Aspergillus fumigatus FhdA Transcription Factor Is Important for Mitochondrial Activity and Codon Usage Regulation during the Caspofungin Paradoxical Effect.

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.

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.

On the lineage of Aspergillus fumigatus isolates in common laboratory use.

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.

Development of a marker-free mutagenesis system using CRISPR-Cas9 in the pathogenic mould Aspergillus fumigatus.

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.

Aspergillus fumigatus strains that evolve resistance to the agrochemical fungicide ipflufenoquin in vitro are also resistant to olorofim.

Widespread use of azole antifungals in agriculture has been linked to resistance in the pathogenic fungus Aspergillus fumigatus. We show that exposure of A. fumigatus to the agrochemical fungicide, ipflufenoquin, in vitro can select for strains that are resistant to olorofim, a first-in-class clinical antifungal with the same mechanism of action. Resistance is caused by non-synonymous mutations within the target of ipflufenoquin/olorofim activity, dihydroorotate dehydrogenase (DHODH), and these variants have no overt growth defects.

Frequency of fungal pathogens in autopsy studies of people who died with HIV in Africa: a scoping review.

BACKGROUND: Fungal infections are common in HIV-infected individuals and significantly contribute to mortality. However, a substantial number of cases are undiagnosed before death. OBJECTIVE: To determine the frequency of fungal pathogens in autopsy studies of people who died with HIV in Africa. METHODS: We conducted a scoping review of autopsy studies conducted in Africa. DATA SOURCES: PubMed, Scopus, Web of Science, Embase, Google Scholar, and African Journal Online. STUDY ELIGIBILITY CRITERIA: The review encompasses studies published from inception to September 2023, and no language restrictions were imposed during the search process. We included studies that reported histopathological or microbiological evidence for the diagnosis of fungal infections and other pathogens. DATA SYNTHESIS: Data were summarized using descriptive statistics and no meta-analysis was performed. RESULTS: We examined 30 articles reporting studies conducted between 1991 and 2019, encompassing a total of 13 066 HIV-infected decedents across ten African countries. In five studies, the autopsy type was not specified. Among those studies with specified autopsy types, 20 involved complete diagnostic autopsies, whereas 5 were categorized as partial or minimally invasive autopsies. There were 2333 pathogens identified, with 946 (40.5%) being mycobacteria, 856 (36.7%) fungal, 231 (3.8%) viral, 208 (8.9%) parasitic, and 92 (3.9%) bacterial. Of the 856 fungal pathogens identified, 654 (28.0%) were Cryptococcus species, 167 (7.2%) Pneumocystis jirovecii, 16 (0.69%) Histoplasma species, 15 (0.64%) Aspergillus species, and 4 (0.17%) Candida species. Other major non-fungal pathogens identified were cytomegalovirus 172 (7.37%) and Toxoplasma gondii 173 (7.42%). CONCLUSIONS: Invasive fungal infections occur in over one-third of people who succumb to HIV in Africa. In addition to cryptococcosis and Pneumocystis jirovecii pneumonia, integrating other priority fungal pathogen detection and management strategies into the broader framework of HIV care in Africa is recommended. This involves increasing awareness regarding the impact of fungal infections in advanced HIV disease and strengthening diagnostic and treatment capacity.

Epithelial uptake leads to fungal killing in vivo and is aberrant in COPD-derived epithelial cells.

Hundreds of spores of Aspergillus fumigatus (Af) are inhaled daily by human beings, representing a constant, possibly fatal, threat to respiratory health. The small size of Af spores suggests that interactions with alveolar epithelial cells (AECs) are frequent; thus, we hypothesized that spore uptake by AECs is important for driving fungal killing and susceptibility to Aspergillus-related disease. Using single-cell approaches to measure spore uptake and its outcomes in vivo, we demonstrate that Af spores are internalized and killed by AECs during whole-animal infection. Moreover, comparative analysis of primary human AECs from healthy and chronic obstructive pulmonary disease (COPD) donors revealed significant alterations in the uptake and killing of spores in COPD-derived AECs. We conclude that AECs contribute to the killing of Af spores and that dysregulation of curative AEC responses in COPD may represent a driver of Aspergillus-related diseases.

Re-estimation of the burden of serious fungal diseases in Uganda.

Background: It is of utmost importance to monitor any change in the epidemiology of fungal diseases that may arise from a change in the number of the at-risk population or the availability of local data. Objective: We sought to update the 2015 publication on the incidence and prevalence of serious fungal diseases in Uganda. Methods: Using the Leading International Fungal Education methodology, we reviewed published data on fungal diseases and drivers of fungal diseases in Uganda. Regional or global data were used where there were no Ugandan data. Results: With a population of ~45 million, we estimate the annual burden of serious fungal diseases at 4,099,357 cases (about 9%). We estimated the burden of candidiasis as follows: recurrent Candida vaginitis (656,340 cases), oral candidiasis (29,057 cases), and esophageal candidiasis (74,686 cases) in HIV-infected people. Cryptococcal meningitis annual incidence is estimated at 5553 cases, Pneumocystis pneumonia at 4604 cases in adults and 2100 cases in children. For aspergillosis syndromes, invasive aspergillosis annual incidence (3607 cases), chronic pulmonary aspergillosis (26,765 annual cases and 63,574 5-year-period prevalent cases), and prevalence of allergic bronchopulmonary aspergillosis at 75,931 cases, and severe asthma with fungal sensitization at 100,228 cases. Tinea capitis is common with 3,047,989 prevalent cases. For other mycoses, we estimate the annual incidence of histoplasmosis to be 646 cases and mucormycosis at 9 cases. Conclusion: Serious fungal diseases affect nearly 9% of Ugandans every year. Tuberculosis and HIV remain the most important predisposition to acute fungal infection necessitating accelerated preventive, diagnostic, and therapeutic interventions for the management of these diseases.

How common are serious fungal infections in Uganda? Why was the study done? This study was conducted to provide an updated understanding of the occurrence and impact of serious fungal diseases in Uganda. The aim was to monitor changes in the epidemiology of fungal diseases related to shifts in the at-risk population or the availability of local data. What did the researchers do? Utilizing the Leading International Fungal Education methodology, the research team systematically reviewed published data on fungal diseases in Uganda. In instances where Ugandan data was unavailable, regional, or global data were incorporated. This method allowed for a thorough examination of the incidence and prevalence of various serious fungal diseases, considering the local context. What did the researchers find? With a population of approximately 45 million, the study estimated that nearly 9% of Ugandans, totalling around 4,099,357 individuals, are affected by serious fungal diseases annually. Notable findings include the prevalence of recurrent Candida vaginitis, oral candidiasis, and oesophageal candidiasis in HIV-infected individuals. Cryptococcal meningitis and Pneumocystis pneumonia were identified as significant contributors, along with various aspergillosis syndromes and widespread cases of tinea capitis. What do the findings mean? These findings underscore the substantial impact of serious fungal diseases on the health of almost 9% of the Ugandan population each year. Recognizing tuberculosis and HIV as major predisposing factors, the study calls for urgent interventions to prevent, diagnose, and treat these diseases effectively. The identified targets, including improved access to essential antifungal medications, training of health care workers on fungal diseases, and increasing access to essential diagnostics. These interventions can significantly contribute to improving public health outcomes in Uganda.

Functional analysis of the Aspergillus fumigatus kinome identifies a druggable DYRK kinase that regulates septal plugging.

More than 10 million people suffer from lung diseases caused by the pathogenic fungus Aspergillus fumigatus. Azole antifungals represent first-line therapeutics for most of these infections but resistance is rising, therefore the identification of antifungal targets whose inhibition synergises with the azoles could improve therapeutic outcomes. Here, we generate a library of 111 genetically barcoded null mutants of Aspergillus fumigatus in genes encoding protein kinases, and show that loss of function of kinase YakA results in hypersensitivity to the azoles and reduced pathogenicity. YakA is an orthologue of Candida albicans Yak1, a TOR signalling pathway kinase involved in modulation of stress responsive transcriptional regulators. We show that YakA has been repurposed in A. fumigatus to regulate blocking of the septal pore upon exposure to stress. Loss of YakA function reduces the ability of A. fumigatus to penetrate solid media and to grow in mouse lung tissue. We also show that 1-ethoxycarbonyl-beta-carboline (1-ECBC), a compound previously shown to inhibit C. albicans Yak1, prevents stress-mediated septal spore blocking and synergises with the azoles to inhibit A. fumigatus growth.

Aspergillus fumigatus mitogen-activated protein kinase MpkA is involved in gliotoxin production and self-protection.

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. The Mitogen-Activated Protein 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. The sensor histidine kinase SlnASln1 is important for modulation of MpkA phosphorylation. Our work emphasizes the importance of MpkA and compartmentalization of cellular events for GT production and self-defense.

Prevalence of chronic pulmonary aspergillosis along the continuum of pulmonary tuberculosis care: A protocol for a living systematic review and meta-analysis.

INTRODUCTION: Chronic pulmonary aspergillosis (CPA) is a debilitating disease estimated to affect over 3 million people worldwide. Pulmonary tuberculosis (PTB) is the most significant risk factor for CPA. However, the true burden of CPA at the time of PTB diagnosis, during, and after PTB treatment remains unknown. In this paper, we present a protocol for a living systematic review aimed at estimating the current burden of CPA along the continuum of PTB care. MATERIALS AND METHODS: We followed the Preferred Reporting Items for Systematic Reviews and Meta-Analyses Protocols (PRISMA-P) guidelines to formulate this protocol, which is registered with the International Prospective Register of Systematic Reviews (PROSPERO: CRD42023453900). We will identify primary literature through various electronic databases, including CINAHL, Ovid MEDLINE, MEDLINE (PubMed), EMBASE, Google Scholar, Cochrane Database of Systematic Reviews, and African Journal Online. The search will encompass articles from inception to December 31st, 2023, using medical subject heading search terms "pulmonary tuberculosis" AND "chronic pulmonary aspergillosis". Two reviewers will independently assess titles, abstracts, and full texts for eligibility using the Covidence web-based software. The eligible studies will comprise original observational research that reports on the prevalence of CPA diagnosed in individuals with PTB, based on established criteria, without language or geographic restriction. We intend to exclude single case reports and case series with fewer than 10 participants, as well as review articles, guidelines, and letters to the editors. Cochrane Risk of Bias Tools (ROB2 and ROBINS-I) will used to assess study quality and risk of bias and the quality of the evidence will be rated using the Grading of Recommendations, Assessment, Development and Evaluations (GRADE) tool. Our data syntheses will encompass meta-analysis and meta-regression, conducted using STATA version 18 and R- Studio version 4.0.2. This systematic review will be updated every 3-5 years as more data emerges. CONCLUSIONS: The findings of this proposed systematic review will summarize the available evidence on the occurrence of CPA, at the time of PTB diagnosis, during and after PTB treatment. The study results have the potential to guide healthcare policies regarding screening for CPA, enhance clinical decision-making, and catalyse further research into understanding the interplay between PTB and CPA. By shedding light on the current burden of CPA along the continuum of PTB care, we aspire to contribute to the betterment of patient care, disease management, and global health outcomes. PROSPERO REGISTRATION: CRD42023453900.

The C2H2 transcription factor SltA is required for germination and hyphal development in Aspergillus fumigatus.

Germination of inhaled Aspergillus fumigatus conidia is a necessary sequitur for infection. Germination of conidia starts with the breaking of dormancy, which is initiated by an increase of the cellular perimeter in a process termed isotropic growth. This swelling phase is followed by polarized growth, resulting in the formation of a germ tube. The multinucleate tubular cells exhibit tip growth from the hyphae, after which lateral branches emerge to form the mycelial network. The regulatory mechanisms governing conidial germination are not well defined. In this study, we identified a novel role for the transcription factor SltA in the orchestration of germination and hyphal development. Conidia lacking sltA fail to appropriately regulate isotropic growth and begin to swell earlier and subsequently switch to polarized growth faster. Additionally, hyphal development is distorted in a ∆sltA isolate as hyphae are hyper-branching and wider, and show branching at the apical tip. ∆sltA conidia are more tolerant to cell wall stressors on minimal medium compared to the wild-type (WT) strain. A transcriptome analysis of different stages of early growth was carried out to assess the regulatory role of SltA. Null mutants generated for three of the most dysregulated genes showed rapid germ tube emergence. Distinct from the phenotype observed for ∆sltA, conidia from these strains lacked defects in isotropic growth, but switched to polarized growth faster. Here, we characterize and describe several genes in the regulon of SltA, highlighting the complex nature of germination.IMPORTANCEAspergillus fumigatus is the main human fungal pathogen causing aspergillosis. For this fungus, azoles are the most commonly used antifungal drugs for treatment of aspergillosis. However, the prevalence of azole resistance is alarmingly increasing and linked with elevated mortality. Germination of conidia is crucial within its asexual life cycle and plays a critical role during the infection in the human host. Precluding germination could be a promising strategy considering the role of germination in Aspergillus spp. pathogenicity. Here, we identify a novel role for SltA in appropriate maintenance of dormancy, germination, and hyphal development. Three genes in the regulon of SltA were also essential for appropriate germination of conidia. With an expanding knowledge of germination and its different morphotypes, more advances can be made toward potential anti-germination targets for therapy.

The cystic fibrosis treatment Trikafta affects the growth, viability, and cell wall of Aspergillus fumigatus biofilms.

IMPORTANCE: PwCF commonly test positive for pathogenic fungi, and more than 90% of the cystic fibrosis patient population is approved for the modulator treatment, Trikafta. Therefore, it is critical to understand how fungal communities, specifically A. fumigatus, respond to Trikafta exposure. Therefore, we sought to determine whether Trikafta impacted the biology of A. fumigatus biofilms. Our data demonstrate that Trikafta reduces biomass in several laboratory strains as well as clinical strains isolated from the expectorated sputum of pwCF. Furthermore, Trikafta reduces fungal viability and the capacity of biofilms to recover following treatment. Of particular importance, Trikafta affects how A. fumigatus biofilms respond to cell wall stressors, suggesting that Trikafta modulates components of the cell wall. Since the cell wall directly affects how a host immune system will respond to and effectively neutralize pathogens, our work, demonstrating that Trikafta impacts the A. fumigatus cell wall, is potentially highly relevant to fungal-induced disease pathogenesis.

Fungal diseases in Africa: Closing the gaps in diagnosis and treatment through implementation research and advocacy.

Fungal diseases impose an escalating burden on public health in Africa, exacerbated by issues such as delayed diagnosis, inadequate therapy, and limited access to healthcare resources, resulting in significant morbidity and mortality. Effectively tackling these challenges demands a comprehensive approach encompassing research, training, and advocacy initiatives. Recent clinical mycology surveys conducted by Global Action for Fungal Infection (GAFFI) and the European Confederation of Medical Mycology/International Society for Human and Animal Mycology (ECMM/ISHAM) have underscored gaps in fungal diagnostics and the availability and accessibility of antifungal therapy in Africa. The World Health Organization (WHO) Fungal Priority Pathogens List (FPPL) identifies fungi of critical or high importance to human health, providing a roadmap for action and highlighting the urgent need for prioritizing fungal diseases and developing targeted interventions within the African context. To enhance diagnosis and treatment, it is imperative to invest in comprehensive training programs for healthcare workers across all levels and disciplines. Equipping them with the necessary knowledge and skills will facilitate early detection, accurate diagnosis, and appropriate management of fungal infections. Moreover, implementation science research in medical mycology assumes a pivotal role in bridging the gap between knowledge and practice. By identifying the barriers and facilitators that influence the adoption of diagnostic techniques and public health interventions, tailored strategies can be formulated to improve their implementation within healthcare settings. Advocacy plays a critical role in raising awareness regarding the profound impact of fungal diseases on public health in Africa. Engaging policymakers, healthcare providers, researchers, industry experts and communities underscore the importance of addressing these diseases and galvanize efforts for change. Substantial investment in surveillance, research and development specifically focused on fungal diseases is indispensable for advancing our understanding of local epidemiology, developing effective interventions, and ultimately improving patient outcomes. In conclusion, closing the gaps in diagnosing and treating fungal diseases in Africa demands concerted research and advocacy initiatives to ensure better healthcare delivery, reduced mortality rates, and improved public health outcomes.

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 to susceptibility to COVID-19-associated pulmonary aspergillosis (CAPA), little is known about the mechanisms underpinning copathogenicity. Here, we analyzed the genomes of 11 A. fumigatus isolates from patients with CAPA in three centers 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 indicate 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 better understand the molecular epidemiology of CAPA and to identify genetic drivers of copathogenicity and antifungal resistance in patients with COVID-19. IMPORTANCE Coronavirus 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 mold 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 analyzed the genomes of 11 A. fumigatus isolates from patients with CAPA in three centers from different European countries, and carried out phenotypic analyses with a view to understanding the pathophysiology of the disease. Our data indicate that A. fumigatus CAPA isolates are genomically diverse but are more similar to each other in their responses to infection-relevant stresses.

Aspergillus fumigatus Can Display 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 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.

Functional analysis of the Aspergillus fumigatus kinome reveals a DYRK kinase involved in septal plugging is a novel antifungal drug target.

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.

Vacuoles and peroxisomes are involved in Aspergillus fumigatus gliotoxin production and self-protection.

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.