RESUMO

Invasive Pulmonary Aspergillosis, which is caused by the filamentous fungus Aspergillus fumigatus, is a life-threatening infection for immunosuppressed patients. Chromatin structure regulation is important for genome stability maintenance and has the potential to drive genome rearrangements and affect virulence and pathogenesis of pathogens. Here, we performed the first A. fumigatus global chromatin profiling of two histone modifications, H3K4me3 and H3K9me3, focusing on the two most investigated A. fumigatus clinical isolates, Af293 and CEA17. In eukaryotes, H3K4me3 is associated with active transcription, while H3K9me3 often marks silent genes, DNA repeats, and transposons. We found that H3K4me3 deposition is similar between the two isolates, while H3K9me3 is more variable and does not always represent transcriptional silencing. Our work uncovered striking differences in the number, locations, and expression of transposable elements between Af293 and CEA17, and the differences are correlated with H3K9me3 modifications and higher genomic variations among strains of Af293 background. Moreover, we further showed that the Af293 strains from different laboratories actually differ in their genome contents and found a frequently lost region in chromosome VIII. For one such Af293 variant, we identified the chromosomal changes and demonstrated their impacts on its secondary metabolites production, growth and virulence. Overall, our findings not only emphasize the influence of genome heterogeneity on A. fumigatus fitness, but also caution about unnoticed chromosomal variations among common laboratory strains.

Assuntos

Aspergillus fumigatus/classificação , Cromossomos Fúngicos/genética , Heterogeneidade Genética , Histonas/metabolismo , Aspergilose Pulmonar/microbiologia , Aspergillus fumigatus/genética , Aspergillus fumigatus/isolamento & purificação , Cromatina , Elementos de DNA Transponíveis , Proteínas Fúngicas/metabolismo , Regulação da Expressão Gênica de Plantas , Aptidão Genética , Código das Histonas , Humanos , Regiões Promotoras Genéticas , Metabolismo Secundário , Virulência

RESUMO

Aspergillus fumigatus is the main causative agent of invasive pulmonary aspergillosis (IPA), a severe disease that affects immunosuppressed patients worldwide. The fungistatic drug caspofungin (CSP) is the second line of therapy against IPA but has increasingly been used against clinical strains that are resistant to azoles, the first line antifungal therapy. In high concentrations, CSP induces a tolerance phenotype with partial reestablishment of fungal growth called CSP paradoxical effect (CPE), resulting from a change in the composition of the cell wall. An increasing number of studies has shown that different isolates of A. fumigatus exhibit phenotypic heterogeneity, including heterogeneity in their CPE response. To gain insights into the underlying molecular mechanisms of CPE response heterogeneity, we analyzed the transcriptomes of two A. fumigatus reference strains, Af293 and CEA17, exposed to low and high CSP concentrations. We found that there is a core transcriptional response that involves genes related to cell wall remodeling processes, mitochondrial function, transmembrane transport, and amino acid and ergosterol metabolism, and a variable response related to secondary metabolite (SM) biosynthesis and iron homeostasis. Specifically, we show here that the overexpression of a SM pathway that works as an iron chelator extinguishes the CPE in both backgrounds, whereas iron depletion is detrimental for the CPE in Af293 but not in CEA17. We next investigated the function of the transcription factor CrzA, whose deletion was previously shown to result in heterogeneity in the CPE response of the Af293 and CEA17 strains. We found that CrzA constitutively binds to and modulates the expression of several genes related to processes involved in CSP tolerance and that crzA deletion differentially impacts the SM production and growth of Af293 and CEA17. As opposed to the ΔcrzACEA17 mutant, the ΔcrzAAf293 mutant fails to activate cell wall remodeling genes upon CSP exposure, which most likely severely affects its macrostructure and extinguishes its CPE. This study describes how heterogeneity in the response to an antifungal agent between A. fumigatus strains stems from heterogeneity in the function of a transcription factor and its downstream target genes.

Assuntos

Aspergillus fumigatus

RESUMO

The fungal zinc finger transcription factor NsdC is named after, and is best known for, its essential role in sexual reproduction (never in sexual development). In previous studies with Aspergillus nidulans, it was also shown to have roles in promotion of vegetative growth and suppression of asexual conidiation. In this study, the function of the nsdC homologue in the opportunistic human pathogen A. fumigatus was investigated. NsdC was again found to be essential for sexual development, with deletion of the nsdC gene in both MAT1-1 and MAT1-2 mating partners of a cross leading to complete loss of fertility. However, a functional copy of nsdC in one mating partner was sufficient to allow sexual reproduction. Deletion of nsdC also led to decreased vegetative growth and allowed conidiation in liquid cultures, again consistent with previous findings. However, NsdC in A. fumigatus was shown to have additional biological functions including response to calcium stress, correct organization of cell wall structure, and response to the cell wall stressors. Furthermore, virulence and host immune recognition were affected. Gene expression studies involving chromatin immunoprecipitation (ChIP) of RNA polymerase II (PolII) coupled to next-generation sequencing (Seq) revealed that deletion of nsdC resulted in changes in expression of over 620 genes under basal growth conditions. This demonstrated that this transcription factor mediates the activity of a wide variety of signaling and metabolic pathways and indicates that despite the naming of the gene, the promotion of sexual reproduction is just one among multiple roles of NsdC.IMPORTANCEAspergillus fumigatus is an opportunistic human fungal pathogen and the main causal agent of invasive aspergillosis, a life-threatening infection especially in immunocompromised patients. A. fumigatus can undergo both asexual and sexual reproductive cycles, and the regulation of both cycles involves several genes and pathways. Here, we have characterized one of these genetic determinants, the NsdC transcription factor, which was initially identified in a screen of transcription factor null mutants showing sensitivity when exposed to high concentrations of calcium. In addition to its known essential roles in sexual reproduction and control of growth rate and asexual reproduction, we have shown in the present study that A. fumigatus NsdC transcription factor has additional previously unrecognized biological functions including calcium tolerance, cell wall stress response, and correct cell wall organization and functions in virulence and host immune recognition. Our results indicate that NsdC can play novel additional biological functions not directly related to its role played during sexual and asexual processes.

Assuntos

Aspergillus fumigatus/genética , Aspergillus fumigatus/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Animais , Aspergilose/microbiologia , Aspergilose/patologia , Aspergillus fumigatus/patogenicidade , Parede Celular , Modelos Animais de Doenças , Feminino , Regulação Fúngica da Expressão Gênica , Genes Fúngicos/genética , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Mutação , Fenótipo , Reprodução Assexuada , Transdução de Sinais , Transcriptoma , Virulência/genética , Fatores de Virulência/genética

RESUMO

Invasive pulmonary aspergillosis is a life-threatening fungal infection especially in the immunocompromised patients. The low diversity of available antifungal drugs coupled with the emergence of antifungal resistance has become a worldwide clinical concern. The echinocandin Caspofungin (CSP) is recommended as a second-line therapy but resistance and tolerance mechanisms have been reported. However, how the fungal cell articulates the response to CSP is not completely understood. This work provides a detailed characterization of ZnfA, a transcription factor (TF) identified in previous screening studies that is involved in the A. fumigatus responses to calcium and CSP. This TF plays an important role in the regulation of iron homeostasis and cell wall organization in response to high CSP concentrations as revealed by Chromatin Immunoprecipitation coupled to DNA sequencing (ChIP-seq) analysis. Furthermore, ZnfA acts collaboratively with the key TF CrzA in modulating the response to calcium as well as cell wall and osmotic stresses. This study therefore describes the existence of an additional, previously unknown TF that bridges calcium signaling and the CSP cellular response and further exposes the complex connections that exist among different pathways which govern stress sensing and signaling in A. fumigatus.

RESUMO

Aspergillus fumigatus is an opportunistic fungal pathogen that secretes an array of immune-modulatory molecules, including secondary metabolites (SMs), which contribute to enhancing fungal fitness and growth within the mammalian host. Gliotoxin (GT) is a SM that interferes with the function and recruitment of innate immune cells, which are essential for eliminating A. fumigatus during invasive infections. We identified a C6 Zn cluster-type transcription factor (TF), subsequently named RglT, important for A. fumigatus oxidative stress resistance, GT biosynthesis and self-protection. RglT regulates the expression of several gli genes of the GT biosynthetic gene cluster, including the oxidoreductase-encoding gene gliT, by directly binding to their respective promoter regions. Subsequently, RglT was shown to be important for virulence in a chemotherapeutic murine model of invasive pulmonary aspergillosis (IPA). Homologues of RglT and GliT are present in eurotiomycete and sordariomycete fungi, including the non-GT-producing fungus A. nidulans, where a conservation of function was described. Phylogenetically informed model testing led to an evolutionary scenario in which the GliT-based resistance mechanism is ancestral and RglT-mediated regulation of GliT occurred subsequently. In conclusion, this work describes the function of a previously uncharacterised TF in oxidative stress resistance, GT biosynthesis and self-protection in both GT-producing and non-producing Aspergillus species.

Assuntos

Aspergilose , Aspergillus fumigatus/patogenicidade , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica/fisiologia , Gliotoxina/biossíntese , Fatores de Transcrição/metabolismo , Animais , Aspergilose/metabolismo , Aspergilose/microbiologia , Aspergillus fumigatus/metabolismo , Camundongos , Estresse Oxidativo/fisiologia , Virulência/fisiologia

RESUMO

Aspergillus fumigatus is the leading cause of pulmonary fungal diseases. Azoles have been used for many years as the main antifungal agents to treat and prevent invasive aspergillosis. However, in the last 10 years there have been several reports of azole resistance in A. fumigatus and new strategies are needed to combat invasive aspergillosis. Caspofungin is effective against other human-pathogenic fungal species, but it is fungistatic only against A. fumigatus Resistance to caspofungin in A. fumigatus has been linked to mutations in the fksA gene that encodes the target enzyme of the drug ß-1,3-glucan synthase. However, tolerance of high caspofungin concentrations, a phenomenon known as the caspofungin paradoxical effect (CPE), is also important for subsequent adaptation and drug resistance evolution. Here, we identified and characterized the transcription factors involved in the response to CPE by screening an A. fumigatus library of 484 null transcription factors (TFs) in CPE drug concentrations. We identified 11 TFs that had reduced CPE and that encoded proteins involved in the basal modulation of the RNA polymerase II initiation sites, calcium metabolism, and cell wall remodeling. One of these TFs, FhdA, was important for mitochondrial respiratory function and iron metabolism. The ΔfhdA mutant showed decreased growth when exposed to Congo red or to high temperature. Transcriptome sequencing (RNA-seq) analysis and further experimental validation indicated that the ΔfhdA mutant showed diminished respiratory capacity, probably affecting several pathways related to the caspofungin tolerance and resistance. Our results provide the foundation to understand signaling pathways that are important for caspofungin tolerance and resistance.IMPORTANCEAspergillus fumigatus, one of the most important human-pathogenic fungal species, is able to cause aspergillosis, a heterogeneous group of diseases that presents a wide range of clinical manifestations. Invasive pulmonary aspergillosis is the most serious pathology in terms of patient outcome and treatment, with a high mortality rate ranging from 50% to 95% primarily affecting immunocompromised patients. Azoles have been used for many years as the main antifungal agents to treat and prevent invasive aspergillosis. However, there were several reports of evolution of clinical azole resistance in the last decade. Caspofungin, a noncompetitive ß-1,3-glucan synthase inhibitor, has been used against A. fumigatus, but it is fungistatic and is recommended as second-line therapy for invasive aspergillosis. More information about caspofungin tolerance and resistance is necessary in order to refine antifungal strategies that target the fungal cell wall. Here, we screened a transcription factor (TF) deletion library for TFs that can mediate caspofungin tolerance and resistance. We have identified 11 TFs that are important for caspofungin sensitivity and/or for the caspofungin paradoxical effect (CPE). These TFs encode proteins involved in the basal modulation of the RNA polymerase II initiation sites, calcium metabolism or cell wall remodeling, and mitochondrial respiratory function. The study of those genes regulated by TFs identified in this work will provide a better understanding of the signaling pathways that are important for caspofungin tolerance and resistance.

Assuntos

Aspergillus fumigatus/efeitos dos fármacos , Aspergillus fumigatus/genética , Caspofungina/farmacologia , Farmacorresistência Fúngica/genética , Proteínas Fúngicas/metabolismo , Fatores de Transcrição/metabolismo , Animais , Antifúngicos/farmacologia , Aspergilose/microbiologia , Feminino , Regulação Fúngica da Expressão Gênica , Biblioteca Gênica , Camundongos , Camundongos Endogâmicos BALB C , Testes de Sensibilidade Microbiana , Transdução de Sinais

RESUMO

Aspergillus fumigatus is an opportunistic fungal pathogen that secretes an array of immune-modulatory molecules, including secondary metabolites (SMs), which contribute to enhancing fungal fitness and growth within the mammalian host. Gliotoxin (GT) is a SM that interferes with the function and recruitment of innate immune cells, which are essential for eliminating A. fumigatus during invasive infections. We identified a C6 Zn cluster-type transcription factor (TF), subsequently named RglT, important for A. fumigatus oxidative stress resistance, GT biosynthesis and self-protection. RglT regulates the expression of several gli genes of the GT biosynthetic gene cluster, including the oxidoreductase-encoding gene gliT, by directly binding to their respective promoter regions. Subsequently, RglT was shown to be important for virulence in a chemotherapeutic murine model of invasive pulmonary aspergillosis (IPA). Homologues of RglT and GliT are present in eurotiomycete and sordariomycete fungi, including the non-GT-producing fungus A. nidulans, where a conservation of function was described. Phylogenetically informed model testing led to an evolutionary scenario in which the GliT-based resistance mechanism is ancestral and RglT-mediated regulation of GliT occurred subsequently. In conclusion, this work describes the function of a previously uncharacterised TF in oxidative stress resistance, GT biosynthesis and self-protection in both GT-producing and non-producing Aspergillus species.