the underlying sequence. A Metropolis-Hastings Markov chain Monte Carlo (MCMC) sampler is constructed for approximating the posterior distribution. Our method is benchmarked using simulated data and is applied to uncover differences in the dynamics of fungal growth from imaging time course data collected from different strains. The source code is available on CRAN., Comment: Accepted to International Journal of Biostatistics